An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Science.gov (United States)

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

OpenAIRE

Le Gall, O; Candresse, T; Brault, V; Dunez, J

1989-01-01

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that o...

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) using pre-steady-state kinetics

Science.gov (United States)

Muftuoglu, Yagmur; Sohl, Christal D.; Mislak, Andrea C.; Mitsuya, Hiroaki; Sarafianos, Stefan G.; Anderson, Karen S.

2014-01-01

The novel antiretroviral 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3′-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3′-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. PMID:24632447

Alkyne- and 1,6-elimination- succinimidyl carbonate – terminated heterobifunctional poly(ethylene glycol) for reversible "Click" PEGylation

OpenAIRE

Xie, Yumei; Duan, Shaofeng; Forrest, M. Laird

2010-01-01

A new heterobifunctional (succinimidyl carbonate, SC)-activated poly(ethylene glycol) (PEG) with a reversible 1,6-elimination linker and a terminal alkyne for "click" chemistry was synthesized with high efficiency and low polydispersity. The α-alkyne-ω-hydroxyl PEG was first prepared using trimethylsilyl-2-propargyl alcohol as an initiator for ring-opening polymerization of ethylene oxide followed by mild deprotection with tetrabutylammonium fluoride. The hydroxy end was then modified with di...

DNA Nucleotide Sequence Restricted by the RI Endonuclease

Science.gov (United States)

Hedgpeth, Joe; Goodman, Howard M.; Boyer, Herbert W.

1972-01-01

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage λ has been determined. The 5′-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in λ DNA was deduced from these data and from the 3′-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5′-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry. PMID:4343974

Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics.

Science.gov (United States)

Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C; Mitsuya, Hiroaki; Sarafianos, Stefan G; Anderson, Karen S

2014-06-01

The novel antiretroviral 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a potent nucleoside HIV-1 reverse transcriptase (RT) inhibitor (NRTI). Unlike other FDA-approved NRTIs, EFdA contains a 3'-hydroxyl. Pre-steady-state kinetics showed RT preferred incorporating EFdA-TP over native dATP. Moreover, RT slowly inserted nucleotides past an EFdA-terminated primer, resulting in delayed chain termination with unaffected fidelity. This is distinct from KP1212, another 3'-hydroxyl-containing RT inhibitor considered to promote viral lethal mutagenesis. New mechanistic features of RT inhibition by EFdA are revealed. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

Science.gov (United States)

Wang, Jianye; Huang, Yu; Zhou, Mingxu; Zhu, Guoqiang

2016-09-01

Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure.

Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

Directory of Open Access Journals (Sweden)

Fowke Keith R

2005-10-01

Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal

Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1.

Science.gov (United States)

Le Gall, O; Candresse, T; Brault, V; Dunez, J

1989-10-11

The nucleotide sequence of the RNA1 of hungarian grapevine chrome mosaic virus, a nepovirus very closely related to tomato black ring virus, has been determined from cDNA clones. It is 7212 nucleotides in length excluding the 3' terminal poly(A) tail and contains a large open reading frame extending from nucleotides 216 to 6971. The presumably encoded polyprotein is 2252 amino acids in length with a molecular weight of 250 kDa. The primary structure of the polyprotein was compared with that of other viral polyproteins, revealing the same general genetic organization as that of other picorna-like viruses (comoviruses, potyviruses and picornaviruses), except that an additional protein is suspected to occupy the N-terminus of the polyprotein.

Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

Energy Technology Data Exchange (ETDEWEB)

Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

2006-05-15

Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

The nucleotide sequence of satellite RNA in grapevine fanleaf virus, strain F13.

Science.gov (United States)

Fuchs, M; Pinck, M; Serghini, M A; Ravelonandro, M; Walter, B; Pinck, L

1989-04-01

The nucleotide sequence of cDNA copies of grapevine fanleaf virus (strain F13) satellite RNA has been determined. The primary structure obtained was 1114 nucleotides in length, excluding the poly(A) tail, and contained only one long open reading frame encoding a 341 residue, highly hydrophilic polypeptide of Mr37275. The coding sequence was bordered by a leader of 14 nucleotides and a 3'-terminal non-coding region of 74 nucleotides. No homology has been found with small satellite RNAs associated with other nepoviruses. Two limited homologies of eight nucleotides have been detected between the satellite RNA in grapevine fanleaf virus and those in tomato black ring virus, and a consensus sequence U.G/UGAAAAU/AU/AU/A at the 5' end of nepovirus RNAs is reported. A less extended consensus exists in this region in comovirus and picornavirus RNA.

Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Madura, K.; Prakash, S.

1986-01-01

The authors determined the nucleotide sequence, mapped the 5' and 3' nRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae. A long open reading frame within the RAD2 transcribed region encodes a protein of 1031 amino acids with a calculated molecular weight of 117,847. A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function. The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame. The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame. The RAD2 3' nRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination. Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light. The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nuclotide-long purine-rich tract. The sequence T G G A G G C A T T A A found at position - 167 to -156 in the RAD2 gene is similar to at sequence present in the 5' flanking regions of the RAD7 and RAD10 genes

Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

International Nuclear Information System (INIS)

Dong Hongping; Zhang Bo; Shi Peiyong

2008-01-01

Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5 binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis

Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila.

Science.gov (United States)

Jeong, Sangyun; Yang, Da-Som; Hong, Young Gi; Mitchell, Sarah P; Brown, Matthew P; Kolodkin, Alex L

2017-09-26

The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. Here, we uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.

An Inhibitory Motif on the 5’UTR of Several Rotavirus Genome Segments Affects Protein Expression and Reverse Genetics Strategies

Science.gov (United States)

Papa, Guido; Eichwald, Catherine; Burrone, Oscar R.

2016-01-01

Rotavirus genome consists of eleven segments of dsRNA, each encoding one single protein. Viral mRNAs contain an open reading frame (ORF) flanked by relatively short untranslated regions (UTRs), whose role in the viral cycle remains elusive. Here we investigated the role of 5’UTRs in T7 polymerase-driven cDNAs expression in uninfected cells. The 5’UTRs of eight genome segments (gs3, gs5-6, gs7-11) of the simian SA11 strain showed a strong inhibitory effect on the expression of viral proteins. Decreased protein expression was due to both compromised transcription and translation and was independent of the ORF and the 3’UTR sequences. Analysis of several mutants of the 21-nucleotide long 5’UTR of gs 11 defined an inhibitory motif (IM) represented by its primary sequence rather than its secondary structure. IM was mapped to the 5’ terminal 6-nucleotide long pyrimidine-rich tract 5’-GGY(U/A)UY-3’. The 5’ terminal position within the mRNA was shown to be essentially required, as inhibitory activity was lost when IM was moved to an internal position. We identified two mutations (insertion of a G upstream the 5’UTR and the U to A mutation of the fifth nucleotide of IM) that render IM non-functional and increase the transcription and translation rate to levels that could considerably improve the efficiency of virus helper-free reverse genetics strategies. PMID:27846320

Molecular cloning and complete nucleotide sequence of a human ventricular myosin light chain 1

Energy Technology Data Exchange (ETDEWEB)

Hoffmann, E; Shi, Q W; Floroff, M; Mickle, D A.G.; Wu, T W; Olley, P M; Jackowski, G

1988-03-25

Human ventricular plasmid library was constructed. The library was screened with the oligonucleotide probe (17-mer) corresponding to a conserve region of myosin light chain 1 near the carboxy terminal. Full length cDNA recombinant plasmid containing 1100 bp insert was isolated. RNA blot hybridization with this insert detected a message of approximately 1500 bp corresponding to the size of VLCl and mRNA. Complete nucleotide sequence of the coding region was determined in M13 subclones using dideoxy chain termination method. With the isolation of this clone (pCD HLVCl), the publication of the complete nucleotide sequence of HVLCl and the predicted secondary structure of this protein will aid in understanding of the biochemistry of myosin and its function in contraction, the evolution of myosin light genes and the genetic, developmental and physiological regulation of myosin genes.

The nucleotide sequence of the right-hand terminus of adenovirus type 5 DNA: Implications for the mechanism of DNA replication

NARCIS (Netherlands)

Steenbergh, P.H.; Sussenbach, J.S.

The nucleotide sequence of the right-hand terminal 3% of adenovirus type 5 (Ad5) DNA has been determined, using the chemical degradation technique developed by Maxam and Gilbert (1977). This region of the genome comprises the 1003 basepair long HindIII-I fragment and the first 75 nucleotides of the

Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

International Nuclear Information System (INIS)

Suzuki, Nobuhiro; Yamazaki, Yasuo; Brown, R. Lane; Fujimoto, Zui; Morita, Takashi; Mizuno, Hiroshi

2008-01-01

The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn 2+ -bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn 2+ ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn 2+ binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels

Structures of pseudechetoxin and pseudecin, two snake-venom cysteine-rich secretory proteins that target cyclic nucleotide-gated ion channels: implications for movement of the C-terminal cysteine-rich domain

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Nobuhiro [Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan); Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Yamazaki, Yasuo [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Brown, R. Lane [Neurological Science Institute, Oregon Health and Science University, Beaverton, Oregon 97006 (United States); Fujimoto, Zui [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); Morita, Takashi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Mizuno, Hiroshi, E-mail: tmorita@my-pharm.ac.jp [Department of Biochemistry, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602 (Japan); VALWAY Technology Center, NEC Soft Ltd, Koto-ku, Tokyo 136-8627 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Central 6, Tsukuba, Ibaraki 305-8566 (Japan); Department of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572 (Japan)

2008-10-01

The structures of pseudechetoxin and pseudecin suggest that both proteins bind to cyclic nucleotide-gated ion channels in a manner in which the concave surface occludes the pore entrance. Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction by retinal photoreceptors and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins belong to a cysteine-rich secretory protein (CRISP) family containing an N-terminal pathogenesis-related proteins of group 1 (PR-1) domain and a C-terminal cysteine-rich domain (CRD). PsTx and Pdc are highly homologous proteins, but their blocking affinities on CNG channels are different: PsTx blocks both the olfactory and retinal channels with ∼15–30-fold higher affinity than Pdc. To gain further insights into their structure and function, the crystal structures of PsTx, Pdc and Zn{sup 2+}-bound Pdc were determined. The structures revealed that most of the amino-acid-residue differences between PsTx and Pdc are located around the concave surface formed between the PR-1 domain and the CRD, suggesting that the concave surface is functionally important for CNG-channel binding and inhibition. A structural comparison in the presence and absence of Zn{sup 2+} ion demonstrated that the concave surface can open and close owing to movement of the CRD upon Zn{sup 2+} binding. The data suggest that PsTx and Pdc occlude the pore entrance and that the dynamic motion of the concave surface facilitates interaction with the CNG channels.

The effect of nucleotides and adenosine on stimulus-evoked glutamate release from rat brain cortical slices

OpenAIRE

Bennett, Gillian C; Boarder, Michael R

2000-01-01

Evidence has previously been presented that P1 receptors for adenosine, and P2 receptors for nucleotides such as ATP, regulate stimulus-evoked release of biogenic amines from nerve terminals in the brain. Here we investigated whether adenosine and nucleotides exert presynaptic control over depolarisation-elicited glutamate release.Slices of rat brain cortex were perfused and stimulated with pulses of 46 mM K+ in the presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxyl...

Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region

International Nuclear Information System (INIS)

Riley, L.K.; Morrow, J.K.; Danton, M.J.; Coleman, M.S.

1988-01-01

Human terminal deoxyribonucleotidyltransferase cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones, containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. TATA and CAAT sequences, respectively, were found to start at nucleotides -185 and -204, -328 and -370, and -465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements

A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template–primer

Science.gov (United States)

Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S.; Rouzaut, Ana; Martinez-Irujo, Juan J.

2007-01-01

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-type and AZT (3′-azido-3′-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template–primer (Kd=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template–primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues. PMID:17355225

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

Science.gov (United States)

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

Immune pressure analysis of protease and reverse transcriptase ...

African Journals Online (AJOL)

/dn) were analyzed for 33 HIV-1 subtype C protease (PR) and reverse transcriptase (RT) nucleotide sequences each from antiretroviral naïve South African chronically infected individuals. The ds/dn ratios were calculated using the ...

Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats

Directory of Open Access Journals (Sweden)

Desirée Villahermosa

2017-05-01

Full Text Available Defective mismatch repair (MMR in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6, which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1, which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3 recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe. Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1. Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.

Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

Science.gov (United States)

Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

2012-01-01

Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

Turn-on fluorescence probes based on pyranine/viologen charge-transfer complexes for the determination of nucleotides

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Schäferling, Michael, E-mail: Michael.schaeferling@utu.fi; Lang, Thomas; Schnettelker, Annette

2014-10-15

The formation of ground state charge-transfer complexes between pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid) and viologen (paraquat) derivatives is utilized for the design of novel fluoroionophores for the determination of phosphate species, particularly of nucleotides. The strong quenching of the pyranine fluorescence by viologen-type charge transfer acceptors can be countermanded if these are functionalized with triethylammonium groups that serve as recognition elements for phosphate anions. We report on the fluorogenic responses of these water-soluble molecular probes in presence of different phosphates. Absorbance measurements give additional information on the charge transfer complex formation and the interaction with nucleotides. The experimental data show that these aggregates form attractive, simple and versatile fluorescence turn-on probes for nucleoside triphosphates. The reversibility of the fluorescence response is demonstrated by means of an enzymatic model assay using ATPase for the decomposition of adenosine triphosphate. - Highlights: • Pyranine/viologen charge-transfer complexes as molecular probe for ATP recognition. • Fluorescence turn on mechanism. • Selective compared to other nucleotides and phosphate anions. • Fast and reversible response applicable to monitor enzymatic reactions.

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

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Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2016-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Science.gov (United States)

Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2016-01-15

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

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Moulin, Morgane; Alguacil, Javier; Gu, Siyi; Mehtougui, Asmaa; Adams, Erin J; Peyrottes, Suzanne; Champagne, Eric

2017-12-01

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

Quantum Coherent Three-Terminal Thermoelectrics: Maximum Efficiency at Given Power Output

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Robert S. Whitney

2016-05-01

Full Text Available This work considers the nonlinear scattering theory for three-terminal thermoelectric devices used for power generation or refrigeration. Such systems are quantum phase-coherent versions of a thermocouple, and the theory applies to systems in which interactions can be treated at a mean-field level. It considers an arbitrary three-terminal system in any external magnetic field, including systems with broken time-reversal symmetry, such as chiral thermoelectrics, as well as systems in which the magnetic field plays no role. It is shown that the upper bound on efficiency at given power output is of quantum origin and is stricter than Carnot’s bound. The bound is exactly the same as previously found for two-terminal devices and can be achieved by three-terminal systems with or without broken time-reversal symmetry, i.e., chiral and non-chiral thermoelectrics.

Nature of unstable insertional mutations and reversions at the cut locus of Drosophila melanogaster: Molecular mechanism for transpositional memory

International Nuclear Information System (INIS)

Mizrokhi, L.Yu.; Georgieva, S.G.; Obolenkova, L.A.; Priimyagi, A.F.; Gerasimova, T.I.; Il'in, Yu.V.

1988-01-01

A segment of the cut locus containing an mdg4 insertion as a result of ct MR and ct MRp10 mutations was cloned. Clones were obtained for the phenotypically different ct MR2 and ct MRpN10 mutants and for stable and unstable revertants. All mutations studied are associated with mdg4 insertion at an identical nucleotide sequence of the cut locus, the same site at which mdg4 is inserted at the ct 6 allele. The ct MRpN line differs from ct MR2 in that the mobile element jockey (3 kbp) is inserted in mdg4. Jockey is represented by about 1,000 copies per genome; it is homogeneous and lacks long terminal repeats (LTRs). In stable ct + reversions, mdg4 is completely excised. In unstable ct + reversions, in which there is a high degree of reverse directed transposition of mdg4 to the cut locus, an LTR of mdg4 is preserved at the site of the mutation. It is a sequence along which new copies of mdg4 or jockey-containing mdg4 are inserted into the genome. The authors discuss a molecular mechanism for transpositional memory involving homologous recombination of the remnant LTR and circular extrachromosomal copies of mdg4

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

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Olivier Martínez

Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

Terminal addition in a cellular world.

Science.gov (United States)

Torday, J S; Miller, William B

2018-07-01

Recent advances in our understanding of evolutionary development permit a reframed appraisal of Terminal Addition as a continuous historical process of cellular-environmental complementarity. Within this frame of reference, evolutionary terminal additions can be identified as environmental induction of episodic adjustments to cell-cell signaling patterns that yield the cellular-molecular pathways that lead to differing developmental forms. Phenotypes derive, thereby, through cellular mutualistic/competitive niche constructions in reciprocating responsiveness to environmental stresses and epigenetic impacts. In such terms, Terminal Addition flows according to a logic of cellular needs confronting environmental challenges over space-time. A reconciliation of evolutionary development and Terminal Addition can be achieved through a combined focus on cell-cell signaling, molecular phylogenies and a broader understanding of epigenetic phenomena among eukaryotic organisms. When understood in this manner, Terminal Addition has an important role in evolutionary development, and chronic disease might be considered as a form of 'reverse evolution' of the self-same processes. Copyright © 2017. Published by Elsevier Ltd.

Concept Layout Model of Transportation Terminals

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Li-ya Yao

2012-01-01

Full Text Available Transportation terminal is the key node in transport systems. Efficient terminals can improve operation of passenger transportation networks, adjust the layout of public transportation networks, provide a passenger guidance system, and regulate the development of commercial forms, as well as optimize the assembly and distribution of modern logistic modes, among others. This study aims to clarify the relationship between the function and the structure of transportation terminals and establish the function layout design. The mapping mechanism of demand, function, and structure was analyzed, and a quantitative relationship between function and structure was obtained from a design perspective. Passenger demand and terminal structure were decomposed into several demand units and structural elements following the principle of reverse engineering. The relationship maps between these two kinds of elements were then analyzed. Function-oriented concept layout model of transportation terminals was established using the previous method. Thus, a technique in planning and design of transportation structures was proposed. Meaningful results were obtained from the optimization of transportation terminal facilities, which guide the design of the functional layout of transportation terminals and improve the development of urban passenger transportation systems.

Myotonic Dystrophy Type 1 RNA Crystal Structures Reveal Heterogeneous 1 × 1 Nucleotide UU Internal Loop Conformations

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Kumar, Amit; Park, HaJeung; Fang, Pengfei; Parkesh, Raman; Guo, Min; Nettles, Kendall W.; Disney, Matthew D. (Scripps)

2012-03-27

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[{und UU}GGGC(C{und U}G){sub 3}GUCC]{sub 2}, refined to 2.20 and 1.52 {angstrom} resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 x 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 {angstrom} structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 x 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 x 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 {angstrom} structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 x 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 x 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides

Science.gov (United States)

Lehninger, Albert L.; Vercesi, Anibal; Bababunmi, Enitan A.

1978-01-01

Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca2+ and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca2+ is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as β-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca2+. Successive cycles of Ca2+ release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca2+-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca2+, mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport. Images PMID:25436

NUCLEOTIDES IN INFANT FEEDING

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L.G. Mamonova

2007-01-01

Full Text Available The article reviews the application of nucleotides-metabolites, playing a key role in many biological processes, for the infant feeding. The researcher provides the date on the nucleotides in the women's milk according to the lactation stages. She also analyzes the foreign experience in feeding newborns with nucleotides-containing milk formulas. The article gives a comparison of nucleotides in the adapted formulas represented in the domestic market of the given products.Key words: children, feeding, nucleotides.

Nucleotide Metabolism

DEFF Research Database (Denmark)

Martinussen, Jan; Willemoës, M.; Kilstrup, Mogens

2011-01-01

Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolis...

Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

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Maria Gaglione

2014-01-01

Full Text Available The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3′ end, carrying 5′-phosphate and 3′-hydroxyl groups (siRNAs. Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3′ overhang recognition by the PAZ domain and the 5′-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA moieties at 5′, and at 3′ positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.

Crystallization and preliminary X-ray crystallographic characterization of a cyclic nucleotide-binding homology domain from the mouse EAG potassium channel

International Nuclear Information System (INIS)

Marques-Carvalho, Maria João; Morais-Cabral, João Henrique

2012-01-01

The crystallization conditions and preliminary crystal characterization of the cytoplasmic cyclic nucleotide-binding homology domain from the mouse EAG potassium channel are reported. The members of the family of voltage-gated KCNH potassium channels play important roles in cardiac and neuronal repolarization, tumour proliferation and hormone secretion. These channels have a C-terminal cytoplasmic domain which is homologous to cyclic nucleotide-binding domains (CNB-homology domains), but it has been demonstrated that channel function is not affected by cyclic nucleotides and that the domain does not bind nucleotides in vitro. Here, the crystallization and preliminary crystallographic analysis of a CNB-homology domain from a member of the KCNH family, the mouse EAG channel, is reported. X-ray diffraction data were collected to 2.2 Å resolution and the crystal belonged to the hexagonal space group P3 1 21

Universal statistics of terminal dynamics before collapse

Science.gov (United States)

Lenner, Nicolas; Eule, Stephan; Wolf, Fred

Recent biological developments have both drastically increased the precision as well as amount of generated data, allowing for a switching from pure mean value characterization of the process under consideration to an analysis of the whole ensemble, exploiting the stochastic nature of biology. We focus on the general class of non-equilibrium processes with distinguished terminal points as can be found in cell fate decision, check points or cognitive neuroscience. Aligning the data to a terminal point (e.g. represented as an absorbing boundary) allows to device a general methodology to characterize and reverse engineer the terminating history. Using a small noise approximation we derive mean variance and covariance of the aligned data for general finite time singularities.

Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH isoforms.

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Elaine C Thomas

Full Text Available We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH, a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii communication occurs between the Bateman and catalytic domains and (iii the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

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Gronenborn Angela M

2010-05-01

Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

International Nuclear Information System (INIS)

Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

1987-01-01

Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

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Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

2011-10-28

Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

A novel peptide-nucleotide dual vaccine of human telomerase reverse transcriptase induces a potent cytotoxic T-cell response in vivo

International Nuclear Information System (INIS)

Guo, Hong; Hao, Jia; Wu, Chao; Shi, Yun; Zhao, Xiao-yan; Fang, Dian-chun

2007-01-01

Human telomerase reverse transcriptase (hTERT) is highly expressed in over 85% of human cancers, which makes it a broadly applicable molecular target for cancer therapy. Several groups have demonstrated that hTERT can efficiently evoke specific cytotoxic T lymphocytes (CTL) responses for malignant tumors. In the present study, we developed a novel virus-like particulate peptide-nucleotide dual vaccine (PNDV) of hTERT, which was composed of a low-affinity epitope variant with encoding full-length gene in the same virus-size particulate. We verified the formation of PNDV by DNA retarding assay, DNase I protection assay and transmission electron microscopy, and confirmed its immunogenicity and transfection activities in mammalian cells. Furthermore, in vivo immunization of HLA-A2.1 transgenic mice generated efficient IFN-γ secretion and hTERT-specific CTLs which are known to cause selective cell death of telomerase positive gastrointestinal cancer cells. To our knowledge, this represents the first report on collocating a low-affinity epitope variant with a full-length hTERT gene for anti-cancer vaccine design. This novel strategy for vaccine design not only enables enhanced immunity to a universal tumor antigen, but also has the potential to generate CTLs effective in telomerase-positive tumor cells of diverse tissue origins. Therefore, our findings bear significant implications for immunotherapy of human cancers

Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV)

International Nuclear Information System (INIS)

Wypijewski, K.; Musial, W.; Augustyniak, J.; Malinowski, T.

1994-01-01

The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription - polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequences of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D. (author). 32 refs, 1 fig., 2 tabs

Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

International Nuclear Information System (INIS)

Steenbergh, P.H.

1979-01-01

The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

Nucleotide excision repair in differentiated cells

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Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

2007-01-03

Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

Delineation of the Exact Transcription Termination Signal for Type 3 Polymerase III

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Zongliang Gao

2018-03-01

Full Text Available Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1. Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of ≥6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1–6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III.

The effect of nucleotides and adenosine on stimulus-evoked glutamate release from rat brain cortical slices.

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Bennett, G C; Boarder, M R

2000-10-01

Evidence has previously been presented that P1 receptors for adenosine, and P2 receptors for nucleotides such as ATP, regulate stimulus-evoked release of biogenic amines from nerve terminals in the brain. Here we investigated whether adenosine and nucleotides exert presynaptic control over depolarisation-elicited glutamate release. Slices of rat brain cortex were perfused and stimulated with pulses of 46 mM K(+) in the presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (0.2 mM). High K(+) substantially increased efflux of glutamate from the slices. Basal glutamate release was unchanged by the presence of nucleotides or adenosine at concentrations of 300 microM. Adenosine, ATP, ADP and adenosine 5'-O-(3-thiotriphoshate) at 300 microM attenuated depolarisation-evoked release of glutamate. However UTP, 2-methylthio ATP, 2-methylthio ADP, and alpha,beta-methylene ATP at 300 microM had no effect on stimulated glutamate efflux. Adenosine deaminase blocked the effect of adenosine, but left the response to ATP unchanged. The A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine antagonised the inhibitory effect of both adenosine and ATP. Cibacron blue 3GA inhibited stimulus-evoked glutamate release when applied alone. When cibacron blue 3GA was present with ATP, stimulus-evoked glutamate release was almost eliminated. However, this P2 antagonist had no effect on the inhibition by adenosine. These results show that the release of glutamate from depolarised nerve terminals of the rat cerebral cortex is inhibited by adenosine and ATP. ATP appears to act directly and not through conversion to adenosine.

The roles of the RIIβ linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIβ protein kinase A: a small angle x-ray and neutron scattering study.

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Blumenthal, Donald K; Copps, Jeffrey; Smith-Nguyen, Eric V; Zhang, Ping; Heller, William T; Taylor, Susan S

2014-10-10

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIβ is structurally unique in that the type IIβ holoenzyme is much more compact than the free RIIβ homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIβ C-terminal deletion mutant (RIIβ(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIβ holoenzyme and the RIIβ domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIβ holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIβ homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIβ isoform of PKA. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

GMP reverses the facilitatory effect of glutamate on inhibitory avoidance task in rats.

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Rubin, M A; Jurach, A; da Costa Júnior, E M; Lima, T T; Jiménez-Bernal, R E; Begnini, J; Souza, D O; de Mello, C F

1996-09-02

Previous studies have demonstrated that post-training intrahippocampal glutamate administration improves inhibitory avoidance task performance in rats. Antagonism of the agonist actions of glutamate by guanine nucleotides has been shown at the molecular and behavioural level. In the present investigation we demonstrate that intrahippocampal co-administration of GMP (guanosine 5'-monophosphate) reverses the facilitatory effect of glutamate on the inhibitory avoidance learning paradigm and inhibits [3H]glutamate binding in hippocampal synaptic plasma membranes. These results suggest that guanine nucleotides may modulate glutamate actions.

Click functionalization of phenyl-capped bithiophene on azide-terminated self-assembled monolayers

International Nuclear Information System (INIS)

Zheng, Yijun; Cui, Jiaxi; Ikeda, Taichi

2015-01-01

Graphical abstract: - Highlights: • Electrochemically-active self-assembled monolayers with phenyl-capped bithiophene were prepared. • Post-functionalization method based on click chemistry solved the solubility issue of phenyl-capped thiophene alkanethiol. • The capture and release of the counter anions during the redox reaction were detectable by E-QCM. - Abstract: We immobilized tetra(ethylene glycol)-substituted phenyl-capped bithiophene with alkyne terminals (Ph2TPh-alkyne) on azide-terminated self-assembled monolayers (N 3 -SAMs) by Cu-catalyzed azide-alkyne cycloaddition reaction. Ph2TPh-functionalized SAMs on a gold substrate showed reversible electrochemical response. The surface densities of the azide groups in N 3 -SAMs and Ph2TPh units in Ph2TPh-functionalized SAMs were estimated to be 7.3 ± 0.3 × 10 −10 mol cm −2 and 4.6 ± 0.3 × 10 −10 mol cm −2 , respectively, by quartz crystal microbalance (QCM). Most of Ph2TPh-alkynes are considered to be anchored on N 3 -SAMs via both terminal groups. Ph2TPh-functionalized SAMs exhibited reversible redox peaks in cyclic voltammetry (CV). In redox reaction, reversible capture and release of the counter anion could be monitored by electrochemical QCM (E-QCM).

Click functionalization of phenyl-capped bithiophene on azide-terminated self-assembled monolayers

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Zheng, Yijun; Cui, Jiaxi [Max Planck Institute for Polymer Research (MPIP), Ackermannweg 10, Mainz 55128 (Germany); Ikeda, Taichi, E-mail: IKEDA.Taichi@nims.go.jp [Max Planck Institute for Polymer Research (MPIP), Ackermannweg 10, Mainz 55128 (Germany); Polymer Materials Unit, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044 (Japan)

2015-11-15

Graphical abstract: - Highlights: • Electrochemically-active self-assembled monolayers with phenyl-capped bithiophene were prepared. • Post-functionalization method based on click chemistry solved the solubility issue of phenyl-capped thiophene alkanethiol. • The capture and release of the counter anions during the redox reaction were detectable by E-QCM. - Abstract: We immobilized tetra(ethylene glycol)-substituted phenyl-capped bithiophene with alkyne terminals (Ph2TPh-alkyne) on azide-terminated self-assembled monolayers (N{sub 3}-SAMs) by Cu-catalyzed azide-alkyne cycloaddition reaction. Ph2TPh-functionalized SAMs on a gold substrate showed reversible electrochemical response. The surface densities of the azide groups in N{sub 3}-SAMs and Ph2TPh units in Ph2TPh-functionalized SAMs were estimated to be 7.3 ± 0.3 × 10{sup −10} mol cm{sup −2} and 4.6 ± 0.3 × 10{sup −10} mol cm{sup −2}, respectively, by quartz crystal microbalance (QCM). Most of Ph2TPh-alkynes are considered to be anchored on N{sub 3}-SAMs via both terminal groups. Ph2TPh-functionalized SAMs exhibited reversible redox peaks in cyclic voltammetry (CV). In redox reaction, reversible capture and release of the counter anion could be monitored by electrochemical QCM (E-QCM).

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

International Nuclear Information System (INIS)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

2015-01-01

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp [National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo, Hokkaido 062-8517 (Japan)

2015-10-23

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

Three-terminal resistive switching memory in a transparent vertical-configuration device

International Nuclear Information System (INIS)

Ungureanu, Mariana; Llopis, Roger; Casanova, Fèlix; Hueso, Luis E.

2014-01-01

The resistive switching phenomenon has attracted much attention recently for memory applications. It describes the reversible change in the resistance of a dielectric between two non-volatile states by the application of electrical pulses. Typical resistive switching memories are two-terminal devices formed by an oxide layer placed between two metal electrodes. Here, we report on the fabrication and operation of a three-terminal resistive switching memory that works as a reconfigurable logic component and offers an increased logic density on chip. The three-terminal memory device we present is transparent and could be further incorporated in transparent computing electronic technologies

The arabidopsis cyclic nucleotide interactome

KAUST Repository

Donaldson, Lara Elizabeth

2016-05-11

Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

Hyperpolarization-activated current (I(h)) in vestibular calyx terminals: characterization and role in shaping postsynaptic events.

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Meredith, Frances L; Benke, Tim A; Rennie, Katherine J

2012-12-01

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na(+) currents, voltage-dependent outward K(+) currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3-45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I(h)) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs(+) (1-5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I(h) was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I(h) was -123 mV, which shifted to -114 mV in the presence of cAMP. Activation of I(h) was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at -139 mV). Activation speeded up significantly (τ=136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I(h) could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I(h) was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin-Huxley type ionic conductances, removal of I(h) similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size

Naringin Reverses Hepatocyte Apoptosis and Oxidative Stress Associated with HIV-1 Nucleotide Reverse Transcriptase Inhibitors-Induced Metabolic Complications

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Oluwafeyisetan O. Adebiyi

2015-12-01

Full Text Available Nucleoside Reverse Transcriptase Inhibitors (NRTIs have not only improved therapeutic outcomes in the treatment of HIV infection but have also led to an increase in associated metabolic complications of NRTIs. Naringin’s effects in mitigating NRTI-induced complications were investigated in this study. Wistar rats, randomly allotted into seven groups (n = 7 were orally treated daily for 56 days with 100 mg/kg zidovudine (AZT (groups I, II III, 50 mg/kg stavudine (d4T (groups IV, V, VI and 3 mL/kg of distilled water (group VII. Additionally, rats in groups II and V were similarly treated with 50 mg/kg naringin, while groups III and VI were treated with 45 mg/kg vitamin E. AZT or d4T treatment significantly reduced body weight and plasma high density lipoprotein concentrations but increased liver weights, plasma triglycerides and total cholesterol compared to controls, respectively. Furthermore, AZT or d4T treatment significantly increased oxidative stress, adiposity index and expression of Bax protein, but reduced Bcl-2 protein expression compared to controls, respectively. However, either naringin or vitamin E significantly mitigated AZT- or d4T-induced weight loss, dyslipidemia, oxidative stress and hepatocyte apoptosis compared to AZT- or d4T-only treated rats. Our results suggest that naringin reverses metabolic complications associated with NRTIs by ameliorating oxidative stress and apoptosis. This implies that naringin supplements could mitigate lipodystrophy and dyslipidemia associated with NRTI therapy.

The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

Science.gov (United States)

Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

2014-08-01

Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Molecular modeling study for interaction between Bacillus subtilis Obg and Nucleotides.

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Yuno Lee

Full Text Available The bacterial Obg proteins (Spo0B-associated GTP-binding protein belong to the subfamily of P-loop GTPase proteins that contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain which is referred as the "Obg fold" and now it is considered as one of the new targets for antibacterial drug. When the Obg protein is associated with GTP, it becomes activated, because conformation of Obg fold changes due to the structural changes of GTPase switch elements in GTP binding site. In order to investigate the effects and structural changes in GTP bound to Obg and GTPase switch elements for activation, four different molecular dynamics (MD simulations were performed with/without the three different nucleotides (GTP, GDP, and GDP + Pi using the Bacillus subtilis Obg (BsObg structure. The protein structures generated from the four different systems were compared using their representative structures. The pattern of C(alpha-C(alpha distance plot and angle between the two Obg fold domains of simulated apo form and each system (GTP, GDP, and GDP+Pi were significantly different in the GTP-bound system from the others. The switch 2 element was significantly changed in GTP-bound system. Also root-mean-square fluctuation (RMSF analysis revealed that the flexibility of the switch 2 element region was much higher than the others. This was caused by the characteristic binding mode of the nucleotides. When GTP was bound to Obg, its gamma-phosphate oxygen was found to interact with the key residue (D212 of the switch 2 element, on the contrary there was no such interaction found in other systems. Based on the results, we were able to predict the possible binding conformation of the activated form of Obg with L13, which is essential for the assembly with ribosome.

Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

DEFF Research Database (Denmark)

Rickhag, Karl Mattias; Owens, WA; Winkler, Marie-Therese

2013-01-01

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate......-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects....

Abrupt climate changes during Termination III in Southern Europe.

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Pérez-Mejías, Carlos; Moreno, Ana; Sancho, Carlos; Bartolomé, Miguel; Stoll, Heather; Cacho, Isabel; Cheng, Hai; Edwards, R Lawrence

2017-09-19

The Late Quaternary glacial-interglacial transitions represent the highest amplitude climate changes over the last million years. Unraveling the sequence of events and feedbacks at Termination III (T-III), including potential abrupt climate reversals similar to those of the last Termination, has been particularly challenging due to the scarcity of well-dated records worldwide. Here, we present speleothem data from southern Europe covering the interval from 262.7 to 217.9 kyBP, including the transition from marine isotope stage (MIS) 8 to MIS 7e. High-resolution δ 13 C, δ 18 O, and Mg/Ca profiles reveal major millennial-scale changes in aridity manifested in changing water availability and vegetation productivity. uranium-thorium dates provide a solid chronology for two millennial-scale events (S8.1 and S8.2) which, compared with the last two terminations, has some common features with Heinrich 1 and Heinrich 2 in Termination I (T-I).

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

Science.gov (United States)

Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2015-01-01

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation

Non-nucleotide Agonists Triggering P2X7 Receptor Activation and Pore Formation

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Francesco Di Virgilio

2018-02-01

Full Text Available The P2X7 receptor (P2X7R is a ligand-gated plasma membrane ion channel belonging to the P2X receptor subfamily activated by extracellular nucleotides. General consensus holds that the physiological (and maybe the only agonist is ATP. However, scattered evidence generated over the last several years suggests that ATP might not be the only agonist, especially at inflammatory sites. Solid data show that NAD+ covalently modifies the P2X7R of mouse T lymphocytes, thus lowering the ATP threshold for activation. Other structurally unrelated agents have been reported to activate the P2X7R via a poorly understood mechanism of action: (a the antibiotic polymyxin B, possibly a positive allosteric P2X7R modulator, (b the bactericidal peptide LL-37, (c the amyloidogenic β peptide, and (d serum amyloid A. Some agents, such as Alu-RNA, have been suggested to activate the P2X7R acting on the intracellular N- or C-terminal domains. Mode of P2X7R activation by these non-nucleotide ligands is as yet unknown; however, these observations raise the intriguing question of how these different non-nucleotide ligands may co-operate with ATP at inflammatory or tumor sites. New information obtained from the cloning and characterization of the P2X7R from exotic mammalian species (e.g., giant panda and data from recent patch-clamp studies are strongly accelerating our understanding of P2X7R mode of operation, and may provide hints to the mechanism of activation of P2X7R by non-nucleotide ligands.

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

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Kondo, Jiro; Westhof, Eric

2011-10-01

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

Solution structure and DNA-binding properties of the C-terminal domain of UvrC from E.coli

NARCIS (Netherlands)

Singh, S.; Folkers, G.E.; Bonvin, A.M.J.J.; Boelens, R.; Wechselberger, R.W.; Niztayev, A.; Kaptein, R.

2002-01-01

The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5' incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix±hairpin±helix (HhH) motifs

Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

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Xiaoqi Zhao

Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

The arabidopsis cyclic nucleotide interactome

KAUST Repository

Donaldson, Lara Elizabeth; Meier, Stuart Kurt; Gehring, Christoph A

2016-01-01

Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

Science.gov (United States)

Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

Science.gov (United States)

Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

Energy Technology Data Exchange (ETDEWEB)

Zeymer, Cathleen, E-mail: cathleen.zeymer@mpimf-heidelberg.mpg.de; Barends, Thomas R. M.; Werbeck, Nicolas D.; Schlichting, Ilme; Reinstein, Jochen, E-mail: cathleen.zeymer@mpimf-heidelberg.mpg.de [Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg (Germany)

2014-02-01

High-resolution crystal structures together with mutational analysis and transient kinetics experiments were utilized to understand nucleotide sensing and the regulation of the ATPase cycle in an AAA+ molecular motor. ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg{sup 2+} ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

International Nuclear Information System (INIS)

Zeymer, Cathleen; Barends, Thomas R. M.; Werbeck, Nicolas D.; Schlichting, Ilme; Reinstein, Jochen

2014-01-01

High-resolution crystal structures together with mutational analysis and transient kinetics experiments were utilized to understand nucleotide sensing and the regulation of the ATPase cycle in an AAA+ molecular motor. ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg 2+ ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis

cis-acting elements at opposite ends of the Citrus tristeza virus genome differ in initiation and termination of subgenomic RNAs

International Nuclear Information System (INIS)

Ayllon, Maria A.; Gowda, Siddarame; Satyanarayana, Tatineni; Dawson, William O.

2004-01-01

Citrus tristeza virus (CTV), a member of the Closteroviridae with a plus-stranded genomic RNA of approximately 20 kb, produces 10 3'-coterminal subgenomic (sg) RNAs that serve as messenger (m)RNAs for its internal genes. In addition, a population of 5'-terminal sgRNAs of approximately 700 nts are highly abundant in infected cells. Previous analysis demonstrated that the controller elements (CE) are responsible for the 3'-terminal mRNAs and the small 5'-terminal sgRNAs differ in the number of additional sgRNAs produced. A feature of both types of CE is production of 5'- and 3'-terminal positive-stranded sgRNAs, but the 3' CEs additionally produce a negative-stranded complement of the 3'-terminal mRNAs. Here, we found that the termination (for 5'-terminal sgRNAs) and initiation (for 3'-terminal sgRNAs) sites of the 5' vs. the 3' CEs occur at opposite ends of the respective minimal active CEs. The initiation site for the 3' CE of the major coat protein gene, and probably those of the p20 and p23 genes, was outside (3' in terms of the genomic RNA) the minimal unit, whereas the termination sites were located within the minimal CE, 30-50 nts upstream of the initiation site (referring to the positive-strand sequence). In contrast, the initiation site for the 5' CE was in the 5' region of the minimal unit, with the termination sites 20-35 nts downstream (referring to the positive-strand sequence). Furthermore, the CEs differ in initiation nucleotide and response to mutagenesis of that nucleotide. The 3' CE initiates sgRNA synthesis from a uridylate, whereas the 5' CE initiates from a cytidylate. We previously found that the 3' CEs were unusually tolerant to mutagenesis of the initiation sites, with initiation proceeding from alternative sites. Mutagenesis of the initiation site of the 5' CE prevented synthesis of either the 5'- or 3'-terminal sgRNAs. Thus, the cis-acting elements at opposite ends of the genome are remarkably different, perhaps having arisen from different

Human immunodeficiency virus-1 protein Tat induces excitotoxic loss of presynaptic terminals in hippocampal cultures.

Science.gov (United States)

Shin, Angela H; Thayer, Stanley A

2013-05-01

Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin-proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Cyclic nucleotides and radioresistnace

International Nuclear Information System (INIS)

Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

1982-01-01

The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

Nucleotide diversity analysis of three major bacterial blight resistance genes in rice.

Directory of Open Access Journals (Sweden)

Waikhom Bimolata

Full Text Available Nucleotide sequence polymorphisms among R gene alleles influence the process of co-evolutionary interaction between host and pathogen by shaping the response of host plants towards invading pathogens. Here, we present the DNA sequence polymorphisms and diversities present among natural alleles of three rice bacterial blight resistance genes, Xa21, Xa26 and xa5. The diversity was examined across different wild relatives and cultivars of Oryza species. Functional significance of selected alleles was evaluated through semi-quantitative reverse transcription polymerase chain reaction and real time PCR. The greatest nucleotide diversity and singleton variable sites (SVS were present in Xa26 (π = 0.01958; SVS = 182 followed by xa5 and Xa21 alleles. The highest frequency of single nucleotide polymorphisms were observed in Xa21 alleles and least in xa5. Transition bias was observed in all the genes and 'G' to 'A' transitions were more favored than other form of transitions. Neutrality tests failed to show the presence of selection at these loci, though negative Tajima's D values indicate the presence of a rare form of polymorphisms. At the interspecies level, O. nivara exhibited more diversity than O. sativa. We have also identified two nearly identical resistant alleles of xa5 and two sequentially identical alleles of Xa21. The alleles of xa5 showed basal levels of expression while Xa21 alleles were functionally not expressed.

An intermediate state of T7 RNA polymerase provides another pathway of nucleotide selection

International Nuclear Information System (INIS)

Wang Zhan-Feng; Liu Yu-Ru; Wang Peng-Ye; Xie Ping

2017-01-01

Phage T7 RNA polymerase is a single-subunit transcription enzyme, transcribing template DNA to RNA. Nucleoside triphosphate (NTP) selection and translocation are two critical steps of the transcription elongation. Here, using all-atom molecular dynamics simulations, we found that between pre- and post-translocation states of T7 RNA polymerase an intermediate state exists, where the O helix C-terminal residue tyrosine 639, which plays important roles in translocation, locates between its pre- and post-translocation positions and the side chain of the next template DNA nucleotide has moved into the active site. NTP selection in this intermediate state was studied, revealing that the selection in the intermediate state can be achieved relying on the effect of Watson–Crick interaction between NTP and template DNA nucleotide, effect of stability of the components near the active site such as the nascent DNA–RNA hybrid and role of tyrosine 639. This indicates that another NTP-selection pathway can also exist besides the main pathway where NTP selection begins at the post-translocation state upon the entry of NTP. (paper)

Laser processing for bevel termination of high voltage pn junction in SiC

International Nuclear Information System (INIS)

Kubiak, A; Ruta, Ł; Rosowski, A; French, P

2016-01-01

Proper edge termination of the p-n junction in silicon carbide is a key requirement in the fabrication of discrete devices able to withstand high voltages in reverse polarization. Due to the hardness of SiC the creation of the bevel termination remains difficult using mechanical machining. The use of laser beam sources with medium wavelength (532 nm) gives new possibilities in the machining of the silicon carbide. The paper presents the fabrication of the bevel termination structure in SiC using a green DPSS laser equipped with scanner and dedicated rotating sample holder. Characterization of the resulting structures proves the high potential of the proposed approach. (paper)

Crystallographic and single-particle analyses of native- and nucleotide-bound forms of the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

Science.gov (United States)

Awayn, N H; Rosenberg, M F; Kamis, A B; Aleksandrov, L A; Riordan, J R; Ford, R C

2005-11-01

Cystic fibrosis, one of the major human inherited diseases, is caused by defects in the CFTR (cystic fibrosis transmembrane conductance regulator), a cell-membrane protein. CFTR acts as a chloride channel which can be opened by ATP. Low-resolution structural studies of purified recombinant human CFTR are described in the present paper. Localization of the C-terminal decahistidine tag in CFTR was achieved by Ni2+-nitriloacetate nanogold labelling, followed by electron microscopy and single-particle analysis. The presence of the gold label appears to improve the single-particle-alignment procedure. Projection structures of CFTR from two-dimensional crystals analysed by electron crystallography displayed two alternative conformational states in the presence of nucleotide and nanogold, but only one form of the protein was observed in the quiescent (nucleotide-free) state.

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1987-01-01

The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

Plasmodium falciparum Hsp70-z, an Hsp110 homologue, exhibits independent chaperone activity and interacts with Hsp70-1 in a nucleotide-dependent fashion.

Science.gov (United States)

Zininga, Tawanda; Achilonu, Ikechukwu; Hoppe, Heinrich; Prinsloo, Earl; Dirr, Heini W; Shonhai, Addmore

2016-05-01

The role of molecular chaperones, among them heat shock proteins (Hsps), in the development of malaria parasites has been well documented. Hsp70s are molecular chaperones that facilitate protein folding. Hsp70 proteins are composed of an N-terminal nucleotide binding domain (NBD), which confers them with ATPase activity and a C-terminal substrate binding domain (SBD). In the ADP-bound state, Hsp70 possesses high affinity for substrate and releases the folded substrate when it is bound to ATP. The two domains are connected by a conserved linker segment. Hsp110 proteins possess an extended lid segment, a feature that distinguishes them from canonical Hsp70s. Plasmodium falciparum Hsp70-z (PfHsp70-z) is a member of the Hsp110 family of Hsp70-like proteins. PfHsp70-z is essential for survival of malaria parasites and is thought to play an important role as a molecular chaperone and nucleotide exchange factor of its cytosolic canonical Hsp70 counterpart, PfHsp70-1. Unlike PfHsp70-1 whose functions are fairly well established, the structure-function features of PfHsp70-z remain to be fully elucidated. In the current study, we established that PfHsp70-z possesses independent chaperone activity. In fact, PfHsp70-z appears to be marginally more effective in suppressing protein aggregation than its cytosol-localized partner, PfHsp70-1. Furthermore, based on coimmunoaffinity chromatography and surface plasmon resonance analyses, PfHsp70-z associated with PfHsp70-1 in a nucleotide-dependent fashion. Our findings suggest that besides serving as a molecular chaperone, PfHsp70-z could facilitate the nucleotide exchange function of PfHsp70-1. These dual functions explain why it is essential for parasite survival.

Terminal Investment Strategies and Male Mate choice: Extreme Tests of Bateman.

Science.gov (United States)

Andrade, Maydianne C B; Kasumovic, Michael M

2005-11-01

Bateman's principle predicts the intensity of sexual selection depends on rates of increase of fecundity with mating success for each sex (Bateman slopes). The sex with the steeper increase (usually males) is under more intense sexual selection and is expected to compete for access to the sex under less intense sexual selection (usually females). Under Bateman and modern refinements of his ideas, differences in parental investment are key to defining Bateman slopes and thus sex roles. Other theories predict sex differences in mating investment, or any expenditures that reduce male potential reproductive rate, can also control sex roles. We focus on sexual behaviour in systems where males have low paternal investment but frequently mate only once in their lifetimes, after which they are often killed by the female. Mating effort (=terminal investment) is high for these males, and many forms of investment theory might predict sex role reversal. We find no qualitative evidence for sex role reversal in a sample of spiders that show this extreme male investment pattern. We also present new data for terminally-investing redback spiders (Latrodectus hasselti). Bateman slopes are relatively steep for male redbacks, and, as predicted by Bateman, there is little evidence for role reversal. Instead, males are competitive and show limited choosiness despite wide variation in female reproductive value. This study supports the proposal that high male mating investment coupled with low parental investment may predispose males to choosiness but will not lead to role reversal. We support the utility of using Bateman slopes to predict sex roles, even in systems with extreme male mating investment.

Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

Science.gov (United States)

Laurie, Diane E; Splan, Rebecca K; Green, Kari; Still, Katherine M; McKown, Robert L; Laurie, Gordon W

2012-09-12

Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

Directory of Open Access Journals (Sweden)

Alexandr P Kornev

2008-04-01

Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

Functional mechanism of C-terminal tail in the enzymatic role of porcine testicular carbonyl reductase: a combined experiment and molecular dynamics simulation study of the C-terminal tail in the enzymatic role of PTCR.

Directory of Open Access Journals (Sweden)

Minky Son

Full Text Available Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C-terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV. Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily.

Signals for the initiation and termination of synthesis of the viral strand of bacteriophage f1

International Nuclear Information System (INIS)

Dotto, G.P.; Horiuchi, K.; Jakes, K.S.; Zinder, N.D.

1983-01-01

In this paper the sequence around the plus origin that is required for efficient plus-strand synthesis as well as that necessary for gene-II-protein recognition is described. Results which demonstrate that the nucleotide sequence of the f1 plus origin contains two overlapping but distinct signals, one for initiation and the other for termination of plus-strand synthesis is presented. 29 references, 6 figures, 1 table

Nucleotide sequence preservation of human mitochondrial DNA

International Nuclear Information System (INIS)

Monnat, R.J. Jr.; Loeb, L.A.

1985-01-01

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately = to 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms

Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase

OpenAIRE

Gaur, Vineet; Vyas, Rajan; Fowler, Jason D.; Efthimiopoulos, Georgia; Feng, Joy Y.; Suo, Zucai

2014-01-01

Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) a...

Paleointensity Behavior and Intervals Between Geomagnetic Reversals in the Last 167 Ma

Science.gov (United States)

Kurazhkovskii, A. Yu.; Kurazhkovskaya, N. A.; Klain, B. I.

2018-01-01

The results of comparative analysis of the behavior of paleointensity and polarity (intervals between reversals) of the geomagnetic field for the last 167 Ma are presented. Similarities and differences in the behavior of these characteristics of the geomagnetic field are discussed. It is shown that bursts of paleointensity and long intervals between reversals occurred at high mean values of paleointensity in the Cretaceous and Paleogene. However, there are differences between the paleointensity behavior and the reversal regime: (1) the characteristic times of paleointensity variations are less than the characteristic times of the frequency of geomagnetic reversals, (2) the achievement of maximum values of paleointensity at the Cretaceous-Paleogene boundary and the termination of paleointensity bursts after the boundary of 45-40 Ma are not marked by explicit features in the geomagnetic polarity behavior.

Palindromic nucleotide analysis in human T cell receptor rearrangements.

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Santosh K Srivastava

Full Text Available Diversity of T cell receptor (TCR genes is primarily generated by nucleotide insertions upon rearrangement from their germ line-encoded V, D and J segments. Nucleotide insertions at V-D and D-J junctions are random, but some small subsets of these insertions are exceptional, in that one to three base pairs inversely repeat the sequence of the germline DNA. These short complementary palindromic sequences are called P nucleotides. We apply the ImmunoSeq deep-sequencing assay to the third complementarity determining region (CDR3 of the β chain of T cell receptors, and use the resulting data to study P nucleotides in the repertoire of naïve and memory CD8(+ and CD4(+ T cells. We estimate P nucleotide distributions in a cross section of healthy adults and different T cell subtypes. We show that P nucleotide frequency in all T cell subtypes ranges from 1% to 2%, and that the distribution is highly biased with respect to the coding end of the gene segment. Classification of observed palindromic sequences into P nucleotides using a maximum conditional probability model shows that single base P nucleotides are very rare in VDJ recombination; P nucleotides are primarily two bases long. To explore the role of P nucleotides in thymic selection, we compare P nucleotides in productive and non-productive sequences of CD8(+ naïve T cells. The naïve CD8(+ T cell clones with P nucleotides are more highly expanded.

Pre-Steady State Kinetic Investigation of the Incorporation of Anti-Hepatitis B Nucleotide Analogs Catalyzed by Non-Canonical Human DNA Polymerases

Science.gov (United States)

Brown, Jessica A.; Pack, Lindsey R.; Fowler, Jason D.; Suo, Zucai

2011-01-01

Antiviral nucleoside analogs have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogs can be limited by drug toxicity because the 5′-triphosphates of these nucleoside analogs (nucleotide analogs) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogs are substrates for the recently discovered human X- and Y-family DNA polymerases. Using pre-steady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogs approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogs were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3′-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogs catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity. PMID:22132702

Formation of carboxy- and amide-terminated alkyl monolayers on silicon(111) investigated by ATR-FTIR, XPS, and X-ray scattering: Construction of photoswitchable surfaces

DEFF Research Database (Denmark)

Rück-Braun, Karola; Petersen, Michael Åxman; Michalik, Fabian

2013-01-01

-FTIR and XPS studies of the fulgimide samples revealed closely covered amide-terminated SAMs. Reversible photoswitching of the headgroup was read out by applying XPS, ATR-FTIR, and difference absorption spectra in the mid-IR. In XPS, we observed a reversible breathing of the amide/imide C1s and N1s signals......We have prepared high-quality, densely packed, self-assembled monolayers (SAMs) of carboxy-terminated alkyl chains on Si(111). The samples were made by thermal grafting of methyl undec-10-enoate under an inert atmosphere and subsequent cleavage of the ester functionality to disclose the carboxylic...... zigzag-like substitution pattern for the ester- and carboxy-terminated monolayer. Hydrolysis of the remaining H-Si(111) bonds at the surface furnished HO-Si(111) groups according to XPS and attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) studies. The amide-terminated alkyl...

Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-shock system.

Science.gov (United States)

Grimshaw, J P; Jelesarov, I; Schönfeld, H J; Christen, P

2001-03-02

DnaK, a Hsp70 acting in concert with its co-chaperones DnaJ and GrpE, is essential for Escherichia coli to survive environmental stress, including exposure to elevated temperatures. Here we explored the influence of temperature on the structure of the individual components and the functional properties of the chaperone system. GrpE undergoes extensive but fully reversible conformational changes in the physiologically relevant temperature range (transition midpoint at approximately 48 degrees C), as observed with both circular dichroism measurements and differential scanning calorimetry, whereas no thermal transitions occur in DnaK and DnaJ between 15 degrees C and 48 degrees C. The conformational changes in GrpE appear to be important in controlling the interconversion of T-state DnaK (ATP-liganded, low affinity for polypeptide substrates) and R-state DnaK (ADP-liganded, high affinity for polypeptide substrates). The rate of the T --> R conversion of DnaK due to DnaJ-triggered ATP hydrolysis follows an Arrhenius temperature dependence. In contrast, the rate of the R --> T conversion due to GrpE-catalyzed ADP/ATP exchange increases progressively less with increasing temperature and even decreases at temperatures above approximately 40 degrees C, indicating a temperature-dependent reversible inactivation of GrpE. At heat-shock temperatures, the reversible structural changes of GrpE thus shift DnaK toward its high-affinity R state.

Supplementary Material for: The arabidopsis cyclic nucleotide interactome

KAUST Repository

Donaldson, Lara; Meier, Stuart; Gehring, Christoph A

2016-01-01

Abstract Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

Science.gov (United States)

Wang, Sihua; Ding, Mingcui; Duan, Xiaoran; Wang, Tuanwei; Feng, Xiaolei; Wang, Pengpeng; Yao, Wu; Wu, Yongjun; Yan, Zhen; Feng, Feifei; Yu, Songcheng; Wang, Wei

2017-09-01

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase ( hTERT ) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme Msp I for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency. © 2017 by the Association of Clinical Scientists, Inc.

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses.

Science.gov (United States)

Brennan, Benjamin; Rezelj, Veronica V; Elliott, Richard M

2017-08-15

SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3' rapid amplification of cDNA ends (RACE), we mapped the 3' end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3' end of the N mRNA terminates upstream of a 5'-GCCAGCC-3' motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy

In silico screening for inhibitors of p-glycoprotein that target the nucleotide binding domains.

Science.gov (United States)

Brewer, Frances K; Follit, Courtney A; Vogel, Pia D; Wise, John G

2014-12-01

Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein.

Science.gov (United States)

Okamura, Hideyasu; Nishikiori, Masaki; Xiang, Hongyu; Ishikawa, Masayuki; Katoh, Etsuko

2011-07-13

ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(∗)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram. Copyright © 2011 Elsevier Ltd. All rights reserved.

Study of 4H-SiC junction barrier Schottky diode using field guard ring termination

International Nuclear Information System (INIS)

Feng-Ping, Chen; Yu-Ming, Zhang; Hong-Liang, Lü; Yi-Men, Zhang; Jian-Hua, Huang

2010-01-01

This paper reports that the 4H-SiC Schottky barrier diode, PiN diode and junction barrier Schottky diode terminated by field guard rings are designed, fabricated and characterised. The measurements for forward and reverse characteristics have been done, and by comparison with each other, it shows that junction barrier Schottky diode has a lower reverse current density than that of the Schottky barrier diode and a higher forward drop than that of the PiN diode. High-temperature annealing is presented in this paper as well to figure out an optimised processing. The barrier height of 0.79 eV is formed with Ti in this work, the forward drop for the Schottky diode is 2.1 V, with an ideality factor of 3.2, and junction barrier Schottky diode with blocking voltage higher than 400 V was achieved by using field guard ring termination. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

Directory of Open Access Journals (Sweden)

Christelle Folio

2017-11-01

Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

Direct Determination of Six Cytokinin Nucleotide Monophosphates in Coconut Flesh by Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry.

Science.gov (United States)

Cao, Zhao-Yun; Ma, You-Ning; Sun, Li-Hua; Mou, Ren-Xiang; Zhu, Zhi-Wei; Chen, Ming-Xue

2017-11-15

Coconut contains many uncharacterized cytokinins that have important physiological effects in plants and humans. In this work, a method based on liquid chromatography-tandem mass spectrometry was developed for identification and quantification of six cytokinin nucleotide monophosphates in coconut flesh. Excellent separation was achieved using a low-coverage C18 bonded-phase column with an acidic mobile phase, which greatly improved the retention of target compounds. To enable high-throughput analysis, a single-step solid-phase extraction using mixed-mode anion-exchange cartridges was employed for sample preparation. This proved to be an effective method to minimize matrix effects and ensure high selectivity. The limits of detection varied from 0.06 to 0.3 ng/mL, and the limits of quantification ranged from 0.2 to 1.0 ng/mL. The linearity was statistically verified over 2 orders of magnitude, giving a coefficient of determination (R 2 ) greater than 0.9981. The mean recoveries were from 81 to 108%; the intraday precision (n = 6) was less than 11%; and the interday precision (n = 11) was within 14%. The developed method was applied to the determination of cytokinin nucleotide monophosphates in coconut flesh samples, and four of them were successfully identified and quantified. The results showed that trans-zeatin riboside-5'-monophosphate was the dominant cytokinin, with a concentration of 2.7-34.2 ng/g, followed by N 6 -isopentenyladenosine-5'-monophosphate (≤12.9 ng/g), while the concentrations of cis-zeatin riboside-5'-monophosphate and dihydrozeatin riboside-5'-monophosphate were less than 2.2 and 4.9 ng/g, respectively.

Protection against β-amyloid neurotoxicity by a non-toxic endogenous N-terminal β-amyloid fragment and its active hexapeptide core sequence.

Science.gov (United States)

Forest, Kelly H; Alfulaij, Naghum; Arora, Komal; Taketa, Ruth; Sherrin, Tessi; Todorovic, Cedomir; Lawrence, James L M; Yoshikawa, Gene T; Ng, Ho-Leung; Hruby, Victor J; Nichols, Robert A

2018-01-01

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application. © 2017 International Society for Neurochemistry.

Segment-specific terminal sequences of Bunyamwera bunyavirus regulate genome replication

International Nuclear Information System (INIS)

Barr, John N.; Elliott, Richard M.; Dunn, Ewan F.; Wertz, Gail W.

2003-01-01

Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are transcribed to give a single mRNA and replicated to generate an antigenome that is the template for synthesis of further genomic RNA strands. We modified an existing cDNA-derived RNA synthesis system to allow identification of BUNV RNA replication and transcription products by direct metabolic labeling. Direct RNA analysis allowed us to distinguish between template activities that affected either RNA replication or mRNA transcription, an ability that was not possible using previous reporter gene expression assays. We generated genome analogs containing the entire nontranslated terminal sequences of the S, M, and L BUNV segments surrounding a common sequence. Analysis of RNAs synthesized from these templates revealed that the relative abilities of BUNV segments to perform RNA replication was M > L > S. Exchange of segment-specific terminal nucleotides identified a 12-nt region located within both the 3' and 5' termini of the M segment that correlated with its high replication ability

Adenine nucleotide depletion from endothelial cells exposed to xanthine oxidase

International Nuclear Information System (INIS)

Aalto, T.K.; Raivio, K.O.

1990-01-01

Hypoxia causes breakdown of cellular nucleotides, accumulation of hypoxanthine (HX), and conversion of xanthine dehydrogenase into xanthine oxidase (XO). Upon reoxygenation, the HX-XO reaction generates free radicals, one potential mechanism of tissue damage. Because endothelial cells contain XO and are exposed to circulating HX, they are a likely target for damage. We studied the effect of XO and/or HX at physiologically relevant concentrations on nucleotide metabolism of cultured endothelial cells from human umbilical veins. Cells were labeled with [14C]adenine and incubated for up to 6 h with HX, XO, or both, in the absence or presence of serum. Adenine nucleotides from cell extracts and nucleotide breakdown products (HX, xanthine, and urate) from the medium were separated and counted. HX alone had no effect. XO (80 mU/ml) alone caused a 70% (no serum) or 40% (with serum) fall in adenine nucleotides and an equivalent increase of xanthine and urate. The combination of HX and XO caused a 90% (no serum) or 70% (with serum) decrease in nucleotides, decrease in energy charge, and detachment of cells from the culture plate. Nucleotide depletion was not accounted for by proteolytic activity in the XO preparation. Albumin was only half as effective as serum in preventing nucleotide loss. Thus exogenous XO, in the presence of endogenous HX, triggers adenine nucleotide catabolism, but endogenous XO activity is too low to influence nucleotide levels even at high exogenous HX concentrations. Serum limits the catabolic effect of XO and thus protects cells from free radical damage

Distribution of ³H within purine nucleotides of Ehrlich mouse ascites tumour cells after intraabdominal injection of myo-[2-³H]inositol

DEFF Research Database (Denmark)

Christensen, Søren; Klenow, H.; Overgaard-Hansen, Kay

2000-01-01

/desorption the nucleotides were dephosphorylated, enriched with [U-14C]adenosine, and exposed to purine-nucleoside specific enzymes. Reverse phase HPLC and radioactivity measurement demonstrated that for adenosine about 82% of total stable 3H label was in ribose and thus about 18% in adenine. For guanosine about 89...

Main: Nucleotide Analysis [KOME

Lifescience Database Archive (English)

Full Text Available Nucleotide Analysis Japonica genome blast search result Result of blastn search against jap...onica genome sequence kome_japonica_genome_blast_search_result.zip kome_japonica_genome_blast_search_result ...

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Directory of Open Access Journals (Sweden)

Linder Markus

2006-03-01

Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription

NARCIS (Netherlands)

Berkhout, B.; Vastenhouw, N. L.; Klasens, B. I.; Huthoff, H.

2001-01-01

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There

Nucleotide sequence analysis of HTLV-I isolated from cerebrospinal fluid of a patient with TSP/HAM: comparison to other HTLV-I isolates.

Science.gov (United States)

Mukhopadhyaya, R; Sadaie, M R

1993-02-01

Human T-cell leukemia virus type I (HTLV-I) has been associated with adult T-cell leukemia/lymphoma and the chronic neurologic disorder tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). To study the genetic structure of the virus associated with TSP/HAM, we have obtained and sequenced a partial genomic clone from an HTLV-I-positive cell line established from cerebrospinal fluid (CSF) of a Jamaican patient with TSP/HAM. This clone consisted of a 4.3-kb viral sequence containing the 5' long terminal repeat (LTR), gag, and N-terminal portion of the pol gene, with an overall 1.3% sequence variation resulting from mostly nucleotide substitutions, as compared to the prototype HTLV-I ATK-1. The gag and pol regions showed only 1.4% and 1.2% nucleotide variations, respectively. However, the U3 region of the LTR showed the highest sequence variation (3.6%), where several changes appear to be common among certain TSP/HAM isolates. Several of these changes reside within the 21-bp boundaries and the Tax-responsive element. It would be important to determine if the observed changes are sufficient to cause neurologic disorders similar to the murine leukemia virus system or simply reflect the divergent pool of HTLV-I from different geographic locations. At this time, we cannot rule out the possibility that the observed changes have either direct or indirect significance for the HTLV-I pathogenesis in TSP/HAM.

Reverse transcriptase inhibitors as microbicides.

Science.gov (United States)

Lewi, Paul; Heeres, Jan; Ariën, Kevin; Venkatraj, Muthusamy; Joossens, Jurgen; Van der Veken, Pieter; Augustyns, Koen; Vanham, Guido

2012-01-01

The CAPRISA 004 study in South Africa has accelerated the development of vaginal and rectal microbicides containing antiretrovirals that target specific enzymes in the reproduction cycle of HIV, especially reverse transcriptase inhibitors (RTI). In this review we discuss the potential relevance of HIV-1 RTIs as microbicides, focusing in the nucleotide RTI tenofovir and six classes of nonnucleoside RTIs (including dapivirine, UC781, urea and thiourea PETTs, DABOs and a pyrimidinedione). Although tenofovir and dapivirine appear to be most advanced in clinical trials as potential microbicides, several issues remain unresolved, e.g., the importance of nonhuman primates as a "gatekeeper" for clinical trials, the emergence and spread of drug-resistant mutants, the combination of microbicides that target different phases of viral reproduction and the accessibility to microbicides in low-income countries. Thus, here we discuss the latest research on RTI as microbicides in the light of the continuing spread of the HIV pandemic from the point of view of medicinal chemistry, virological, and pharmaceutical studies.

Dimensional testing for reverse k-nearest neighbor search

DEFF Research Database (Denmark)

Casanova, Guillaume; Englmeier, Elias; Houle, Michael E.

2017-01-01

Given a query object q, reverse k-nearest neighbor (RkNN) search aims to locate those objects of the database that have q among their k-nearest neighbors. In this paper, we propose an approximation method for solving RkNN queries, where the pruning operations and termination tests are guided...... by a characterization of the intrinsic dimensionality of the data. The method can accommodate any index structure supporting incremental (forward) nearest-neighbor search for the generation and verification of candidates, while avoiding impractically-high preprocessing costs. We also provide experimental evidence...

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

Science.gov (United States)

Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

2010-11-19

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

Bacterial nucleotide-based second messengers.

Science.gov (United States)

Pesavento, Christina; Hengge, Regine

2009-04-01

In all domains of life nucleotide-based second messengers transduce signals originating from changes in the environment or in intracellular conditions into appropriate cellular responses. In prokaryotes cyclic di-GMP has emerged as an important and ubiquitous second messenger regulating bacterial life-style transitions relevant for biofilm formation, virulence, and many other bacterial functions. This review describes similarities and differences in the architecture of the cAMP, (p)ppGpp, and c-di-GMP signaling systems and their underlying signaling principles. Moreover, recent advances in c-di-GMP-mediated signaling will be presented and the integration of c-di-GMP signaling with other nucleotide-based signaling systems will be discussed.

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

Science.gov (United States)

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA.

OpenAIRE

Adachi, A; Sakai, K; Kitamura, N; Nakanishi, S; Niwa, O; Matsuyama, M; Ishimoto, A

1984-01-01

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. ...

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus.

Science.gov (United States)

Hammond, R W; Crosslin, J M

1995-04-01

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.

Posterior Reversible Encephalopathy Syndrome (PRES Associated with Eclampsia: A Case Study

Directory of Open Access Journals (Sweden)

Maasoumeh Mirzamoradi

2017-03-01

Full Text Available The occurrence of posterior reversible encephalopathy syndrome (PRES in patients with eclampsia is a rare condition. PRES is a reversible syndrome characterized by headache, seizure, altered mentation and loss of vision associated with white matter changes on imaging. The lesions in PRES are thought to be due to vasogenic oedema, predominantly in the posterior cerebral hemispheres. This study reports a 16-year-old pregnant woman who presented with blindness and seizure. The MRI of her brain showed abnormal signal intensity in the white matter of the occipital and frontal lobes. She was treated successfully with pregnancy termination, anti-hypertensives, anticonvulsants, and supportive care. It is concluded that early diagnosis is important to prevent permanent neurologic damage and mortality.

Nucleotide supplementation: a randomised double-blind placebo controlled trial of IntestAidIB in people with Irritable Bowel Syndrome [ISRCTN67764449

Directory of Open Access Journals (Sweden)

Attree EA

2006-06-01

Full Text Available Abstract Background Dietary nucleotide supplementation has been shown to have important effects on the growth and development of cells which have a rapid turnover such as those in the immune system and the gastrointestinal tract. Work with infants has shown that the incidence and duration of diarrhoea is lower when nucleotide supplementation is given, and animal work shows that villi height and crypt depth in the intestine is increased as a result of dietary nucleotides. Dietary nucleotides may be semi-essential under conditions of ill-health, poor diet or stress. Since people with Irritable Bowel Syndrome tend to fulfil these conditions, we tested the hypothesis that symptoms would be improved with dietary nucleotide supplementation. Methods Thirty-seven people with a diagnosis of Irritable Bowel gave daily symptom severity ratings for abdominal pain, diarrhoea, urgency to have a bowel movement, incomplete feeling of evacuation after a bowel movement, bloating, flatulence and constipation for 28 days (baseline. They were then assigned to either placebo (56 days followed by experimental (56 days or the reverse. There was a four week washout period before crossover. During the placebo and experimental conditions participants took one 500 mg capsule three times a day; in the experimental condition the capsule contained the nutroceutical substances. Symptom severity ratings and psychological measures (anxiety, depression, illness intrusiveness and general health were obtained and analysed by repeated measures ANOVAs. Results Symptom severity for all symptoms (except constipation were in the expected direction of baseline>placebo>experimental condition. Symptom improvement was in the range 4 – 6%. A feeling of incomplete evacuation and abdominal pain showed the most improvement. The differences between conditions for diarrhoea, bloating and flatulence were not significant at the p Conclusion Dietary nucleotide supplementation improves some of the

Structure of long terminal repeats of transcriptionally active and inactive copies of Drosophila mobile dispersed genetic elements mdg3

International Nuclear Information System (INIS)

Dzhumagaliev, E.B.; Mazo, A.N.; Baev, A.A. Jr.; Gorelova, T.V.; Arkhipova, I.R.; Shuppe, N.G.; Il'in, Yu.V.

1986-01-01

The authors have determined the nucleotide sequences of long terminal repeats (LTRS) and adjacent regions in the transcribed and nontranscribed variants of the mobile dispersed gene mdg3. In its main characteristics the mdg3 is similar to other mdg. Its integration into chromosomal DNA brings about duplication of the 4 bp of the host DNA, no specificity of the mdg integration at the nucleotide level being detected. The mdg3 is flanked by a 5 bp inverted repeat. The variations in the length of the LTR in different mdg copies is mainly due to duplication of certain sequences in the U3 and R regions. mdg3 copies with a LTR length of 267 bp are the most abundant and are completely conservative in their primary structure. They are transcribed in the cells of the 67J25D culture, but not transcribed in the K/sub c/ line, where another mdg3 variant with a LTR length of 293 bp is transcriptionally active. The SI mapping of transcription initiation and termination sites has shown that in both mdg3 variants they are localized in the same LTR regions, and that the LTR itself has a characteristic U3-R-U5 structure-like retroviral LTRs. The possible factors involved in the regulation of mdg transcription are discussed

Enzymatic Incorporation of Modified Purine Nucleotides in DNA.

Science.gov (United States)

Abu El Asrar, Rania; Margamuljana, Lia; Abramov, Mikhail; Bande, Omprakash; Agnello, Stefano; Jang, Miyeon; Herdewijn, Piet

2017-12-14

A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N 8 -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reversal thyristor-relay direct current commutator

International Nuclear Information System (INIS)

Ivanenko, A.I.

1982-01-01

A thyristor-relay commutator used for alteration of the leading magnetic field direction in experiments with polarized neutrons is described. The commutator flowsheet is presented. Thyristors, connected so as to allow the relay trigger operation mode, are used as controllable electronic relay. Two connected in series coils with the total inductance of the order of 0.28 H serve as the electronic relay load. The arc-free current commutation is effected at the moment of the minimal current across the load terminals, which allows to easily reverse the current up to 10 A at a volatage, v <= 150 V. The experience gained within a year of operation has shown that the commutator meets the requirements of reliability and tuning

Nucleotides, micro- and macro-nutrients, limonoids, flavonoids, and hydroxycinnamates composition in the phloem sap of sweet orange.

Science.gov (United States)

Hijaz, Faraj; Manthey, John A; Van der Merwe, Deon; Killiny, Nabil

2016-06-02

Currently, the global citrus production is declining due to the spread of Huanglongbing (HLB). HLB, otherwise known as citrus greening, is caused by Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllids (ACP), Diaphorina citri Kuwayama. ACP transmits CLas bacterium while feeding on the citrus phloem sap. Multiplication of CLas in the phloem of citrus indicates that the sap contains all the essential nutrients needed for CLas. In this study, we investigated the micro- and macro-nutrients, nucleotides, and others secondary metabolites of phloem sap from pineapple sweet orange. The micro- and macro-nutrients were analyzed using inductively coupled plasma-mass spectroscopy (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES). Nucleotides and other secondary metabolites analysis was accomplished by reversed phase HPLC coupled with UV, fluorescence detection, or negative mode electrospray ionization mass spectrometry (ESI-MS). Calcium (89 mM) was the highest element followed by potassium (38.8 mM) and phosphorous (24 mM). Magnesium and sulfur were also abundant and their concentrations were 15 and 9 mM, respectively. The rest of the elements were found in low amounts (sap.

Ionic interaction of myosin loop 2 with residues located beyond the N-terminal part of actin probed by chemical cross-linking.

Science.gov (United States)

Pliszka, Barbara; Martin, Brian M; Karczewska, Emilia

2008-02-01

To probe ionic contacts of skeletal muscle myosin with negatively charged residues located beyond the N-terminal part of actin, myosin subfragment 1 (S1) and actin split by ECP32 protease (ECP-actin) were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have found that unmodified S1 can be cross-linked not only to the N-terminal part, but also to the C-terminal 36 kDa fragment of ECP-actin. Subsequent experiments performed on S1 cleaved by elastase or trypsin indicate that the cross-linking site in S1 is located within loop 2. This site is composed of Lys-636 and Lys-637 and can interact with negatively charged residues of the 36 kDa actin fragment, most probably with Glu-99 and Glu-100. Cross-links are formed both in the absence and presence of MgATP.P(i) analog, although the addition of nucleotide decreases the efficiency of the cross-linking reaction.

Identification of cyclic nucleotide gated channels using regular expressions

KAUST Repository

Zelman, Alice K.; Dawe, Adam Sean; Berkowitz, Gerald A.

2013-01-01

Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one- pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin

The reverse boomerang sign: a marker for first-trimester transposition of great arteries.

Science.gov (United States)

Bravo-Valenzuela, Nathalie Jeanne; Peixoto, Alberto Borges; Araujo Júnior, Edward; Da Silva Costa, Fabricio; Meagher, Simon

2017-10-12

To describe a new sonographic marker of transposition of great arteries (TGA) during the first-trimester screening. We reviewed six cases of TGA from 2013 to 2016 in which an antenatal diagnosis of TGA at first-trimester screening (11-13 + 6 weeks of gestation) was confirmed postnatally. We specifically assessed images obtained by scanning the fetal heart in three vessels (3V) and three-vessel with trachea (3VT) views using color Doppler. The "reverse boomerang" sign was defined as a reverse curvature of right ventricle outflow tract (RVOT) at level of the 3VT view. We described six cases of confirmed TGA, five singletons and one twin pregnancy, among which only two vessels and the reverse curvature of RVOT (reverse boomerang sign) was demonstrated in the first-trimester screening at level of 3VT view. Ventricular septal defects were observed in three cases, and double outlet right ventricle in one case. No other cardiac or extracardiac anomalies were identified. Termination of pregnancy was not performed in any case. Our series case suggests that the reverse boomerang sign may improve the early prenatal screening for TGA.

Nucleotide Metabolism and its Control in Lactic Acid Bacteria

DEFF Research Database (Denmark)

Kilstrup, Mogens; Hammer, Karin; Jensen, Peter Ruhdal

2005-01-01

Most metabolic reactions are connected through either their utilization of nucleotides or their utilization of nucleotides or their regulation by these metabolites. In this review the biosynthetic pathways for pyrimidine and purine metabolism in lactic acid bacteria are described including...... the interconversion pathways, the formation of deoxyribonucleotides and the salvage pathways for use of exogenous precursors. The data for the enzymatic and the genetic regulation of these pathways are reviewed, as well as the gene organizations in different lactic acid bacteria. Mutant phenotypes and methods...... for manipulation of nucleotide pools are also discussed. Our aim is to provide an overview of the physiology and genetics of nucleotide metabolism and its regulation that will facilitate the interpretation of data arising from genetics, metabolomics, proteomics, and transcriptomics in lactic acid bacteria....

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.

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Robart, Aaron R; O'Connor, Catherine M; Collins, Kathleen

2010-03-01

Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

Effects of hypokinesia on cyclic nucleotides and hormonal regulation ...

African Journals Online (AJOL)

PTH), calcitonin (CT), cyclic nucleotides (cAMP, cGMP) and calcium in the blood of rats, while in urine - phosphate, calcium and cyclic nucleotides. Design: Laboratory based experiment. Setting: Laboratory in the Department of Biochemistry, ...

Structural studies of a bacterial tRNA(HIS guanylyltransferase (Thg1-like protein, with nucleotide in the activation and nucleotidyl transfer sites.

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Samantha J Hyde

Full Text Available All nucleotide polymerases and transferases catalyze nucleotide addition in a 5' to 3' direction. In contrast, tRNA(His guanylyltransferase (Thg1 enzymes catalyze the unusual reverse addition (3' to 5' of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3'-5' addition activity to add G-1 to the 5'-end of tRNA(His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5'-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA(His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA(His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP. The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3'-5' reaction with 5'-monophosphorylated tRNA necessitates first an activation step, generating a 5'-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5'-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

Nucleotide sequence of the 3' ends of the double-stranded RNAs of grapevine chrome mosaic nepovirus.

Science.gov (United States)

Le Gall, O; Candresse, T; Dunez, J

1988-02-01

Attempts were made to label the termini of dsRNAs corresponding to the two genomic RNAs of grapevine chrome mosaic nepovirus (GCMV). It was not possible to label the 5' ends of the dsRNAs with [gamma-32P]ATP, which suggests that a genome-linked protein blocks their 5' ends. Both dsRNA species were labelled at their 3' ends with pCp. The 3'-terminal sequences were determined by 'wandering spot' or by partial enzymic cleavage analysis. One strand (presumably positive) ended in a poly(A) 30 to 50 nucleotides long whereas the other (presumably negative) ended in 3'-ACCUUUUAAAAAG (RNA1) or 3'-ACCUUUUAAUAAAG (RNA2). The sequences resemble closely those complementary to the 5' ends of the RNAs of tomato black ring virus (strain S), which is distantly related to GCMV.

N-terminally truncated POM121C inhibits HIV-1 replication.

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Hideki Saito

Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

The nucleotide sequence of parsnip yellow fleck virus: a plant picorna-like virus.

Science.gov (United States)

Turnbull-Ross, A D; Reavy, B; Mayo, M A; Murant, A F

1992-12-01

The complete sequence of 9871 nucleotides (nts) of parsnip yellow fleck virus (PYFV; isolate P-121) was determined from cDNA clones and by direct sequencing of viral RNA. The RNA contains a large open reading frame between nts 279 and 9362 which encodes a polyprotein of 3027 amino acids with a calculated M(r) of 336212 (336K). A PYFV polyclonal antiserum reacted with the proteins expressed from phage carrying cDNA clones from the 5' half of the PYFV genome. Comparison of the polyprotein sequence of PYFV with other viral polyprotein sequences reveals similarities to the putative NTP-binding and RNA polymerase domains of cowpea mosaic comovirus, tomato black ring nepovirus and several animal picornaviruses. The 3' untranslated region of PYFV RNA is 509 nts long and does not have a poly(A) tail. The 3'-terminal 121 nts may form a stem-loop structure which resembles that formed in the genomic RNA of mosquito-borne flaviviruses.

Application of a global magnetospheric-ionospheric current model for dayside and terminator Pi2 pulsations

Science.gov (United States)

Imajo, S.; Yoshikawa, A.; Uozumi, T.; Ohtani, S.; Nakamizo, A.; Chi, P. J.

2017-12-01

Pi2 magnetic oscillations on the dayside are considered to be produced by the ionospheric current that is driven by Pi2-associated electric fields from the high-latitude region, but this idea has not been quantitatively tested. The present study numerically tested the magnetospheric-ionospheric current system for Pi2 consisting of field-aligned currents (FACs) localized in the nightside auroral region, the perpendicular magnetospheric current flowing in the azimuthal direction, and horizontal ionospheric currents driven by the FACs. We calculated the spatial distribution of the ground magnetic field produced by these currents using the Biot-Savart law in a stationary state. The calculated magnetic field reproduced the observational features reported by previous studies; (1) the sense of the H component does not change a wide range of local time sectors at low latitudes; (2) the amplitude of the H component on the dayside is enhanced at the equator; (3) The D component reverses its phase near the dawn and dusk terminators; (4) the meridian of the D-component phase reversal near the dusk terminator is shifted more sunward than that near the dawn terminator; (5) the amplitude of the D component in the morning is larger than that in the early evening. We also derived the global distributions of observed equivalent currents for two Pi2 events. The spatial patterns of dayside equivalent currents were similar to the spatial pattern of numerically derived equivalent currents. The results indicate that the oscillation of the magnetospheric-ionospheric current system is a plausible explanation of Pi2s on the dayside and near the terminator. These results are included in an accepted paper by Imajo et al. [2017JGR, DOI: 10.1002/2017JA024246].

Nucleotide Selectivity at a Preinsertion Checkpoint of T7 RNA Polymerase Transcription Elongation.

Science.gov (United States)

E, Chao; Duan, Baogen; Yu, Jin

2017-04-20

Nucleotide selection is crucial for transcription fidelity control, in particular, for viral T7 RNA polymerase (RNAP) lack of proofreading activity. It has been recognized that multiple kinetic checkpoints exist prior to full nucleotide incorporation. In this work, we implemented intensive atomistic molecular dynamics (MD) simulations to quantify how strong the nucleotide selection is at the initial checkpoint of an elongation cycle of T7 RNAP. The incoming nucleotides bind into a preinsertion site where a critical tyrosine residue locates nearby to assist the nucleotide selection. We calculated the relative binding free energy between a noncognate nucleotide and a cognate one at a preinsertion configuration via alchemical simulations, showing that a small selection free energy or the binding free energy difference (∼3 k B T) exists between the two nucleotides. Indeed, another preinsertion configuration favored by the noncognate nucleotides was identified, which appears to be off path for further nucleotide insertion and additionally assists the nucleotide selection. By chemical master equation (CME) approach, we show that the small selection free energy at the preinsertion site along with the off-path noncognate nucleotide filtering can help substantially to reduce the error rate and to maintain the elongation rate high in the T7 RNAP transcription.

Single Nucleotide Polymorphism

DEFF Research Database (Denmark)

Børsting, Claus; Pereira, Vania; Andersen, Jeppe Dyrberg

2014-01-01

Single nucleotide polymorphisms (SNPs) are the most frequent DNA sequence variations in the genome. They have been studied extensively in the last decade with various purposes in mind. In this chapter, we will discuss the advantages and disadvantages of using SNPs for human identification...... of SNPs. This will allow acquisition of more information from the sample materials and open up for new possibilities as well as new challenges....

Condensing the information in DNA with double-headed nucleotides

DEFF Research Database (Denmark)

Hornum, Mick; Sharma, Pawan K; Reslow-Jacobsen, Charlotte

2017-01-01

A normal duplex holds as many Watson-Crick base pairs as the number of nucleotides in its constituent strands. Here we establish that single nucleotides can be designed to functionally imitate dinucleotides without compromising binding affinity. This effectively allows sequence information...

Energy efficient and fast reversal of a fixed skyrmion two-terminal memory with spin current assisted by voltage controlled magnetic anisotropy

Science.gov (United States)

Bhattacharya, Dhritiman; Mamun Al-Rashid, Md; Atulasimha, Jayasimha

2017-10-01

Recent work (P-H Jang et al 2015 Appl. Phys. Lett. 107 202401, J. Sampaio et al 2016 Appl. Phys. Lett. 108 112403) suggests that ferromagnetic reversal with spin transfer torque (STT) requires more current in a system in the presence of Dzyaloshinskii-Moriya interaction (DMI) than switching a typical ferromagnet of the same dimensions and perpendicular magnetic anisotropy (PMA). However, DMI promotes the stabilization of skyrmions and we report that when perpendicular anisotropy is modulated (reduced) for both the skyrmion and ferromagnet, it takes a much smaller current to reverse the fixed skyrmion than to reverse the ferromagnet in the same amount of time, or the skyrmion reverses much faster than the ferromagnet at similar levels of current. We show with rigorous micromagnetic simulations that skyrmion switching proceeds along a different path at very low PMA, which results in a significant reduction in the spin current or time required for reversal. This can offer potential for memory applications where a relatively simple modification of the standard STT-RAM (to include a heavy metal adjacent to the soft magnetic layer and with appropriate design of the tunnel barrier) can lead to an energy efficient and fast magnetic memory device based on the reversal of fixed skyrmions.

Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein

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Monaco Regina

2009-01-01

Full Text Available Aim: The xeroderma pigmentosum D (XPD protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER and transcription-coupled repair (TCR. The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln. Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. Materials and Methods: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. Results: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. Conclusion: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

The nucleotide sequence of RNA1 of Lettuce big-vein virus, genus Varicosavirus, reveals its relation to nonsegmented negative-strand RNA viruses.

Science.gov (United States)

Sasaya, Takahide; Ishikawa, Koichi; Koganezawa, Hiroki

2002-06-05

The complete nucleotide sequence of RNA1 from Lettuce big-vein virus (LBVV), the type member of the genus Varicosavirus, was determined. LBVV RNA1 consists of 6797 nucleotides and contains one large ORF that encodes a large (L) protein of 2040 amino acids with a predicted M(r) of 232,092. Northern blot hybridization analysis indicated that the LBVV RNA1 is a negative-sense RNA. Database searches showed that the amino acid sequence of L protein is homologous to those of L polymerases of nonsegmented negative-strand RNA viruses. A cluster dendrogram derived from alignments of the LBVV L protein and the L polymerases indicated that the L protein is most closely related to the L polymerases of plant rhabdoviruses. Transcription termination/polyadenylation signal-like poly(U) tracts that resemble those in rhabdovirus and paramyxovirus RNAs were present upstream and downstream of the coding region. Although LBVV is related to rhabdoviruses, a key distinguishing feature is that the genome of LBVV is segmented. The results reemphasize the need to reconsider the taxonomic position of varicosaviruses.

Critical role of DNA intercalation in enzyme-catalyzed nucleotide flipping

Science.gov (United States)

Hendershot, Jenna M.; O'Brien, Patrick J.

2014-01-01

Nucleotide flipping is a common feature of DNA-modifying enzymes that allows access to target sites within duplex DNA. Structural studies have identified many intercalating amino acid side chains in a wide variety of enzymes, but the functional contribution of these intercalating residues is poorly understood. We used site-directed mutagenesis and transient kinetic approaches to dissect the energetic contribution of intercalation for human alkyladenine DNA glycosylase, an enzyme that initiates repair of alkylation damage. When AAG flips out a damaged nucleotide, the void in the duplex is filled by a conserved tyrosine (Y162). We find that tyrosine intercalation confers 140-fold stabilization of the extrahelical specific recognition complex, and that Y162 functions as a plug to slow the rate of unflipping by 6000-fold relative to the Y162A mutant. Surprisingly, mutation to the smaller alanine side chain increases the rate of nucleotide flipping by 50-fold relative to the wild-type enzyme. This provides evidence against the popular model that DNA intercalation accelerates nucleotide flipping. In the case of AAG, DNA intercalation contributes to the specific binding of a damaged nucleotide, but this enhanced specificity comes at the cost of reduced speed of nucleotide flipping. PMID:25324304

Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

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Tobias Fromme

2018-02-01

Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

Structural determination of functional units of the nucleotide binding domain (NBD94 of the reticulocyte binding protein Py235 of Plasmodium yoelii.

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Ardina Grüber

2010-02-01

Full Text Available Invasion of the red blood cells (RBC by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94 of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547, NBD94(566-663 and NBD94(674-793, respectively. Using fluorescence correlation spectroscopy NBD94(444-547 has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547 in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663 and NBD94(674-793 enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663 consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793 in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the

Field-Reversed Configuration Formation Scheme Utilizing a Spheromak and Solenoid Induction

International Nuclear Information System (INIS)

Gerhardt, S.P.; Belova, E.V.; Yamada, M.; Ji, H.; Ren, Y.; McGeehan, B.; Inomoto, M.

2008-01-01

A new field-reversed configuration (FRC) formation technique is described, where a spheromak transitions to a FRC with inductive current drive. The transition is accomplished only in argon and krypton plasmas, where low-n kink modes are suppressed; spheromaks with a lighter majority species, such as neon and helium, either display a terminal tilt-mode, or an n=2 kink instability, both resulting in discharge termination. The stability of argon and krypton plasmas through the transition is attributed to the rapid magnetic diffusion of the currents that drive the kink-instability. The decay of helicity during the transition is consistent with that expected from resistivity. This observation indicates a new scheme to form a FRC plasma, provided stability to low-n modes is maintained, as well as a unique situation where the FRC is a preferred state

Crystal structures of HIV-1 nonnucleoside reverse transcriptase inhibitors: N-benzyl-4-methyl-benzimidazoles

Science.gov (United States)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2009-07-01

HIV-1 nonnucleoside reverse transcriptase inhibitors are potentially specific and effective drugs in AIDS therapy. The presence of two aromatic systems with an angled orientation in the molecule of the inhibitor is crucial for interactions with HIV-1 RT. The inhibitor drives like a wedge into the cluster of aromatic residues of RT HIV-1 and restrains the enzyme in a conformation that blocks the chemical step of nucleotide incorporation. Structural studies provide useful information for designing new, more active inhibitors. The crystal structures of four NNRTIs are presented here. The investigated compounds are derivatives of N-benzyl-4-methyl-benzimidazole with various aliphatic and aromatic substituents at carbon 2 positions and a 2,6-dihalogeno-substituted N-benzyl moiety. Structural data reported here show that the conformation of the investigated compounds is relatively rigid. Such feature is important for the nonnucleoside inhibitor binding to HIV-1 reverse transcriptase.

DNA Nucleotides Detection via capacitance properties of Graphene

Science.gov (United States)

Khadempar, Nahid; Berahman, Masoud; Yazdanpanah, Arash

2016-05-01

In the present paper a new method is suggested to detect the DNA nucleotides on a first-principles calculation of the electronic features of DNA bases which chemisorbed to a graphene sheet placed between two gold electrodes in a contact-channel-contact system. The capacitance properties of graphene in the channel are surveyed using non-equilibrium Green's function coupled with the Density Functional Theory. Thus, the capacitance properties of graphene are theoretically investigated in a biological environment, and, using a novel method, the effect of the chemisorbed DNA nucleotides on electrical charges on the surface of graphene is deciphered. Several parameters in this method are also extracted including Electrostatic energy, Induced density, induced electrostatic potential, Electron difference potential and Electron difference density. The qualitative and quantitative differences among these parameters can be used to identify DNA nucleotides. Some of the advantages of this approach include its ease and high accuracy. What distinguishes the current research is that it is the first experiment to investigate the capacitance properties of gaphene changes in the biological environment and the effect of chemisorbed DNA nucleotides on the surface of graphene on the charge.

HIV-1 Promoter Single Nucleotide Polymorphisms Are Associated with Clinical Disease Severity.

Directory of Open Access Journals (Sweden)

Michael R Nonnemacher

Full Text Available The large majority of human immunodeficiency virus type 1 (HIV-1 markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs contained within the viral promoter or long terminal repeat (LTR in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count.

In-silico single nucleotide polymorphisms (SNP) mining of Sorghum ...

African Journals Online (AJOL)

Single nucleotide polymorphisms (SNPs) may be considered the ultimate genetic markers as they represent the finest resolution of a DNA sequence (a single nucleotide), and are generally abundant in populations with a low mutation rate. SNPs are important tools in studying complex genetic traits and genome evolution.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

Directory of Open Access Journals (Sweden)

Parvathi Chary

Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

Effect of Reverse Bias Stress on Leakage Currents and Breakdown Voltages of Solid Tantalum Capacitors

Science.gov (United States)

Teverovsky, Alexander A.

2011-01-01

The majority of solid tantalum capacitors are produced by high-temperature sintering of a fine tantalum powder around a tantalum wire followed by electrolytic anodization that forms a thin amorphous Ta2O5 dielectric layer and pyrolysis of manganese nitrite on the oxide to create a conductive manganese dioxide electrode. A contact to tantalum wire is used as anode terminal and to the manganese layer as a cathode terminal of the device. This process results in formation of an asymmetric Ta -- Ta2O5 -- MnO2 capacitor that has different characteristics at forward (positive bias applied to tantalum) and reverse (positive bias applied to manganese cathode) voltages. Reverse bias currents might be several orders of magnitude larger than forward leakage currents so I-V characteristics of tantalum capacitors resemble characteristics of semiconductor rectifiers. Asymmetric I-V characteristics of Ta -- anodic Ta2O5 systems have been observed at different top electrode materials including metals, electrolytes, conductive polymers, and manganese oxide thus indicating that this phenomenon is likely related to the specifics of the Ta -- Ta2O5 interface. There have been multiple attempts to explain rectifying characteristics of capacitors employing anodic tantalum pentoxide dielectrics. A brief review of works related to reverse bias (RB) behavior of tantalum capacitors shows that the mechanism of conduction in Ta -- Ta2O5 systems is still not clear and more testing and analysis is necessary to understand the processes involved. If tantalum capacitors behave just as rectifiers, then the assessment of the safe reverse bias operating conditions would be a relatively simple task. Unfortunately, these parts can degrade with time under reverse bias significantly, and this further complicates analysis of the I-V characteristics and establishing safe operating areas of the parts. On other hand, time dependence of reverse currents might provide additional information for investigation of

Statistical properties and fractals of nucleotide clusters in DNA sequences

International Nuclear Information System (INIS)

Sun Tingting; Zhang Linxi; Chen Jin; Jiang Zhouting

2004-01-01

Statistical properties of nucleotide clusters in DNA sequences and their fractals are investigated in this paper. The average size of nucleotide clusters in non-coding sequence is larger than that in coding sequence. We investigate the cluster-size distribution P(S) for human chromosomes 21 and 22, and the results are different from previous works. The cluster-size distribution P(S 1 +S 2 ) with the total size of sequential Pu-cluster and Py-cluster S 1 +S 2 is studied. We observe that P(S 1 +S 2 ) follows an exponential decay both in coding and non-coding sequences. However, we get different results for human chromosomes 21 and 22. The probability distribution P(S 1 ,S 2 ) of nucleotide clusters with the size of sequential Pu-cluster and Py-cluster S 1 and S 2 respectively, is also examined. In the meantime, some of the linear correlations are obtained in the double logarithmic plots of the fluctuation F(l) versus nucleotide cluster distance l along the DNA chain. The power spectrums of nucleotide clusters are also discussed, and it is concluded that the curves are flat and hardly changed and the 1/3 frequency is neither observed in coding sequence nor in non-coding sequence. These investigations can provide some insights into the nucleotide clusters of DNA sequences

Reversible flowchart languages and the structured reversible program theorem

DEFF Research Database (Denmark)

Yokoyama, Tetsuo; Axelsen, Holger Bock; Glück, Robert

2008-01-01

Many irreversible computation models have reversible counterparts, but these are poorly understood at present. We introduce reversible flowcharts with an assertion operator and show that any reversible flowchart can be simulated by a structured reversible flowchart using only three control flow...... operators. Reversible flowcharts are r- Turing-complete, meaning that they can simuluate reversible Turing machines without garbage data. We also demonstrate the injectivization of classical flowcharts into reversible flowcharts. The reversible flowchart computation model provides a theoretical...

Simulation of conditional diffusions via forward-reverse stochastic representations

KAUST Repository

Bayer, Christian

2015-01-01

We derive stochastic representations for the finite dimensional distributions of a multidimensional diffusion on a fixed time interval,conditioned on the terminal state. The conditioning can be with respect to a fixed measurement point or more generally with respect to some subset. The representations rely on a reverse process connected with the given (forward) diffusion as introduced by Milstein, Schoenmakers and Spokoiny in the context of density estimation. The corresponding Monte Carlo estimators have essentially root-N accuracy, and hence they do not suffer from the curse of dimensionality. We also present an application in statistics, in the context of the EM algorithm.

Simulation of conditional diffusions via forward-reverse stochastic representations

KAUST Repository

Bayer, Christian

2015-01-07

We derive stochastic representations for the finite dimensional distributions of a multidimensional diffusion on a fixed time interval,conditioned on the terminal state. The conditioning can be with respect to a fixed measurement point or more generally with respect to some subset. The representations rely on a reverse process connected with the given (forward) diffusion as introduced by Milstein, Schoenmakers and Spokoiny in the context of density estimation. The corresponding Monte Carlo estimators have essentially root-N accuracy, and hence they do not suffer from the curse of dimensionality. We also present an application in statistics, in the context of the EM algorithm.

Osteogenic cell differentiation on H-terminated and O-terminated nanocrystalline diamond films

Directory of Open Access Journals (Sweden)

Liskova J

2015-01-01

Full Text Available Jana Liskova,1 Oleg Babchenko,2 Marian Varga,2 Alexander Kromka,2 Daniel Hadraba,1 Zdenek Svindrych,1 Zuzana Burdikova,1 Lucie Bacakova1 1Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic; 2Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Abstract: Nanocrystalline diamond (NCD films are promising materials for bone implant coatings because of their biocompatibility, chemical resistance, and mechanical hardness. Moreover, NCD wettability can be tailored by grafting specific atoms. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition and grafted by hydrogen atoms (H-termination or oxygen atoms (O-termination. Human osteoblast-like Saos-2 cells were used for biological studies on H-terminated and O-terminated NCD films. The adhesion, growth, and subsequent differentiation of the osteoblasts on NCD films were examined, and the extracellular matrix production and composition were quantified. The osteoblasts that had been cultivated on the O-terminated NCD films exhibited a higher growth rate than those grown on the H-terminated NCD films. The mature collagen fibers were detected in Saos-2 cells on both the H-terminated and O-terminated NCD films; however, the quantity of total collagen in the extracellular matrix was higher on the O-terminated NCD films, as were the amounts of calcium deposition and alkaline phosphatase activity. Nevertheless, the expression of genes for osteogenic markers – type I collagen, alkaline phosphatase, and osteocalcin – was either comparable on the H-terminated and O-terminated films or even lower on the O-terminated films. In conclusion, the higher wettability of the O-terminated NCD films is promising for adhesion and growth of osteoblasts. In addition, the O-terminated surface also seems to support the deposition of extracellular matrix proteins and extracellular matrix

The reversion phenomenon in irradiated fern prothalli: Effects of acute or chronic anoxia and let

International Nuclear Information System (INIS)

Hendry, J.H.; Cowie, F.G.; Wangenheim, K.H. von

1985-01-01

In several systems a paradoxical reduction of radiation damage with increasing dose, termed reversion, has been observed. In the fern Osmunda regalis the percentage of cells which does not die but stays alive, although reproductively sterile, increases with dose. The assumed mechanism of this effect is a continuation of cytoplasmic growth during radiation-induced mitotic delay which induces terminal differentiation (early differentiation) thus preventing mitosis and the expression of chromosomal injury. Suppression of cytoplasmic growth after irradiation should abrogate reversion. This was tested using anoxia. Reversion was suppressed by storage of the sporelings in nitrogen for 8 h or more after X-rays, but was not suppressed by storage in 0.27 μM oxygen nor by a 60-min exposure to air after irradiation and before storage in nitrogen. Anoxia before irradiation in air had no effect.

Detection of DNA nucleotides on pretreated boron doped diamond electrodes

Energy Technology Data Exchange (ETDEWEB)

Garbellini, Gustavo S.; Uliana, Carolina V.; Yamanaka, Hideko [UNESP, Araraquara, SP (Brazil). Inst. de Quimica

2011-07-01

The individual detection and equimolar mixture of DNA nucleotides guanosine monophosphate (GMP), adenosine monophosphate (AMP), thymidine (TMP) and cytidine (CMP) 5'-monophosphate using square wave voltammetry was performed on boron doped diamond (BDD) electrodes cathodically (Red-DDB) and anodically (Oxi-DDB) pretreated. The oxidation of individual DNA nucleotides was more sensitive on Oxi-BDD electrode. In a simultaneous detection of nucleotides, the responses of GMP, AMP, TMP and CMP were very adequate on both treated electrodes. Particularly, more sensitive and separate peaks for TMP and CMP on Oxi-BDD and Red-BDD electrodes, respectively, were observed after deconvolution procedure. The detection of nucleotides in aqueous solutions will certainly contribute for genotoxic evaluation of substances and hybridization reactions by immobilizing ss or ds-DNA on BDD surface. (author)

Organizational Relationship Termination Competence

DEFF Research Database (Denmark)

Ritter, Thomas; Geersbro, Jens

2011-01-01

termination are found to significantly affect a firm's relationship termination competence. The findings suggest that managers should regard termination as a legitimate option in customer relationship management. In order to decrease the number of unwanted customers, managers must accept termination......Most firms are involved in a number of customer relationships that drain the firm's resources. However, many firms are hesitant to address this problem. This paper investigates customer relationship termination at the organizational level. We develop and analyze the organizational dimensions...... of organizational termination in order to improve our understanding of the management of termination. The impact of these termination dimensions on the percentage of unwanted customers is developed and tested using PLS on data gathered from a cross-sectional survey of more than 800 sales representatives. We find...

Kitimat LNG terminal

International Nuclear Information System (INIS)

Schmaltz, I.; Boulton, R.

2007-01-01

Kitimat Liquefied Natural Gas (LNG) terminal is a terminal development company owned by Galveston LNG, a privately owned Canadian energy development company. This presentation provided information on Kitimat LNG with particular reference to its terminal located in Bish Cove on the Douglas Channel in British Columbia. This LNG terminal is reported to be the only fully permitted regasification terminal on the west coast of Canada and the United States. The presentation addressed market fundamentals including several graphs, such as world natural gas proved reserves in 2006; LNG supplements to Canadian gas supplies; global LNG demand for 2005-2020; average annual United States LNG imports; and global LNG liquefaction projects. Other market fundamentals were described, including that Kitimat is the only other approved terminal aside from the Costa Azul terminal in Mexico; Kitimat is the only west coast LNG import terminal that connects to midwest and eastern North American markets through existing gas pipelines; LNG producers are looking for destination diversification; and markets and marketers are looking for supply diversification. The authors noted that by 2010, western Canadian gas demand will exceed Californian demand. Other topics that were discussed in the presentation included Canadian natural gas field receipts; unadjusted bitumen production outlook; oil sands gas demand; forward basis fundamentals; and the commercial drivers of the Kitimat LNG terminal. The presentation also discussed the pacific trail pipelines, a partnership between Galveston LNG and Pacific Northern Gas to develop the natural gas transmission line from Kitimat to Summit. The presentation concluded with a discussion of the benefits of Kitimat LNG terminal such as providing access to the largest natural gas markets in the world via major gas transmission lines with spare capacity. figs

The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway

International Nuclear Information System (INIS)

Mitsuda, Hisateru; Nakajima, Kenji; Nadamoto, Tomonori

1977-01-01

In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashbyii. The added purines, at 10 -4 M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed. (auth.)

Genetic variation of classical swine fever virus based on palindromic nucleotide substitutions, a genetic marker in the 5' untranslated region of RNA

Directory of Open Access Journals (Sweden)

Massimo Giangaspero

2008-06-01

Full Text Available Forty-three strains of classical swine fever (hog cholera virus (CSFV from outbreaks in pigs in Europe, Asia and America, two strains from commercial CSFV modified live vaccines and a strain isolated from a diseased lamb from Spain were subjected to analyses of nucleotide sequence variations in the 5’ terminal region of the genome. These isolates were divided into three clusters, namely: CSFV-1, CSFV-2, and CSFV-3, based on palindromic nucleotide substitutions in the 5’ untranslated region (UTR. The homology degree, according to nucleotide base pairing variation in the secondary palindromic structure of the three variable loci V1, V2 and V3, was 60% in the CSFV species, with a mean divergence value of 6.19 base pairs (bp. relatedness within genotypes ranged from 71.11% to 100%, with mean divergence values from 5.5 to 0.73 base pairs. Subgenotypes showed a divergence ranging from 1 to 9 base pairs within the genotype. Genotype CSFV-1 revealed 15 base pair combinations with 13 divergent base pairs, resulting in 4 subgenotypes with 6 variants in subgenotype CSFV-1.1, including the reference strain Brescia and 6 variants in subgenotype CSFV-1.2, including the Alfort reference strain. Subgenotypes CSFV-1.3 and CSFV-1.4 comprised one and two variants, respectively. Genotype CSFV-2 was represented by the Spanish ovine isolate 5440/99 and the genotype CSFV-3 included the Japanese strains Okinawa/86 and Kanagawa/74. CSFV genotypes revealed a strong relationship with Border disease virus strains, showing relatively low divergence values when compared to other pestivirus species. Evaluation of nucleotide base pair divergence among genotypes and expression of evolutionary changes in the CSFV species led to the construction of a phylogenetic tree based on secondary structure.

Electrical detection and quantification of single and mixed DNA nucleotides in suspension

Science.gov (United States)

Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

2016-09-01

High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

Reverse Algols

Science.gov (United States)

Leung, K. C.

1989-01-01

Reverse Algols, binary systems with a semidetached configuration in which the more massive component is in contact with the critical equipotential surface, are examined. Observational evidence for reverse Algols is presented and the parameters of seven reverse Algols are listed. The evolution of Algols and reverse Algols is discussed. It is suggested that, because reverse Algols represent the premass-reversal semidetached phase of close binary evolution, the evolutionary time scale between regular and reverse Algols is the ratio of the number of confirmed systems of these two Algol types.

The possible role of human milk nucleotides as sleep inducers.

Science.gov (United States)

Sánchez, Cristina L; Cubero, Javier; Sánchez, Javier; Chanclón, Belén; Rivero, Montserrat; Rodríguez, Ana B; Barriga, Carmen

2009-02-01

Breast-milk contains a potent mixture of diverse components, such as the non-protein nitrogen fraction which includes nucleotides, whose variation in levels is evident throughout lactation. In addition, these substances play an important role in sleep homeostasis. In the present study, human milk samples were analyzed using a capillary electrophoresis system. The rhythmicity of each nucleotide was studied by cosinor analysis. It was found that the nucleotides 5'AMP, 5'GMP, 5'CMP, and 5'IMP have significant (P inducing the 'hypnotic' action of breast-milk at night in the infant.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

Science.gov (United States)

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-04-17

The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

Directory of Open Access Journals (Sweden)

Roe Bruce A

2007-04-01

Full Text Available Abstract Background The long bone abnormality (lbab mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

Sustained neurochemical plasticity in central terminals of mouse DRG neurons following colitis.

Science.gov (United States)

Benson, Jessica R; Xu, Jiameng; Moynes, Derek M; Lapointe, Tamia K; Altier, Christophe; Vanner, Stephen J; Lomax, Alan E

2014-05-01

Sensitization of dorsal root ganglia (DRG) neurons is an important mechanism underlying the expression of chronic abdominal pain caused by intestinal inflammation. Most studies have focused on changes in the peripheral terminals of DRG neurons in the inflamed intestine but recent evidence suggests that the sprouting of central nerve terminals in the dorsal horn is also important. Therefore, we examine the time course and reversibility of changes in the distribution of immunoreactivity for substance P (SP), a marker of the central terminals of DRG neurons, in the spinal cord during and following dextran sulphate sodium (DSS)-induced colitis in mice. Acute and chronic treatment with DSS significantly increased SP immunoreactivity in thoracic and lumbosacral spinal cord segments. This increase developed over several weeks and was evident in both the superficial laminae of the dorsal horn and in lamina X. These increases persisted for 5 weeks following cessation of both the acute and chronic models. The increase in SP immunoreactivity was not observed in segments of the cervical spinal cord, which were not innervated by the axons of colonic afferent neurons. DRG neurons dissociated following acute DSS-colitis exhibited increased neurite sprouting compared with neurons dissociated from control mice. These data suggest significant colitis-induced enhancements in neuropeptide expression in DRG neuron central terminals. Such neurotransmitter plasticity persists beyond the period of active inflammation and might contribute to a sustained increase in nociceptive signaling following the resolution of inflammation.

The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

Energy Technology Data Exchange (ETDEWEB)

Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

2014-02-26

For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA gamma.

Science.gov (United States)

Gustafson, G; Hunter, B; Hanau, R; Armour, S L; Jackson, A O

1987-06-01

The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.

Managing Reverse Logistics or Reversing Logistics Management?

OpenAIRE

Brito, Marisa

2004-01-01

textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse logistics. The thesis brings insights on reverse logistics decision-making and it lays down theoretical principles for reverse logistics as a research field.In particular it puts together a framework ...

An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function

Science.gov (United States)

Reynafarje, Baltazar; Lehninger, Albert L.

1978-01-01

This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP4- (out) for ADP3- (in) + 0.5 phosphate2- (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated. PMID:283393

Nucleotide excision repair in yeast

NARCIS (Netherlands)

Eijk, Patrick van

2012-01-01

Nucleotide Excision Repair (NER) is a conserved DNA repair pathway capable of removing a broad spectrum of DNA damage. In human cells a defect in NER leads to the disorder Xeroderma pigmentosum (XP). The yeast Saccharomyces cerevisiae is an excellent model organism to study the mechanism of NER. The

The Coding of Biological Information: From Nucleotide Sequence to Protein Recognition

Science.gov (United States)

Štambuk, Nikola

The paper reviews the classic results of Swanson, Dayhoff, Grantham, Blalock and Root-Bernstein, which link genetic code nucleotide patterns to the protein structure, evolution and molecular recognition. Symbolic representation of the binary addresses defining particular nucleotide and amino acid properties is discussed, with consideration of: structure and metric of the code, direct correspondence between amino acid and nucleotide information, and molecular recognition of the interacting protein motifs coded by the complementary DNA and RNA strands.

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

Science.gov (United States)

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

Nucleotide diversity and phylogenetic relationships among ...

Indian Academy of Sciences (India)

2017-03-03

Mar 3, 2017 ... 2Department of Botany, D. S. B. Campus, Kumaun University, Nainital 263 001, India ... Rana T. S. 2017 Nucleotide diversity and phylogenetic relationships ... Anderson and Park 1989). ..... Edgewood Press, Edgewood, USA.

Free amino acids and 5'-nucleotides in Finnish forest mushrooms.

Science.gov (United States)

Manninen, Hanna; Rotola-Pukkila, Minna; Aisala, Heikki; Hopia, Anu; Laaksonen, Timo

2018-05-01

Edible mushrooms are valued because of their umami taste and good nutritional values. Free amino acids, 5'-nucleotides and nucleosides were analyzed from four Nordic forest mushroom species (Lactarius camphoratus, Boletus edulis, Cantharellus cibarius, Craterellus tubaeformis) using high precision liquid chromatography analysis. To our knowledge, these taste components were studied for the first time from Craterellus tubaeformis and Lactarius camphoratus. The focus was on the umami amino acids and 5'-nucleotides. The free amino acid and 5'-nucleotide/nucleoside contents of studied species differed from each other. In all studied samples, umami amino acids were among five major free amino acids. The highest concentration of umami amino acids was on L. camphoratus whereas B. edulis had the highest content of sweet amino acids and C. cibarius had the highest content of bitter amino acids. The content of umami enhancing 5'-nucleotides were low in all studied species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

International Nuclear Information System (INIS)

Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

1991-01-01

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

A gold nanoparticles-based colorimetric test to detect single nucleotide polymorphisms for improvement of personalized therapy of psoriasis

Science.gov (United States)

Marsella, Alessandra; Valentini, Paola; Tarantino, Paolo; Congedo, Maurizio; Pompa, Pier Paolo

2016-04-01

We report a simple, rapid and low-cost test, based on gold nanoparticles, for the naked-eye colorimetric detection of a signature of single nucleotide polymorphisms (SNPs) relevant for the personalized medicine of psoriasis patients. We validated the colorimetric assay on real-world DNA samples from a cohort of 30 psoriasis patients and we compared the results, in double-blind, with those obtained with two state-of-the-art instrumental techniques, namely reverse dot blotting and direct sequencing, finding 100% agreement. We demonstrated high accuracy, sensitivity and specificity of the colorimetric test that can be easily adapted for the genotypization of different SNPs, important for the pharmacogenomics of various diseases, and in other fields, such as food traceability and population structure analysis.

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

Science.gov (United States)

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Complexes of Escherichia coli adenylate kinase and nucleotides: 1H NMR studies of the nucleotide sites in solution

International Nuclear Information System (INIS)

Vetter, I.R.; Reinstein, J.; Roesch, P.

1990-01-01

One- and two-dimensional nuclear magnetic resonance (NMR) studies, in particular substrate-protein nuclear Overhauser effect (NOESY) measurements, as well as nucleotide and P 1 ,P 5 -bis-(5'-adenosyl) pentaphosphate (AP 5 A) titrations and studies of the temperature-dependent unfolding of the tertiary structure of Escherichia coli adenylate kinase (AK EC ) were performed. These experiments and comparison with the same type of experiments performed with the porcine enzyme led them to the following conclusions: (1) at pH 8 and concentrations of approximately 2.5-3 mM, AK EC is partially unfolded at 318 K; (2) ATP·Mg 2+ binds to the ATP site with a dissociation constant of approximately 40 μM under the assumption that ATP binds to one nucleotide site only; (3) AP 5 A·Mg 2+ binds to both nucleotide sites and thus simulates the active complex; (4) the ATP·Mg 2+ adenine in the AK EC ·AP 5 A·Mg 2+ complex is located close to His 134 and Phe 19 ; (5) the AK EC G-loop with bound ATP·Mg 2+ is structurally highly homologous to the loop region in the oncogene product p21 with bound GTP·Mg 2+

Detection of Brazilian hantavirus by reverse transcription polymerase chain reaction amplification of N gene in patients with hantavirus cardiopulmonary syndrome

OpenAIRE

Marcos Lázaro Moreli; Ricardo Luiz Moro de Sousa; Luiz Tadeu Moraes Figueiredo

2004-01-01

We report a nested reverse transcription-polymerase chain reaction (RT-PCR) assay for hantavirus using primers selected to match high homology regions of hantavirus genomes detected from the whole blood of hantavirus cardiopulmonary syndrome (HCPS) patients from Brazil, also including the N gene nucleotide sequence of Araraquara virus. Hantavirus genomes were detected in eight out of nine blood samples from the HCPS patients by RT-PCR (88.9% positivity) and in all 9 blood samples (100% positi...

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10).

Science.gov (United States)

Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-08-26

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.

Identification of a Negative Regulatory Region for the Exchange Activity and Characterization of T332I Mutant of Rho Guanine Nucleotide Exchange Factor 10 (ARHGEF10)*

Science.gov (United States)

Chaya, Taro; Shibata, Satoshi; Tokuhara, Yasunori; Yamaguchi, Wataru; Matsumoto, Hiroshi; Kawahara, Ichiro; Kogo, Mikihiko; Ohoka, Yoshiharu; Inagaki, Shinobu

2011-01-01

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1–332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant. PMID:21719701

Nucleotides, micro- and macro-nutrients, limonoids, flavonoids, and hydroxycinnamates composition in the phloem sap of sweet orange

Science.gov (United States)

Hijaz, Faraj; Manthey, John A.; Van der Merwe, Deon; Killiny, Nabil

2016-01-01

ABSTRACT Currently, the global citrus production is declining due to the spread of Huanglongbing (HLB). HLB, otherwise known as citrus greening, is caused by Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllids (ACP), Diaphorina citri Kuwayama. ACP transmits CLas bacterium while feeding on the citrus phloem sap. Multiplication of CLas in the phloem of citrus indicates that the sap contains all the essential nutrients needed for CLas. In this study, we investigated the micro- and macro-nutrients, nucleotides, and others secondary metabolites of phloem sap from pineapple sweet orange. The micro- and macro-nutrients were analyzed using inductively coupled plasma-mass spectroscopy (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES). Nucleotides and other secondary metabolites analysis was accomplished by reversed phase HPLC coupled with UV, fluorescence detection, or negative mode electrospray ionization mass spectrometry (ESI-MS). Calcium (89 mM) was the highest element followed by potassium (38.8 mM) and phosphorous (24 mM). Magnesium and sulfur were also abundant and their concentrations were 15 and 9 mM, respectively. The rest of the elements were found in low amounts (flavonoids. In addition, several hydroxycinnamates were detected. The results of this study will increase our knowledge about the nature and the chemical composition of citrus phloem sap. PMID:27171979

FTO, m6 Am , and the hypothesis of reversible epitranscriptomic mRNA modifications.

Science.gov (United States)

Mauer, Jan; Jaffrey, Samie R

2018-05-12

The fate of mRNA is regulated by epitranscriptomic nucleotide modifications, the most abundant of which is N 6 -methyladenosine (m 6 A). Although the pattern and distribution of m 6 A in mRNA is mediated by specific methyltransferases, a recent hypothesis is that specific demethylases or 'erasers' allow m 6 A to be dynamically reversed by signaling pathways. In this Review, we discuss the data in support and against this model. New insights into the function of fat mass and obesity-associated protein (FTO), the original enzyme thought to be an m 6 A eraser, reveal that its physiologic target is not m 6 A, but instead is N 6 ,2'-O-dimethyladenosine (m 6 A m ). Another m 6 A demethylase, ALKBH5, appears to have functions limited to sperm development in normal mice. Overall, the majority of the data suggest that m 6 A is generally not reversible, although m 6 A may be susceptible to demethylation in pathophysiological states such as cancer. © 2018 Federation of European Biochemical Societies.

CONTAINER TERMINALS IN EUROPE

Directory of Open Access Journals (Sweden)

Bart W. WIEGMANS

2001-01-01

Full Text Available This paper aims to address the linkage between logistics (in particular, the management of marketing channel flows and transport markets, while also the interaction between these two markets and intermodal container terminals is analysed. The marketing channel theory is used to describe all relevant actors and flows that run through marketing channels, starting with customer needs and ending with customer satisfaction. Porter's theory of competitive advantages is used to review competitive forces in both markets. Finally, a competitor analysis is performed for the logistics and transport market. These theories are applied so as to be able to determine the competitive position of intermodal container terminals with a view to the management of marketing channel flows and the physical transport of freight flows. Hence, the central question of this paper is: Which markets are served by intermodal container terminals and with whom are they competing? At present, neither the maritime container terminals nor the continental container terminals appear to have a significant influence in the logistics service market; they concentrate mainly on the physical movement of containers (transshipment. Furthermore, maritime container terminals and continental container terminals are not dominant players in the transport service market. Our conclusion is that continental terminals are predominantly competing with unimodal road transport, with neighbouring continental terminals and with barge transport companies.

Reverse Logistics

OpenAIRE

Kulikova, Olga

2016-01-01

This thesis was focused on the analysis of the concept of reverse logistics and actual reverse processes which are implemented in mining industry and finding solutions for the optimization of reverse logistics in this sphere. The objective of this paper was the assessment of the development of reverse logistics in mining industry on the example of potash production. The theoretical part was based on reverse logistics and mining waste related literature and provided foundations for further...

Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

Directory of Open Access Journals (Sweden)

Ajay Kumar

2010-01-01

Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

Science.gov (United States)

Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

2013-07-01

Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

Visual communication and terminal equipment

International Nuclear Information System (INIS)

Kang, Cheol Hui

1988-06-01

This book is divided two parts about visual communication and terminal equipment. The first part introduces visual communication, which deals with foundation of visual communication, technique of visual communication, equipment of visual communication, a facsimile and pictorial image system. The second part contains terminal equipment such as telephone, terminal equipment for data transmission on constitution and constituent of terminal equipment for data transmission, input device and output device, terminal device and up-to-date terminal device.

Visual communication and terminal equipment

Energy Technology Data Exchange (ETDEWEB)

Kang, Cheol Hui

1988-06-15

This book is divided two parts about visual communication and terminal equipment. The first part introduces visual communication, which deals with foundation of visual communication, technique of visual communication, equipment of visual communication, a facsimile and pictorial image system. The second part contains terminal equipment such as telephone, terminal equipment for data transmission on constitution and constituent of terminal equipment for data transmission, input device and output device, terminal device and up-to-date terminal device.

Detection by reverse transcriptase-polymerase chain reaction and molecular characterization of subtype B avian metapneumovirus isolated in Brazil.

Science.gov (United States)

Chacón, Jorge Luis; Brandão, Paulo E; Buim, Marcos; Villarreal, Laura; Ferreira, Antonio J Piantino

2007-10-01

Subtype B avian metapneumovirus (aMPV) was isolated and detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in Brazilian commercial laying chicken flocks with no history of vaccination against aMPV and presenting respiratory signs and decreased egg production. RT-PCR results from samples from three affected flocks revealed that the three isolates were subtype B. Partial sequence analysis of the G glycoprotein gene confirmed that the samples belonged to subtype B and were not of the vaccine type. Comparison of nucleotide and amino acid sequences of the G gene of the three Brazilian aMPV samples with subtype B isolates from other countries revealed 95.1% to 96.1% identity. Nucleotide sequences showed 100% identity among the Brazilian subtype B samples and 95.6% identity with the subtype B vaccine strain used in Brazil. This work describes the circulation of subtype B aMPV in Brazil and discusses its importance in terms of disease epidemiology.

A novel two-nucleotide deletion in the ATP7A gene associated with delayed infantile onset of Menkes disease.

Science.gov (United States)

Wada, Takahito; Haddad, Marie Reine; Yi, Ling; Murakami, Tomomi; Sasaki, Akiko; Shimbo, Hiroko; Kodama, Hiroko; Osaka, Hitoshi; Kaler, Stephen G

2014-04-01

Determining the relationship between clinical phenotype and genotype in genetic diseases is important in clinical practice. In general, frameshift mutations are expected to produce premature termination codons, leading to production of mutant transcripts destined for degradation by nonsense-mediated decay. In X-linked recessive diseases, male patients with frameshift mutations typically have a severe or even lethal phenotype. We report a case of a 17-month-old boy with Menkes disease (NIM #309400), an X-linked recessive copper metabolism disorder caused by mutations in the ATP7A copper transporter gene. He exhibited an unexpectedly late onset and experienced milder symptoms. His genomic DNA showed a de novo two-nucleotide deletion in exon 4 of ATP7A, predicting a translational frameshift and premature stop codon, and a classic severe phenotype. Characterization of his ATP7A mRNA showed no abnormal splicing. We speculate that translation reinitiation could occur downstream to the premature termination codon and produce a partially functional ATP7A protein. Study of the child's fibroblasts found no evidence of translation reinitiation; however, the possibility remains that this phenomenon occurred in neural tissues and influenced the clinical phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Direct detection of single-nucleotide polymorphisms in bacterial DNA by SNPtrap

DEFF Research Database (Denmark)

Grønlund, Hugo Ahlm; Moen, Birgitte; Hoorfar, Jeffrey

2011-01-01

A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technolo...

Posterior reversible encephalopathy syndrome: diagnosis with CT and MRI

International Nuclear Information System (INIS)

Wang Guangbin; Zhao Bin; Yang Zhenzhen; Shi Hao; Chiu, L.C.; Shan Ruiqin

2006-01-01

Objective: To evaluate the role of CT and MRI in the diagnosis of posterior reversible encephalopathy syndrome (PRES). Methods: Eight women with PRES (6 pregnant women, 1 case after chemotherapy, and 1 patient with hypertension)were enrolled in our study. All of them had MR imaging (T 1 WI, T 2 WI, FLAIR, DWI), and five cases underwent post-contrast T 1 WI and three dimensional contrast enhanced MR angiography (3D CEMRA). Two cases also had CT scan. Results: MRV in all 8 patients showed no evidence of stenosis, dilation, or thrombosis in cranial veins and sinuses. MRI demonstrated multiple lesions located in bilateral parieto-occipital lobes (8 cases), bilateral basal ganglia (2 cases), and bilateral frontal lobes (4 cases). The lesions were prominent within white matter, some of them involved gray matter (3 cases). Lesions appeared as hyperintense signals on FLAIR and T 2 -weighted images, isointense or mildly hypointense signals on T 1 -weighted images, normal or decreased intensity on DWI, and isointensity or hyperintensity on apparent diffusion coefficient (ADC) maps. Post-contrast T 1 WI showed mild reversible enhancement and 3D CEMRA displayed numerous reversible 'grape-like' enhancements in terminal arterial branches along the middle cerebral artery (MCA), anterior cerebral artery (ACA) and posterior cerebral artery (PCA). Follow-up scan showed decreased abnormal signals. Conclusion: Lesions of PRES are usually located in parieto-occipital lobes, especially in white matter, but they can also be seen in frontal lobes and basal ganglia bilaterally. Post-contrast T 1 WI and 3D enhanced MRA can provide useful information in the manifestation of reversible enhancement. MRI has advantages to display lesion in PRES. (authors)

Nucleotide Excision Repair in Cellular Chromatin: Studies with Yeast from Nucleotide to Gene to Genome

Directory of Open Access Journals (Sweden)

Simon Reed

2012-09-01

Full Text Available Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes.

Nucleotide excision repair in the test tube.

NARCIS (Netherlands)

N.G.J. Jaspers (Nicolaas); J.H.J. Hoeijmakers (Jan)

1995-01-01

textabstractThe eukaryotic nucleotide excision-repair pathway has been reconstituted in vitro, an achievement that should hasten the full enzymological characterization of this highly complex DNA-repair pathway.

Ion heating and MHD dynamo fluctuations in the reversed field pinch

International Nuclear Information System (INIS)

Scime, E.; Hokin, S.; Watts, C.; Mattor, N.

1992-01-01

Ion temperature measurements, time resolved to 10 μs, have been made in the Madison Symmetric Torus reversed-field pinch with a five channel charge exchange analyzer. The ion temperature, T i ∼ 200 eV for I = 350 kA, increases by as much as 100% during discrete dynamo bursts in MST discharges. Magnetic field fluctuations in the range 0.5--5 MHz were also measured. Structure in the fluctuation frequency spectrum at the ion cyclotron frequency appears as the bursts terminate, suggesting that the mechanism of ion heating involves the dissipation of dynamo fluctuations at ion gyro-orbit scales

Antecedents of Customer Relationship Termination

DEFF Research Database (Denmark)

Geersbro, Jens; Ritter, Thomas

To end business relationships, or to more actively terminate relationships, has long been acknowledged as part of customer relationship management. However, compared to other elements such as initiation and maintenance of relationships, little is known about the termination of business...... relationships as a managerial task. This paper contributes by (1) developing a conceptualization of relationship termination competence and (2) analyzing its antecedents. The empirical results identify termination acceptance, definition non-customers, organizational relationship termination routines......, and motivation as significant antecedents. Because of this, managers need to develop their organizations in order to use relationship termination as a vital strategy....

Experimental profile evolution of a high-density field-reversed configuration

International Nuclear Information System (INIS)

Ruden, E. L.; Zhang, Shouyin; Intrator, T. P.; Wurden, G. A.

2006-01-01

A field-reversed configuration (FRC) gains angular momentum over time, eventually resulting in an n=2 rotational instability (invariant under rotation by π) terminating confinement. To study this, a laser interferometer probes the time history of line integrated plasma density along eight chords of the high-density (∼10 17 cm -3 ) field-reversed configuration experiment with a liner. Abel and tomographic inversions provide density profiles during the FRC's azimuthally symmetric phase, and over a period when the rotational mode has saturated and rotates with a roughly fixed profile, respectively. During the latter part of the symmetric phase, the FRC approximates a magnetohydrodynamic (MHD) equilibrium, allowing the axial magnetic-field profile to be calculated from pressure balance. Basic FRC properties such as temperature and poloidal flux are then inferred. The subsequent two-dimensional n=2 density profiles provide angular momentum information needed to set bounds on prior values of the stability relevant parameter α (rotational to ion diamagnetic drift frequency ratio), in addition to a view of plasma kinematics useful for benchmarking plasma models of higher order than MHD

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

Science.gov (United States)

Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

Multifactor dimensionality reduction analysis identifies specific nucleotide patterns promoting genetic polymorphisms

Directory of Open Access Journals (Sweden)

Arehart Eric

2009-03-01

Full Text Available Abstract Background The fidelity of DNA replication serves as the nidus for both genetic evolution and genomic instability fostering disease. Single nucleotide polymorphisms (SNPs constitute greater than 80% of the genetic variation between individuals. A new theory regarding DNA replication fidelity has emerged in which selectivity is governed by base-pair geometry through interactions between the selected nucleotide, the complementary strand, and the polymerase active site. We hypothesize that specific nucleotide combinations in the flanking regions of SNP fragments are associated with mutation. Results We modeled the relationship between DNA sequence and observed polymorphisms using the novel multifactor dimensionality reduction (MDR approach. MDR was originally developed to detect synergistic interactions between multiple SNPs that are predictive of disease susceptibility. We initially assembled data from the Broad Institute as a pilot test for the hypothesis that flanking region patterns associate with mutagenesis (n = 2194. We then confirmed and expanded our inquiry with human SNPs within coding regions and their flanking sequences collected from the National Center for Biotechnology Information (NCBI database (n = 29967 and a control set of sequences (coding region not associated with SNP sites randomly selected from the NCBI database (n = 29967. We discovered seven flanking region pattern associations in the Broad dataset which reached a minimum significance level of p ≤ 0.05. Significant models (p Conclusion The present study represents the first use of this computational methodology for modeling nonlinear patterns in molecular genetics. MDR was able to identify distinct nucleotide patterning around sites of mutations dependent upon the observed nucleotide change. We discovered one flanking region set that included five nucleotides clustered around a specific type of SNP site. Based on the strongly associated patterns identified in

Adsorption of nucleotides onto Fe-Mg-Al rich swelling clays

Science.gov (United States)

Feuillie, Cécile; Daniel, Isabelle; Michot, Laurent J.; Pedreira-Segade, Ulysse

2013-11-01

Mineral surfaces may have played a role in the origin of the first biopolymers, by concentrating organic monomers from a dilute ocean. Swelling clays provide a high surface area for the concentration of prebiotic monomers, and have therefore been the subject of numerous investigations. In that context, montmorillonite, the most abundant swelling clay in modern environments, has been extensively studied with regard to adsorption and polymerization of nucleic acids. However, montmorillonite was probably rather marginal on the primitive ocean floor compared to iron-magnesium rich phyllosilicates such as nontronite that results from the hydrothermal alteration of a mafic or ultramafic oceanic crust. In the present paper, we study the adsorption of nucleotides on montmorillonite and nontronite, at various pH and ionic strength conditions plausible for Archean sea-water. A thorough characterization of the mineral surfaces shows that nucleotide adsorb mainly on the edge faces of the smectites by ligand exchange between the phosphate groups of the nucleotides and the -OH groups from the edge sites over a wide pH range (4-10). Nontronite is more reactive than montmorillonite. At low pH, additional ion exchange may play a role as the nucleotides become positively charged.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides

Energy Technology Data Exchange (ETDEWEB)

Shin, Joong-Won, E-mail: jshin@govst.edu [Division of Science, Governors State University, University Park, Illinois 60484-0975 (United States); Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States); Bernstein, Elliot R., E-mail: erb@lamar.colostate.edu [Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872 (United States)

2014-01-28

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

Vacuum ultraviolet photoionization of carbohydrates and nucleotides

International Nuclear Information System (INIS)

Shin, Joong-Won; Bernstein, Elliot R.

2014-01-01

Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5 ′ -monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results

Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome.

Science.gov (United States)

Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K; Drewell, Robert A

2016-01-01

A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.

The International Nucleotide Sequence Database Collaboration.

Science.gov (United States)

Cochrane, Guy; Karsch-Mizrachi, Ilene; Nakamura, Yasukazu

2011-01-01

Under the International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org), globally comprehensive public domain nucleotide sequence is captured, preserved and presented. The partners of this long-standing collaboration work closely together to provide data formats and conventions that enable consistent data submission to their databases and support regular data exchange around the globe. Clearly defined policy and governance in relation to free access to data and relationships with journal publishers have positioned INSDC databases as a key provider of the scientific record and a core foundation for the global bioinformatics data infrastructure. While growth in sequence data volumes comes no longer as a surprise to INSDC partners, the uptake of next-generation sequencing technology by mainstream science that we have witnessed in recent years brings a step-change to growth, necessarily making a clear mark on INSDC strategy. In this article, we introduce the INSDC, outline data growth patterns and comment on the challenges of increased growth.

Age-dependent terminal declines in reproductive output in a wild bird.

Directory of Open Access Journals (Sweden)

Martijn Hammers

Full Text Available In many iteroparous species individual fitness components, such as reproductive output, first increase with age and then decline during late-life. However, individuals differ greatly in reproductive lifespan, but reproductive declines may only occur in the period just before their death as a result of an age-independent decline in physiological condition. To fully understand reproductive senescence it is important to investigate to what extent declines in late-life reproduction can be explained by age, time until death, or both. However, the study of late-life fitness performance in natural populations is challenging as the exact birth and death dates of individuals are often not known, and most individuals succumb to extrinsic mortality before reaching old age. Here, we used an exceptional long-term longitudinal dataset of individuals from a natural, closed, and predator-free population of the Seychelles warbler (Acrocephalus sechellensis to investigate reproductive output, both in relation to age and to the time until the death of an individual (reverse-age approach. We observed an initial age-dependent increase in reproductive output that was followed by a decline in old age. However, we found no significant decline in reproductive output in the years directly preceding death. Although post-peak reproductive output declined with age, this pattern differed between terminal and non-terminal reproductive attempts, and the age-dependence of the terminal breeding attempt explained much of the variation in age-specific reproductive output. In fact, terminal declines in reproductive output were steeper in very old individuals. These results indicate that not only age-dependent, but also age-independent factors, such as physiological condition, need to be considered to understand reproductive senescence in wild-living animals.

Subnanomole detection and quantitation of high specific activity 32P-nucleotides

International Nuclear Information System (INIS)

Coniglio, C.; Pappas, G.; Gill, W.J.; Kashdan, M.; Maniscalco, M.

1991-01-01

Microbore liquid chromatography utilizes conventional HPLC and ultraviolet detection principles to determine subnanomole mass quantities of biologically significant molecules. This system takes advantage of specifically designed microflow equipment to analyze ultraviolet absorbing species at the picomole range. 32P-labeled nucleotides are examples of compounds routinely used at picomole quantities that are extremely difficult to accurately quantify using standard mass measurement techniques. The procedure described in this paper has the capability of measuring nucleotides down to 10 pmol using commercially available microbore ultraviolet detection equipment. The technique can be used to accurately measure the specific activity of as little as 10 microCi of an aqueous 32P-nucleotide solution

The nucleotide sequence of the RNA-2 of an isolate of the English serotype of tomato black ring virus: RNA recombination in the history of nepoviruses.

Science.gov (United States)

Le Gall, O L; Lanneau, M; Candresse, T; Dunez, J

1995-05-01

The RNA-2 of a carrot isolate from the English serotype of tomato black ring nepovirus (TBRV-ED) has been sequenced. It is 4618 nucleotides long and contains one open reading frame encoding a polypeptide of 1344 amino acids. The 5' non-coding region contains three repetitions of a stem-loop structure also conserved in TBRV-Scottish and grapevine chrome mosaic nepovirus (GCMV). The coat protein domain was mapped to the carboxy-terminal one-third of the polyprotein. Sequence comparisons indicate that TBRV-ED RNA-2 probably arose by an RNA recombination event that resulted in the exchange of the putative movement protein gene between TBRV and GCMV.

Therapeutic efficiency of nucleotides and nucleosides in UV radiation edema of mice

Energy Technology Data Exchange (ETDEWEB)

Kutta, I

1973-01-01

The influence of several nucleotides and nucleosides on UV radiation edemas of mice was studied with the aid of a staining test. In the first test series, amounts equimolar to 20 mg thymidine were injected i.p. It was found that thymidine, ATP, ADP and A5'MP had a significant influence which uridine did not have. The NAD dose of 54.8 mg was lethal in all 10 animals and the ATP dose of 42 mg in three out of 10 animals, while ADP and A5'MP had the effect of a reversible retardation of movements. The most effective substances of this series were ATP and ADP. In the second test series, the substances were equimolar to 1.8 mg thymidine. All substances tested, i.e. thymidine, adenosin, adenosin-cyclophosphate, NAD, NADH, ATP, ADP and A5'MP had a significant effect. Except for NAD, to which the animals reacted with a slight retardation, all substances were well tolerated. NAD and ADP were the most effective. In a third test series, dose-efficiency curves were established for thymidine and ATP. ATP was significantly more effective in equimolar doses. This finding is discussed.

De novo synthesis of adenine nucleotides in different skeletal muscle fiber types

International Nuclear Information System (INIS)

Tullson, P.C.; John-Alder, H.B.; Hood, D.A.; Terjung, R.L.

1988-01-01

Management of adenine nucleotide catabolism differs among skeletal muscle fiber types. This study evaluated whether there are corresponding differences in the rates of de novo synthesis of adenine nucleotide among fiber type sections of skeletal muscle using an isolated perfused rat hindquarter preparation. Label incorporation into adenine nucleotides from the [1-14C]glycine precursor was determined and used to calculate synthesis rates based on the intracellular glycine specific radioactivity. Results show that intracellular glycine is closely related to the direct precursor pool. Rates of de novo synthesis were highest in fast-twitch red muscle (57.0 +/- 4.0, 58.2 +/- 4.4 nmol.h-1.g-1; deep red gastrocnemius and vastus lateralis), relatively high in slow-twitch red muscle (47.0 +/- 3.1; soleus), and low in fast-twitch white muscle (26.1 +/- 2.0 and 21.6 +/- 2.3; superficial white gastrocnemius and vastus lateralis). Rates for four mixed muscles were intermediate, ranging between 32.3 and 37.3. Specific de novo synthesis rates exhibited a strong correlation (r = 0.986) with muscle section citrate synthase activity. Turnover rates (de novo synthesis rate/adenine nucleotide pool size) were highest in high oxidative muscle (0.82-1.06%/h), lowest in low oxidative muscle (0.30-0.35%/h), and intermediate in mixed muscle (0.44-0.55%/h). Our results demonstrate that differences in adenine nucleotide management among fiber types extends to the process of de novo adenine nucleotide synthesis

A novel Y-xylosidase, nucleotide sequence encoding it and use thereof.

NARCIS (Netherlands)

Graaff, de L.H.; Peij, van N.N.M.E.; Broeck, van den H.C.; Visser, J.

1996-01-01

A nucleotide sequence is provided which encodes a peptide having beta-xylosidase activity and exhibits at least 30mino acid identity with the amino acid sequence shown in SEQ ID NO. 1 or hybridises under stringent conditions with a nucleotide sequence shown in SEQ ID NO. 1, or a part thereof having

Improvements in DC Current-Ioltage (I-V) Characteristics of n-GaN Schottky Diode using Metal Overlap Edge Termination

International Nuclear Information System (INIS)

Munir, T.; Aziz, A. A.; Abdullah, M. J.; Ain, M. F.

2010-01-01

Practical design of GaN Schottky diodes incorporating a field plate necessitates an understanding of how the addition of such plate affects the diode performance. In this paper, we investigated the effects on DC current-voltage (I-V) characteristics of n-GaN schottky diode by incorporating metal overlap edge termination. The thickness of the oxide film varies from 0.001 to 1 micron. Two-dimensional Atlas/Blaze simulations revealed that severe electric field crowding across the metal semiconductor contact will cause reliability concern and limit device breakdown voltage. DC current-voltage (I-V) measurements indicate that the forward currents are higher for thinner oxide film schottky diodes with metal overlap edge termination than those of unterminated schottky diodes. The forward current increased due to formation of an accumulation layer underneath the oxide layer. Extending the field plate to beyond periphery regions of schottky contact does not result in any significant increase in forward current. The new techniques of ramp oxide metal overlap edge termination have been implemented to increase the forward current of n-GaN schottky diode. In reverse bias, breakdown voltage increased with edge termination oxide up to a certain limit of oxide thickness.

Reversible alkyne insertion in the benzannulation reaction of Fischer carbene complexes with alkynes

Energy Technology Data Exchange (ETDEWEB)

Waters, M.L.; Bos, M.E.; Wulff, W.D. [Univ. of Chicago, IL (United States)

1995-12-31

The benzannulation reaction of Fischer carbene complexes with alkynes to give phenols is highly regioselective with terminal alkynes, and reasonably regioselective with internal alkynes. This has been attributed to steric factors in intermediates, where one form is favored due to close contact between the R substituent and a cis-CO ligand. Whether alkyne insertion is kinetically or thermodynamically controlled has not been determined. The authors now have evidence from regioselectivity studies that alkyne insertion into the metal-carbon bond is reversible. Implications of these results and further mechanistic considerations will be presented.

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide γ-phosphate derivative

Science.gov (United States)

Hansen, Connie J.; Wu, Lydia; Fox, Jeffrey D.; Arezi, Bahram; Hogrefe, Holly H.

2011-01-01

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure. PMID:21062827

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide gamma-phosphate derivative.

Science.gov (United States)

Hansen, Connie J; Wu, Lydia; Fox, Jeffrey D; Arezi, Bahram; Hogrefe, Holly H

2011-03-01

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.

LNG TERMINAL SAFE OPERATION MANAGEMENT

Directory of Open Access Journals (Sweden)

Andrzej ADAMKIEWICZ

2012-07-01

Full Text Available This article presents the significance of LNG terminal safety issues in natural gas sea transport. It shows particular requirements for LNG transmission installations resulting from the specific properties of LNG. Out of the multi‐layer critical safety areas comprising structural elements of the terminal safety system, possibilities to decrease the risk of emergency occurrence on LNG terminals have been selected. Tasks performed by the LNG terminal, together with its own personnel and the outside one, have been defined. General theses for LNG terminal safety have been formulated.

Reversible Thermoset Adhesives

Science.gov (United States)

Mac Murray, Benjamin C. (Inventor); Tong, Tat H. (Inventor); Hreha, Richard D. (Inventor)

2016-01-01

Embodiments of a reversible thermoset adhesive formed by incorporating thermally-reversible cross-linking units and a method for making the reversible thermoset adhesive are provided. One approach to formulating reversible thermoset adhesives includes incorporating dienes, such as furans, and dienophiles, such as maleimides, into a polymer network as reversible covalent cross-links using Diels Alder cross-link formation between the diene and dienophile. The chemical components may be selected based on their compatibility with adhesive chemistry as well as their ability to undergo controlled, reversible cross-linking chemistry.

A functional polymorphism in the prodynorphin gene affects cognitive flexibility and brain activation during reversal learning.

Directory of Open Access Journals (Sweden)

Mikhail eVotinov

2015-07-01

Full Text Available Whether the opioid system plays a role in the ability to flexibly adapt behavior is still unclear. We used fMRI to investigate the effect of a nucleotide tandem repeat (68-bp VNTR functional polymorphism of the prodynorphin gene on cerebral activation during a reversal learning task in which participants had to flexibly adapt stimulus-response associations. Past studies suggested that alleles with 3 or 4 repeats (HH genotype of this polymorphism are associated with higher levels of dynorphin peptides than alleles with 1 or 2 repeats (LL genotype. On the behavioral level, the HH group made more perseverative errors than the LL group. On the neural level, the HH group demonstrated less engagement of left orbitofrontal cortex (lOFC and cortico-striatal circuitry, and lower effective connectivity of lOFC with anterior midcingulate cortex and anterior insula/ventrolateral prefrontal cortex during reversal learning and processing negative feedback. This points to a lower ability of the HH genotype to monitor or adapt to changes in reward contingencies. These findings provide first evidence that dynorphins may contribute to individual differences in reversal learning, and that considering the opioid system may shed new light on the neurochemical correlates of decision-making and behavioral regulation.

Nonleaking battery terminals.

Science.gov (United States)

Snider, W. E.; Nagle, W. J.

1972-01-01

Three different terminals were designed for usage in a 40 ampere/hour silver zinc battery which has a 45% KOH by weight electrolyte in a plastic battery case. Life tests, including thermal cycling, electrical charge and discharge for up to three years duration, were conducted on these three different terminal designs. Tests for creep rate and tensile strength were conducted on the polyphenylene oxide plastic battery cases. Some cases were unused and others containing KOH electrolyte were placed on life tests. The design and testing of nonleaking battery terminals for use with a KOH electrolyte in a plastic case are considered.

Plastid: nucleotide-resolution analysis of next-generation sequencing and genomics data.

Science.gov (United States)

Dunn, Joshua G; Weissman, Jonathan S

2016-11-22

Next-generation sequencing (NGS) informs many biological questions with unprecedented depth and nucleotide resolution. These assays have created a need for analytical tools that enable users to manipulate data nucleotide-by-nucleotide robustly and easily. Furthermore, because many NGS assays encode information jointly within multiple properties of read alignments - for example, in ribosome profiling, the locations of ribosomes are jointly encoded in alignment coordinates and length - analytical tools are often required to extract the biological meaning from the alignments before analysis. Many assay-specific pipelines exist for this purpose, but there remains a need for user-friendly, generalized, nucleotide-resolution tools that are not limited to specific experimental regimes or analytical workflows. Plastid is a Python library designed specifically for nucleotide-resolution analysis of genomics and NGS data. As such, Plastid is designed to extract assay-specific information from read alignments while retaining generality and extensibility to novel NGS assays. Plastid represents NGS and other biological data as arrays of values associated with genomic or transcriptomic positions, and contains configurable tools to convert data from a variety of sources to such arrays. Plastid also includes numerous tools to manipulate even discontinuous genomic features, such as spliced transcripts, with nucleotide precision. Plastid automatically handles conversion between genomic and feature-centric coordinates, accounting for splicing and strand, freeing users of burdensome accounting. Finally, Plastid's data models use consistent and familiar biological idioms, enabling even beginners to develop sophisticated analytical workflows with minimal effort. Plastid is a versatile toolkit that has been used to analyze data from multiple NGS assays, including RNA-seq, ribosome profiling, and DMS-seq. It forms the genomic engine of our ORF annotation tool, ORF-RATER, and is readily

A new general method for simultaneous fitting of temperature and concentration dependence of reaction rates yields kinetic and thermodynamic parameters for HIV reverse transcriptase specificity.

Science.gov (United States)

Li, An; Ziehr, Jessica L; Johnson, Kenneth A

2017-04-21

Recent studies have demonstrated the dominant role of induced fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking, and equilibrium thermodynamic methods are of no avail because the key parameters can only be determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37 °C, and the raw data were fit globally to derive a single set of rate constants at 37 °C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding ( K d , Δ G , Δ H , and Δ S at 37 °C) and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis.

Science.gov (United States)

Pisithkul, Tippapha; Jacobson, Tyler B; O'Brien, Thomas J; Stevenson, David M; Amador-Noguez, Daniel

2015-09-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using (13)C-labeled sugars and [(15)N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. Copyright © 2015, Pisithkul et al.

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

Science.gov (United States)

Pisithkul, Tippapha; Jacobson, Tyler B.; O'Brien, Thomas J.; Stevenson, David M.

2015-01-01

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using 13C-labeled sugars and [15N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals. PMID:26070680

Zero field reversal probability in thermally assisted magnetization reversal

Science.gov (United States)

Prasetya, E. B.; Utari; Purnama, B.

2017-11-01

This paper discussed about zero field reversal probability in thermally assisted magnetization reversal (TAMR). Appearance of reversal probability in zero field investigated through micromagnetic simulation by solving stochastic Landau-Lifshitz-Gibert (LLG). The perpendicularly anisotropy magnetic dot of 50×50×20 nm3 is considered as single cell magnetic storage of magnetic random acces memory (MRAM). Thermally assisted magnetization reversal was performed by cooling writing process from near/almost Curie point to room temperature on 20 times runs for different randomly magnetized state. The results show that the probability reversal under zero magnetic field decreased with the increase of the energy barrier. The zero-field probability switching of 55% attained for energy barrier of 60 k B T and the reversal probability become zero noted at energy barrier of 2348 k B T. The higest zero-field switching probability of 55% attained for energy barrier of 60 k B T which corespond to magnetif field of 150 Oe for switching.

Homopolar machine for reversible energy storage and transfer systems

International Nuclear Information System (INIS)

Stillwagon, R.E.

1978-01-01

A homopolar machine designed to operate as a generator and motor in reversibly storing and transferring energy between the machine and a magnetic load coil for a thermonuclear reactor is described. The machine rotor comprises hollow thin-walled cylinders or sleeves which form the basis of the system by utilizing substantially all of the rotor mass as a conductor thus making it possible to transfer substantially all the rotor kinetic energy electrically to the load coil in a highly economical and efficient manner. The rotor is divided into multiple separate cylinders or sleeves of modular design, connected in series and arranged to rotate in opposite directions but maintain the supply of current in a single direction to the machine terminals

AVP-stimulated nucleotide secretion in perfused mouse medullary thick ascending limb and cortical collecting duct

DEFF Research Database (Denmark)

Odgaard, Elvin V. P.; Prætorius, Helle; Leipziger, Jens Georg

2009-01-01

is stimulated remain elusive. Here, we investigate the phenomenon of nucleotide secretion in intact, perfused mouse medullary thick ascending limb (mTAL) and cortical collecting duct (CCD). The nucleotide secretion was monitored by a biosensor adapted to register nucleotides in the tubular outflow...

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

Science.gov (United States)

De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

2016-07-27

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Reverse Osmosis

Indian Academy of Sciences (India)

many applications, one of which is desalination of seawater. The inaugural Nobel Prize in Chemistry was awarded in 1901 to van 't Hoff for his seminal work in this area. The present article explains the principle of osmosis and reverse osmosis. Osmosis and Reverse Osmosis. As the name suggests, reverse osmosis is the ...

The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae.

Science.gov (United States)

Woolfitt, A R; Kellett, G L; Hoggett, J G

1988-01-29

The binding of glucose, ADP and AdoPP[NH]P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence. The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and [L]0.5, the concentration of ligand at half-maximal quenching. No changes in fluorescence were observed with free enzyme and nucleotide alone. In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and [L]0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species. Qmax induced by glucose alone was between 22 and 25%, while [L]0.5 was approx. 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer. In the presence of 6 mM ADP or 2 mM AdoPP[NH]P, Qmax for glucose was increased by up to 4% and [L]0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer. The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.

Intrinsic terminators in Mycoplasma hyopneumoniae transcription.

Science.gov (United States)

Fritsch, Tiago Ebert; Siqueira, Franciele Maboni; Schrank, Irene Silveira

2015-04-08

Mycoplasma hyopneumoniae, an important pathogen of swine, exhibits a low guanine and cytosine (GC) content genome. M. hyopneumoniae genome is organised in long transcriptional units and promoter sequences have been mapped upstream of all transcription units. These analysis provided insights into the gene organisation and transcription initiation at the genome scale. However, the presence of transcriptional terminator sequences in the M. hyopneumoniae genome is poorly understood. In silico analyses demonstrated the presence of putative terminators in 82% of the 33 monocistronic units (mCs) and in 74% of the 116 polycistronic units (pCs) considering different classes of terminators. The functional activity of 23 intrinsic terminators was confirmed by RT-PCR and qPCR. Analysis of all terminators found by three software algorithms, combined with experimental results, allowed us to propose a pattern of RNA hairpin formation during the termination process and to predict the location of terminators in the M. hyopneumoniae genome sequence. The stem-loop structures of intrinsic terminators of mycoplasma diverge from the pattern of terminators found in other bacteria due the low content of guanine and cytosine. In M. hyopneumoniae, transcription can end after a transcriptional unit and before its terminator sequence and can also continue past the terminator sequence with RNA polymerases gradually releasing the RNA.

LNG TERMINAL SAFE OPERATION MANAGEMENT

OpenAIRE

Andrzej ADAMKIEWICZ; Włodzimierz KAMIŃSKI

2012-01-01

This article presents the significance of LNG terminal safety issues in natural gas sea transport. It shows particular requirements for LNG transmission installations resulting from the specific properties of LNG. Out of the multi‐layer critical safety areas comprising structural elements of the terminal safety system, possibilities to decrease the risk of emergency occurrence on LNG terminals have been selected. Tasks performed by the LNG terminal, together with its own personnel and the out...

The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP.

Science.gov (United States)

Querol-Audí, Jordi; Casañas, Arnau; Usón, Isabel; Luque, Daniel; Castón, José R; Fita, Ignasi; Verdaguer, Nuria

2009-11-04

Vaults are ubiquitous ribonucleoprotein complexes involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. The vault particle shows a barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein MVP. Earlier data indicated that vault halves can dissociate at acidic pH. The crystal structure of the vault particle solved at 8 A resolution, together with the 2.1-A structure of the seven N-terminal domains (R1-R7) of MVP, reveal the interactions governing vault association and provide an explanation for a reversible dissociation induced by low pH. The structural comparison with the recently published 3.5 A model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2.

Optimization time synthesis of nucleotide labelled [γ-32P]-ATP

International Nuclear Information System (INIS)

Rahman, Wira Y; Sarmini, Endang; Herlina; Lubis, Hotman; Triyanto; Hambali

2013-01-01

Adenosine triphosphate-labelled with γ- 32 P([γ- 32 p]-ATP) has been widely used in the biotechnology research, usually as a tracer to study aspects of physiological and pathological processes. In order to support biotechnology research in Indonesia, a process for production of [γ- 32 P]-ATP with enzymatic reaction was used as precursors DL-glyceraldehydde 3-phosphate, Adenosine Diphosphate (ADP) and H 3 32 PO 4 , and enzyme glyceraldehid 3-phosphate dehydrogenase, 3-phosphoglyceryc phosphokinase and lactate dehydrogenase. Optimization of incubation time labeled nucleotide synthesis process is performed to find the optimum conditions, in terms of the most advantageous time in the synthesis process. With the success of the synthesis and optimization is done incubation time of synthesis labeled nucleotide, the result suggested can be used for producing [γ- 32 P] -ATP to support the provision of radiolabeled nucleotide for biotechnology research in Indonesia. (author)

Termination unit

Energy Technology Data Exchange (ETDEWEB)

Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

2016-05-03

Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

Efficacy of the chelating agent CaEDTA in reversing lead-induced changes in behavior.

Science.gov (United States)

Cory-Slechta, D A; Weiss, B

1989-01-01

The chelating agent CaEDTA has been reported to reverse the deficits in intellectual function and performance associated with Pb (lead) exposure in children. However, such studies have not included rigorous controls for the intervention procedures per se. The experiments reported here examined reversibility of performance changes in a rat model based on behavior sensitive to low-level Pb exposure. Rats were exposed to 50 ppm sodium or Pb acetate in drinking water from weaning. Performance maintained under a Fixed-Interval schedule of food reinforcement began at 55 days of age. Following the onset of the characteristic increase in short interresponse times (IRTs) associated with low-level Pb exposure after 35 experimental sessions, Pb treatment was terminated. Animals within both the control and Pb groups were then matched on the basis of performance indices and injected daily for 5 days with either saline, 75 mg/kg or 150 mg/kg CaEDTA. Subsequent changes in F1 performance were monitored for 35-60 sessions. No consistent effects of CaEDTA were detected in control animals. CaEDTA treatment failed to reverse the behavioral effects in Pb-exposed animals. If anything, it tended to further increase the proportion of short IRTs. These data suggest that better controlled clinical studies are warranted to evaluate the efficacy of CaEDTA in reversing Pb-induced behavioral effects before its application for these purposes becomes widespread.

WEB-server for search of a periodicity in amino acid and nucleotide sequences

Science.gov (United States)

E Frenkel, F.; Skryabin, K. G.; Korotkov, E. V.

2017-12-01

A new web server (http://victoria.biengi.ac.ru/splinter/login.php) was designed and developed to search for periodicity in nucleotide and amino acid sequences. The web server operation is based upon a new mathematical method of searching for multiple alignments, which is founded on the position weight matrices optimization, as well as on implementation of the two-dimensional dynamic programming. This approach allows the construction of multiple alignments of the indistinctly similar amino acid and nucleotide sequences that accumulated more than 1.5 substitutions per a single amino acid or a nucleotide without performing the sequences paired comparisons. The article examines the principles of the web server operation and two examples of studying amino acid and nucleotide sequences, as well as information that could be obtained using the web server.

Reversible photocapture of a [2]rotaxane harnessing a barbiturate template.

Science.gov (United States)

Tron, Arnaud; Thornton, Peter J; Lincheneau, Christophe; Desvergne, Jean-Pierre; Spencer, Neil; Tucker, James H R; McClenaghan, Nathan D

2015-01-16

Photoirradiation of a hydrogen-bonded molecular complex comprising acyclic components, namely, a stoppered thread (1) with a central barbiturate motif and an optimized doubly anthracene-terminated acyclic Hamilton-like receptor (2b), leads to an interlocked architecture, which was isolated and fully characterized. The sole isolated interlocked photoproduct (Φ = 0.06) is a [2]rotaxane, with the dimerized anthracenes assuming a head-to-tail geometry, as evidenced by NMR spectroscopy and consistent with molecular modeling (PM6). A different behavior was observed on irradiating homologous molecular complexes 1⊂2a, 1⊂2b, and 1⊂2c, where the spacers of 2a, 2b, and 2c incorporated 3, 6, and 9 methylene units, respectively. While no evidence of interlocked structure formation was observed following irradiation of 1⊂2a, a kinetically labile rotaxane was obtained on irradiating the complex 1⊂2c, and ring slippage was revealed. A more stable [2]rotaxane was formed on irradiating 1⊂2b, whose capture is found to be fully reversible upon heating, thereby resetting the system, with some fatigue (38%) after four irradiation–thermal reversion cycles.

Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

Science.gov (United States)

Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

2011-03-01

The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Managing Reverse Logistics or Reversing Logistics Management?

NARCIS (Netherlands)

M.P. de Brito (Marisa)

2004-01-01

textabstractIn the past, supply chains were busy fine-tuning the logistics from raw material to the end customer. Today an increasing flow of products is going back in the chain. Thus, companies have to manage reverse logistics as well.This thesis contributes to a better understanding of reverse

A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff

Science.gov (United States)

Cingolani, Pablo; Platts, Adrian; Wang, Le Lily; Coon, Melissa; Nguyen, Tung; Wang, Luan; Land, Susan J.; Lu, Xiangyi; Ruden, Douglas M.

2012-01-01

We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w1118; iso-2; iso-3 strain and the reference y1; cn1 bw1 sp1 strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5′UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5′ and 3′ UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory. PMID:22728672

The therapeutic efficiency of nucleotides and nucleosides in UV radiation edema of mice

International Nuclear Information System (INIS)

Kutta, I.

1973-01-01

The influence of several nucleotides and nucleosides on UV radiation edemas of mice was studied with the aid of a staining test. In the first test series, amounts equimolar to 20 mg thymidine were injected i.p. It was found that thymidine, ATP, ADP and A5'MP had a significant influence which uridine did not have. The NAD dose of 54.8 mg was lethal in all 10 animals and the ATP dose of 42 mg in three out of 10 animals, while ADP and A5'MP had the effect of a reversible retardation of movements. The most effective substances of this series were ATP and ADP. In the second test series, the substances were equimolar to 1.8 mg thymidine. All substances tested, i.e. thymidine, adenosin, adenosin-cyclophosphate, NAD, NADH, ATP, ADP and A5'MP had a significant effect. Except for NAD, to which the animals reacted with a slight retardation, all substances were well tolerated. NAD and ADP were the most effective. In a third test series, dose-efficiency curves were established for thymidine and ATP. ATP was significantly more effective in equimolar doses. This finding is discussed. (orig.) [de

Reverse transcriptase inhibitors as potential colorectal microbicides.

Science.gov (United States)

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J

2009-05-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides.

A single nucleotide polymorphism (SNP) assay for population ...

African Journals Online (AJOL)

A single nucleotide polymorphism (SNP) assay for population stratification test ... phenotypes and unlinked candidate loci in case-control and cohort studies of ... Key words: Chinese, Japanese, population stratification, ancestry informative ...

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

Science.gov (United States)

Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

2014-01-01

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

Carboxylic Terminated Thermo-Responsive Copolymer Hydrogel and Improvement in Peptide Release Profile

Directory of Open Access Journals (Sweden)

Zi-Kun Rao

2018-02-01

Full Text Available To improve the release profile of peptide drugs, thermos-responsive triblock copolymer poly (ε-caprolactone-co-p-dioxanone-b-poly (ethylene glycol-b-poly (ε-caprolactone-co-p-dioxanone (PECP was prepared and end capped by succinic anhydride to give its carboxylic terminated derivative. Both PCEP block copolymer and its end group modified derivative showed temperature-dependent reversible sol-gel transition in water. The carboxylic end group could significantly decrease the sol-gel transition temperature by nearly 10 °C and strengthen the gel due to enhanced intermolecular force among triblock copolymer chains. Furthermore, compared with the original PECP triblock copolymer, HOOC–PECP–COOH copolymer displayed a retarded and sustained release profile for leuprorelin acetate over one month while effectively avoiding the initial burst. The controlled release was believed to be related to the formation of conjugated copolymer-peptide pair by ionic interaction and enhanced solubility of drug molecules into the hydrophobic domains of the hydrogel. Therefore, carboxyl terminated HOOC–PECP–COOH hydrogel was a promising and well-exhibited sustained release carrier for peptide drugs with the advantage of being able to develop injectable formulation by simple mixing.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

Science.gov (United States)

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

Characterization of cDNA for human tripeptidyl peptidase II: The N-terminal part of the enzyme is similar to subtilisin

International Nuclear Information System (INIS)

Tomkinson, B.; Jonsson, A-K

1991-01-01

Tripeptidyl peptidase II is a high molecular weight serine exopeptidase, which has been purified from rat liver and human erythrocytes. Four clones, representing 4453 bp, or 90% of the mRNA of the human enzyme, have been isolated from two different cDNA libraries. One clone, designated A2, was obtained after screening a human B-lymphocyte cDNA library with a degenerated oligonucleotide mixture. The B-lymphocyte cDNA library, obtained from human fibroblasts, were rescreened with a 147 bp fragment from the 5' part of the A2 clone, whereby three different overlapping cDNA clones could be isolated. The deduced amino acid sequence, 1196 amino acid residues, corresponding to the longest open rading frame of the assembled nucleotide sequence, was compared to sequences of current databases. This revealed a 56% similarity between the bacterial enzyme subtilisin and the N-terminal part of tripeptidyl peptidase II. The enzyme was found to be represented by two different mRNAs of 4.2 and 5.0 kilobases, respectively, which probably result from the utilziation of two different polyadenylation sites. Futhermore, cDNA corresponding to both the N-terminal and C-terminal part of tripeptidyl peptidase II hybridized with genomic DNA from mouse, horse, calf, and hen, even under fairly high stringency conditions, indicating that tripeptidyl peptidase II is highly conserved

klax Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kprc Terminal Aerodrome Forecast

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katl Terminal Aerodrome Forecast

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kmcn Terminal Aerodrome Forecast

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kogb Terminal Aerodrome Forecast

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kama Terminal Aerodrome Forecast

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ptkk Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kiwa Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kavp Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdca Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbwg Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdfw Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kssi Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pahn Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksrq Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpvd Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kisp Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kttd Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pmdy Terminal Aerodrome Forecast

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kmgm Terminal Aerodrome Forecast

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khib Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pavd Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kfar Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kluk Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kwwr Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klse Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksts Terminal Aerodrome Forecast

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koth Terminal Aerodrome Forecast

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kbfl Terminal Aerodrome Forecast

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ksgf Terminal Aerodrome Forecast

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klch Terminal Aerodrome Forecast

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kpkb Terminal Aerodrome Forecast

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krog Terminal Aerodrome Forecast

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kbjc Terminal Aerodrome Forecast

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ksea Terminal Aerodrome Forecast

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kbwi Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kftw Terminal Aerodrome Forecast

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kpuw Terminal Aerodrome Forecast

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kabq Terminal Aerodrome Forecast

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ksny Terminal Aerodrome Forecast

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khio Terminal Aerodrome Forecast

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klaf Terminal Aerodrome Forecast

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kfoe Terminal Aerodrome Forecast

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ksmx Terminal Aerodrome Forecast

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kipt Terminal Aerodrome Forecast

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ktrk Terminal Aerodrome Forecast

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kwmc Terminal Aerodrome Forecast

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katy Terminal Aerodrome Forecast

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tjmz Terminal Aerodrome Forecast

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kdet Terminal Aerodrome Forecast

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National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcxp Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbur Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krkd Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pawg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kloz Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcec Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdec Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paor Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kavl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdrt Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kstl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbfi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khsv Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pafa Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kekn Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

tncm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kith Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgnv Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktoi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgso Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

nstu Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpbi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgdv Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcmx Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdls Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

koaj Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krhi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbpk Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khuf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kont Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kyng Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcwa Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kflg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktup Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktop Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kink Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krut Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbli Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kaoo Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klit Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

panc Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktcl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pgwt Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpsp Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbih Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdnl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kart Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kilm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpne Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kabi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ptpn Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kblf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kosh Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpdt Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kewr Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kiso Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpga Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbkw Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmyl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krbg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kril Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksus Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

padq Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbil Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krfd Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdug Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktix Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcod Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kslk Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgfl Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kguc Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmlu Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbff Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksmn Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdro Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmce Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktpa Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmot Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcre Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klws Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kotm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khqm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kabr Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klal Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kelp Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kecg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khbg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpbf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

konp Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pkwa Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ktvf Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paga Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khks Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kdsm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpsm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgrb Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kgmu Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

papg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbgm Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pamc Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klrd Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

ksan Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

patk Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kowb Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klru Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kfxe Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kjct Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcrg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paaq Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kaex Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

klbx Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmia Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kpit Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcrw Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

paen Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kast Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kuin Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kmht Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krsw Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kbpi Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kcys Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kflo Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kphx Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pakn Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

pabt Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

krdg Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

khdn Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

kjac Terminal Aerodrome Forecast

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — TAF (terminal aerodrome forecast or terminal area forecast) is a format for reporting weather forecast information, particularly as it relates to aviation. TAFs are...

Adipogenic differentiation and EGFP gene transfection of amniotic fluid-derived stem cells from goat fetus at terminal gestational age.

Science.gov (United States)

He, Xiao-Ying; Zheng, Yue-Mao; Qiu, Shuang; Qi, Ying-Pei; Zhang, Yong

2011-08-01

The aims of this study were to determine whether stem cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and to determine if these stem cells could differentiate into adipogenic cells and be transfected with a reporter gene, EGFP (enhanced green fluorescent protein). The stem cells were isolated from amniotic fluid of goat fetus at terminal gestational age, induced to differentiate into adipogenic cells in vitro and transfected with the EGFP gene using lipofection. Markers associated with undifferentiated AFS (amniotic fluid-derived stem) cells were tested by RT (reverse transcription)-PCR. The results demonstrated that AFS cells could be isolated from amniotic fluid of goat fetus at terminal gestational age and could differentiate into adipogenic cells. The EGFP gene was transfected into AFS cells successfully. EGFP gene transfection efficiency of the three groups of transgenic AFS cells were 26.0, 29.9 and 30.5%, respectively. Both transgenic and wild-type AFS cells could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4) and Nanog.

Scambio, a novel guanine nucleotide exchange factor for Rho

Directory of Open Access Journals (Sweden)

Groffen John

2004-04-01

Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

Purine nucleotide synthesis from exogenous adenine and guanine in rodent small intestine

International Nuclear Information System (INIS)

Gross, C.J.; Karlberg, P.K.; Savaiano, D.A.

1986-01-01

14 C-Adenine and 14 C-guanine uptake was studied in isolated guinea pig enterocytes. Cells were incubated in Hank's buffer and separated from the medium by centrifugation through silicone oil into 1M PCA. Uptake was temperature and concentration dependent. Both compounds were incorporated into nucleotides as measured by HPLC and HVE. Adenine was more extensively incorporated into nucleotides than was guanine. Adenine nucleotides accounted for about 70% of the intracellular label after 30 min with a majority being ADP and ATP (medium concentration = 10 μM). Guanine nucleotides accounted for only 30% of the intracellular label after 30 min. Labeled intracellular free adenine or guanine were not detected. Significantly more guanine vs. adenine was converted to uric acid. After 30 min, 11.5 +/- 3.9% (n=3) and 83.0 +/- 8.4% (n=4) of the label was present as uric acid in the medium when adenine and guanine, respectively, were the substrate. After 1 min, 34.8 +/- 3.4% (n=4) of the label in the medium was present as uric acid when guanine was the substrate. Decreasing the concentration of adenine resulted in an increase in the percent of uric acid in the medium. 14 C-adenine (75 nmol) was injected into 1 gm segments of rat jejunum. After 5 min., segments were quickly flushed and the tissue homogenized in 1M PCA. Only uric acid was present after 5 min (n=6). In contrast, in animals fasted 3 to 5 days, less conversion to uric acid was observed in the intestinal content (50-80% of the same dose was still present as adenine after 5 min) and nucleotide formation was observed in the tissue. The results indicate that uric acid and nucleotide synthesis from exogenous adenine and guanine are concentration dependent and affected by nutritional state

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

Science.gov (United States)

Lawrence, C W; O'Brien, T; Bond, J

1984-01-01

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

Ports and Terminals : Planning and Functional Design

NARCIS (Netherlands)

Groenveld, R.; Velsink, H.

1993-01-01

1. Maritime transport, means and commodities 3. Principles of integrated port planning 4. Planning and design of a port's water areas 5. Port terminals - introduction 6. Conventional general cargo terminals 7. Container terminals 8. Oil & liquid gas terminals 9. Dry bulk cargo terminals 10. Fishery

Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

Directory of Open Access Journals (Sweden)

Po-Yuan Yeh

2014-07-01

Full Text Available It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−-strand] complement. However, the cis-acting elements on the positive-strand [(+-strand] sgmRNA required for (−-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV sgmRNA 7 required for the synthesis of its (−-strand counterpart by deletion mutagenesis. The major findings are as follows. (1 Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−-strand sgmRNA complement. (2 Deletions of the 3' untranslated region (UTR bulged stem-loop showed no effect on (−-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−-strand sgmRNA. (3 Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−-strand sgmRNA synthesis. (4 Nucleotide species at the 3'-most position (−1 of sgmRNA 7 is correlated to the efficiency of (−-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−-strand sgmRNA synthesis in BCoV.

Reversible assembly of protein-DNA nanostructures triggered by mediated electron transfer

International Nuclear Information System (INIS)

Vogt, Stephan; Wenderhold-Reeb, Sabine; Nöll, Gilbert

2017-01-01

Stable protein-DNA nanostructures have been assembled by reconstitution of the multi-ligand binding flavoprotein dodecin on top of flavin-terminated dsDNA monolayers on gold electrodes. These structures could be disassembled by electrochemical flavin reduction via mediated electron transfer. For this purpose a negative potential was applied at the Au working electrode in the presence of the redox mediator bis-(ammoniumethyl)-4,4′-bipyridinium tetrabromide. The stepwise formation of the flavin-terminated dsDNA monolayers as well as the binding and electrochemically triggered release of apododecin were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. The assembly and disassembly of the protein-DNA nanostructures were fully reversible processes, which could be carried out multiple times at the same flavin-dsDNA modified surface. When a negative potential was applied in the absence of a redox mediator apododecin could not be released, i.e. direct electron transfer was not possible. As alternative redox mediators also methylene blue and phenosafranine were studied, but in the presence of these molecules apododecin was released without applying a potential, probably because the tricyclic aromatic compounds are able to replace the flavins at the binding sites.

Tandem Terminal Ion Source

International Nuclear Information System (INIS)

Harper, G.C.; Lindner, C.E.; Myers, A.W.; Wechel, T.D. van

2000-01-01

OAK-B135 Tandem Terminal Ion Source. The terminal ion source (TIS) was used in several experiments during this reporting period, all for the 7 Be(γ) 8 B experiment. Most of the runs used 1 H + at terminal voltages from 0.3 MV to 1.5 MV. One of the runs used 2 H + at terminal voltage of 1.4 MV. The other run used 4 He + at a terminal voltage of 1.37 MV. The list of experiments run with the TIS to date is given in table 1 below. The tank was opened four times for unscheduled source repairs. On one occasion the tank was opened to replace the einzel lens power supply which had failed. The 10 kV unit was replaced with a 15 kV unit. The second time the tank was opened to repair the extractor supply which was damaged by a tank spark. On the next occasion the tank was opened to replace a source canal which had sputtered away. Finally, the tank was opened to replace the discharge bottle which had been coated with aluminum sputtered from the exit canal

Tandem Terminal Ion Source

International Nuclear Information System (INIS)

None

2000-01-01

OAK-B135 Tandem Terminal Ion Source. The terminal ion source (TIS) was used in several experiments during this reporting period, all for the(sup 7)Be((gamma))(sup 8)B experiment. Most of the runs used(sup 1)H(sup+) at terminal voltages from 0.3 MV to 1.5 MV. One of the runs used(sup 2)H(sup+) at terminal voltage of 1.4 MV. The other run used(sup 4)He(sup+) at a terminal voltage of 1.37 MV. The list of experiments run with the TIS to date is given in table 1 below. The tank was opened four times for unscheduled source repairs. On one occasion the tank was opened to replace the einzel lens power supply which had failed. The 10 kV unit was replaced with a 15 kV unit. The second time the tank was opened to repair the extractor supply which was damaged by a tank spark. On the next occasion the tank was opened to replace a source canal which had sputtered away. Finally, the tank was opened to replace the discharge bottle which had been coated with aluminum sputtered from the exit canal

Study on the change of cyclic nucleotide in mice with yang vacuity disease

International Nuclear Information System (INIS)

Zhu Xinhua; Shen Ling; Wang Shuguang

2002-01-01

To study the relation between Yang Vacuity disease happening, development and cyclic nucleotide response, and prove curative effects of some assisting Yang drug, the plasma cAMP, cGMP and cAMP/cGMP levels were detected by radioimmunoassay in the Yang Vacuity group and curing group. Results: showed: (1) Yang Vacuity group: the symptoms were clear, death rate was high, the plasma cAMP and cAMP/cGMP increased obviously, it suggests that cyclic nucleotide was imbalance. (2) Curing group: the symptoms of Yang Vacuity disease were improved obviously, death rate dropped, cAMP declined, cGMP increased, while cAMP/cGMP reached the normal level, it showed that cyclic nucleotide of the body had altered greatly. (3) It is a reference target for Yang Vacuity. (4) Assisting yang drug (Sini Decoction) had a close relation with correcting imbalance of cyclic nucleotide

Understanding specificity in metabolic pathways-Structural biology of human nucleotide metabolism

International Nuclear Information System (INIS)

Welin, Martin; Nordlund, Paer

2010-01-01

Interactions are the foundation of life at the molecular level. In the plethora of activities in the cell, the evolution of enzyme specificity requires the balancing of appropriate substrate affinity with a negative selection, in order to minimize interactions with other potential substrates in the cell. To understand the structural basis for enzyme specificity, the comparison of structural and biochemical data between enzymes within pathways using similar substrates and effectors is valuable. Nucleotide metabolism is one of the largest metabolic pathways in the human cell and is of outstanding therapeutic importance since it activates and catabolises nucleoside based anti-proliferative drugs and serves as a direct target for anti-proliferative drugs. In recent years the structural coverage of the enzymes involved in human nucleotide metabolism has been dramatically improved and is approaching completion. An important factor has been the contribution from the Structural Genomics Consortium (SGC) at Karolinska Institutet, which recently has solved 33 novel structures of enzymes and enzyme domains in human nucleotide metabolism pathways and homologs thereof. In this review we will discuss some of the principles for substrate specificity of enzymes in human nucleotide metabolism illustrated by a selected set of enzyme families where a detailed understanding of the structural determinants for specificity is now emerging.

Nucleotide sequence of a cDNA coding for the amino-terminal region of human prepro. alpha. 1(III) collagen

Energy Technology Data Exchange (ETDEWEB)

Toman, P D; Ricca, G A [Rorer Biotechnology, Inc., Springfield, VA (USA); de Crombrugghe, B [National Institutes of Health, Bethesda, MD (USA)

1988-07-25

Type III Collagen is synthesized in a variety of tissues as a precursor macromolecule containing a leader sequence, a N-propeptide, a N-telopeptide, the triple helical region, a C-telopeptide, and C-propeptide. To further characterize the human type III collagen precursor, a human placental cDNA library was constructed in gt11 using an oligonucleotide derived from a partial cDNA sequence corresponding to the carboxy-terminal part of the 1(III) collagen. A cDNA was identified which contains the leader sequence, the N-propeptide and N-telopeptide regions. The DNA sequence of these regions are presented here. The triple helical, C-telopeptide and C-propeptide amino acid sequence for human type III collagen has been determined previously. A comparison of the human amino acid sequence with mouse, chicken, and calf sequence shows 81%, 81%, and 92% similarity, respectively. At the DNA level, the sequence similarity between human and mouse or chicken type III collagen sequences in this area is 82% and 77%, respectively.

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

Science.gov (United States)

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Kajian Kinerja Terminal Batu Ampar Kota Balikpapan

Directory of Open Access Journals (Sweden)

Randha Alief Chikita

2018-01-01

Full Text Available Batu Ampar Terminal Balikpapan is the only type A passenger terminal in Balikpapan City. The purpose of this study is to determine the operational performance of the terminal at this time and also to determine the level of service in the terminal. The results of this study indicate that for FIFO queue discipline analysis on AKDP and AKAP bus lines, it is known that ρ <1 means that there is currently no queue in the terminal. For the analysis of terminal facilities it is known that there are still some terminal facilities that are not yet available from the main facilities and supporting facilities, therefore the need for additional facilities in order to meet the standard of passenger terminal type A. In the next 15 years analysis for traffic intensity value is approaching 1 which means in the future will cause the queue in the terminal, so it is necessary for the improvement of terminal performance. For the service performance with IPA method there are 35 variables that there are 7 variables that enter in quadrant I. In the next step to do analysis to know the priority of handling by using QFD method. Keywords: Batu Ampar Terminal Balikpapan, IPA, terminal performance, QFD

25 CFR 11.1114 - Termination.

Science.gov (United States)

2010-04-01

... Minor-in-Need-of-Care Procedure § 11.1114 Termination. (a) Parental rights to a child may be terminated... rights if, following efforts to prevent or eliminate the need to remove the minor, it finds such efforts... alternatives: (1) If parental rights to a child are terminated, the children's court shall place the minor in a...

St. James marine terminal facility description

Energy Technology Data Exchange (ETDEWEB)

1993-10-01

The US Department of Energy (DOE) currently owns and operates a marine terminal on the west bank of the Mississippi River at St. James, Louisiana. The St. James facility was constructed by the Department to provide marine services associated with the fill and drawdown of the Strategic Petroleum Reserve (SPR) crude oil storage facilities located at Bayou Choctaw and Weeks Island, Louisiana. Although strategic to the mission of the SPR in the event of a national emergency, the St. James terminal is situated such that it has a high potential to also serve the commercial industry`s needs for crude oil terminalling and storage. The St. James terminal is located approximately 45 miles west of New Orleans and 30 miles southeast of Baton Rouge, and approximately 160 miles upstream from the mouth of the Mississippi River. Construction of the St. James terminal was initiated in 1978 and was completed in 1980. Since then, the terminal has received and transferred over 125 million barrels of crude oil to the SPR sites for storage. For crude oil distribution, the St. James terminal was connected to the neighboring LOCAP terminal by a 0.1 mile 36-inch pipeline in 1981 and to the Capline terminal by a 0.5 mile 30-inch pipeline in 1988. The terminal also has a 30-inch pipeline connection to the Koch oil terminal which was used for initial fill purposes; however, this pipeline has been disconnected and is currently inactive. A complete description of the St. James terminal facilities, operational capabilities, operational certifications, and future Government requirements are presented in Sections 2, 3, 4, and 5 respectively.

Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

Science.gov (United States)

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-09-12

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9*

Science.gov (United States)

Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

2014-01-01

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al3+ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell. PMID:25028514

Mechanism of improving forward and reverse blocking voltages in AlGaN/GaN HEMTs by using Schottky drain

International Nuclear Information System (INIS)

Zhao Sheng-Lei; Mi Min-Han; Luo Jun; Wang Yi; Dai Yang; Zhang Jin-Cheng; Ma Xiao-Hua; Hao Yue; Hou Bin

2014-01-01

In this paper, we demonstrate that a Schottky drain can improve the forward and reverse blocking voltages (BVs) simultaneously in AlGaN/GaN high-electron mobility transistors (HEMTs). The mechanism of improving the two BVs is investigated by analysing the leakage current components and by software simulation. The forward BV increases from 72 V to 149 V due to the good Schottky contact morphology. During the reverse bias, the buffer leakage in the Ohmicdrain HEMT increases significantly with the increase of the negative drain bias. For the Schottky-drain HEMT, the buffer leakage is suppressed effectively by the formation of the depletion region at the drain terminal. As a result, the reverse BV is enhanced from −5 V to −49 V by using a Schottky drain. Experiments and the simulation indicate that a Schottky drain is desirable for power electronic applications. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29.

Science.gov (United States)

Testi, M G; Croce, R; Polverino-De Laureto, P; Bassi, R

1996-12-16

Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

Biochemistry of an olfactory purinergic system: dephosphorylation of excitatory nucleotides and uptake of adenosine

Energy Technology Data Exchange (ETDEWEB)

Trapido-Rosenthal, H G; Carr, W E; Gleeson, R A

1987-10-01

The olfactory organ of the spiny lobster, Panulirus argus, is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of (/sup 3/H)-adenosine is saturable with increasing concentration, linear with time for up to 3 h, sodium dependent, insensitive to moderate pH changes and has a Km of 7.1 microM and a Vmax of 5.2 fmol/sensillum/min (573 fmol/micrograms of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; /sup 3/H from adenine-labeled AMP or ATP is internalized, whereas 32P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; /sup 3/H from AMP is internalized at the same rate as /sup 3/H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog alpha, beta-methylene ADP. Collectively, these results indicate that the enzymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.

Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

Science.gov (United States)

Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

2013-02-01

Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

DEFF Research Database (Denmark)

Leonardsen, L; DeClue, J E; Lybaek, H

1996-01-01

The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino ac...... and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive....

Aging and the shape of cognitive change before death: terminal decline or terminal drop?

Science.gov (United States)

MacDonald, Stuart W S; Hultsch, David F; Dixon, Roger A

2011-05-01

Relative to typical age-related cognitive decrements, the terms "terminal decline" and "terminal drop" refer to the phenomenon of increased cognitive decline in proximity to death. Given that these terms are not necessarily synonymous, we examined the important theoretical distinction between the two alternative trajectories or shapes of changes they imply. We used 12-year (5-wave) data from the Victoria Longitudinal Study to directly test whether pre-death cognitive decrements follow a terminal decline (generally gradual) or a terminal drop (more abrupt) shape. Pre-death trajectories of cognitive decline for n=265 decedents (Mage = 72.67 years, SD = 6.44) were examined separately for 5 key cognitive constructs (verbal speed, working memory, episodic memory, semantic memory, and crystallized ability). Several classes of linear mixed models evaluated whether cognitive decline increased per additional year closer to death. Findings indicated that the shape of pre-death cognitive change was predominantly characterized by decline that is steeper as compared with typical aging-related change, but still best described as slow and steady decline, especially as compared with precipitous drop. The present findings suggest that terminal decline and terminal drop trajectories may not be mutually exclusive but could rather reflect distinct developmental trajectories within the same individual.

42 CFR 489.53 - Termination by CMS.

Science.gov (United States)

2010-10-01

... 42 Public Health 5 2010-10-01 2010-10-01 false Termination by CMS. 489.53 Section 489.53 Public... Reinstatement After Termination § 489.53 Termination by CMS. (a) Basis for termination of agreement with any provider. CMS may terminate the agreement with any provider if CMS finds that any of the following failings...

Lazy Productivity via Termination

NARCIS (Netherlands)

Endrullis, J.; Hendriks, R.D.A.

2011-01-01

We present a procedure for transforming strongly sequential constructor-based term rewriting systems (TRSs) into context-sensitive TRSs in such a way that productivity of the input system is equivalent to termination of the output system. Thereby automated termination provers become available for

Nontraumatic terminal ileal perforation

Directory of Open Access Journals (Sweden)

Wani Rauf A

2006-03-01

Full Text Available Abstract Background There is still confusion and controversy over the diagnosis and optimal surgical treatment of non traumatic terminal ileal perforation-a cause of obscure peritonitis. Methods This study was a prospective study aimed at evaluating the clinical profile, etiology and optimal surgical management of patients with nontraumatic terminal ileal perforation. Results There were 79 cases of nontraumatic terminal ileal perforation; the causes for perforation were enteric fever(62%, nonspecific inflammation(26%, obstruction(6%, tuberculosis(4% and radiation enteritis (1%. Simple closure of the perforation (49% and end to side ileotransverse anastomosis(42% were the mainstay of the surgical management. Conclusion Terminal ileal perforation should be suspected in all cases of peritonitis especially in developing countries and surgical treatment should be optimized taking various accounts like etiology, delay in surgery and operative findings into consideration to reduce the incidence of deadly complications like fecal fistula.

Reverse genetics with animal viruses. NSV reverse genetics

International Nuclear Information System (INIS)

Mebatsion, T.

2005-01-01

New strategies to genetically manipulate the genomes of several important animal pathogens have been established in recent years. This article focuses on the reverse genetics techniques, which enables genetic manipulation of the genomes of non-segmented negative-sense RNA viruses. Recovery of a negative-sense RNA virus entirely from cDNA was first achieved for rabies virus in 1994. Since then, reverse genetic systems have been established for several pathogens of medical and veterinary importance. Based on the reverse genetics technique, it is now possible to design safe and more effective live attenuated vaccines against important viral agents. In addition, genetically tagged recombinant viruses can be designed to facilitate serological differentiation of vaccinated animals from infected animals. The approach of delivering protective immunogens of different pathogens using a single vector was made possible with the introduction of the reverse genetics system, and these novel broad-spectrum vaccine vectors have potential applications in improving animal health in developing countries. (author)

2',3'-Dideoxycytidine and human immunodeficiency virus reverse transcriptase

International Nuclear Information System (INIS)

Starnes, M.C.

1988-01-01

2',3'-Dideoxycytidine (ddCyd) is a candidate for clinical trial in the treatment of Acquired Immunodeficiency Syndrome, as a result of its potent inhibition of Human Immunodeficiency Virus (HIV) replication. The cellular metabolism and cytotoxicity of ddCyd are, as well as the interaction of ddCTP and other nucleotide and pyrophosphate analogs with mammalian DNA polymerases and HIV reverse transcriptase (RT). In addition, some structural and functional characteristics of HIV RT are described. 5 μM ddCyd reduced Molt 4 cell division by 50% during a 48 h continuous exposure; however, a 24 h exposure to 0.5 μM ddCyd reduced clonogenic survival by 50%. [ 14 C]-dThd incorporation into DNA was reduced during exposure to ddCyd. Acid-soluble ddCyd metabolites were ddCMP, ddCDP, and ddCTP. Initial ddCyd phosphorylation was catalyzed primarily by cytoplasmic dCyd kinase, and ddCyd was not a substrate for human Cyd-dCyd deaminase. Metabolism of ddCyd was identical in mock and HIV infected H9 cells

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

Science.gov (United States)

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The practice of terminal discharge.

Science.gov (United States)

Radha Krishna, Lalit Kumar; Murugam, Vengadasalam; Quah, Daniel Song Chiek

2017-01-01

'Terminal discharges' are carried out in Singapore for patients who wish to die at home. However, if due diligence is not exercised, parallels may be drawn with euthanasia. We present a theoretical discussion beginning with the definition of terminal discharges and the reasons why they are carried out in Singapore. By considering the intention behind terminal discharges and utilising a multidisciplinary team to deliberate on the clinical, social and ethical intricacies with a patient- and context-specific approach, euthanasia is avoided. It is hoped that this will provide a platform for professionals in palliative medicine to negotiate challenging issues when arranging a terminal discharge, so as to avoid the pitfall of committing euthanasia in a country such as Singapore where euthanasia is illegal. It is hoped that a set of guidelines for terminal discharges may someday be realised to assist professionals in Singapore and around the world.

Homopolar machine for reversible energy storage and transfer systems

Science.gov (United States)

Stillwagon, Roy E.

1978-01-01

A homopolar machine designed to operate as a generator and motor in reversibly storing and transferring energy between the machine and a magnetic load coil for a thermo-nuclear reactor. The machine rotor comprises hollow thin-walled cylinders or sleeves which form the basis of the system by utilizing substantially all of the rotor mass as a conductor thus making it possible to transfer substantially all the rotor kinetic energy electrically to the load coil in a highly economical and efficient manner. The rotor is divided into multiple separate cylinders or sleeves of modular design, connected in series and arranged to rotate in opposite directions but maintain the supply of current in a single direction to the machine terminals. A stator concentrically disposed around the sleeves consists of a hollow cylinder having a number of excitation coils each located radially outward from the ends of adjacent sleeves. Current collected at an end of each sleeve by sleeve slip rings and brushes is transferred through terminals to the magnetic load coil. Thereafter, electrical energy returned from the coil then flows through the machine which causes the sleeves to motor up to the desired speed in preparation for repetition of the cycle. To eliminate drag on the rotor between current pulses, the brush rigging is designed to lift brushes from all slip rings in the machine.

Homopolar machine for reversible energy storage and transfer systems

International Nuclear Information System (INIS)

Stillwagon, R.E.

1981-01-01

A homopolar machine designed to operate as a generator and motor in reversibly storing and transferring energy between the machine and a magnetic load coil for a thermo-nuclear reactor. The machine rotor comprises hollow thin-walled cylinders or sleeves which form the basis of the system by utilizing substantially all of the rotor mass as a conductor thus making it possible to transfer substantially all the rotor kinetic energy electrically to the load coil in a highly economical and efficient manner. The rotor is divided into multiple separate cylinders or sleeves of modular design, connected in series and arranged to rotate in opposite directions but maintain the supply of current in a single direction to the machine terminals. A stator concentrically disposed around the sleeves consists of a hollow cylinder having a number of excitation coils each located radially outward from the ends of adjacent sleeves. Current collected at an end of each sleeve by sleeve slip rings and brushes is transferred through terminals to the magnetic load coil. Thereafter, electrical energy returned from the coil then flows through the machine which causes the sleeves to motor up to the desired speed in preparation for repetition of the cycle. To eliminate drag on the rotor between current pulses, the brush rigging is designed to lift brushes from all slip rings in the machine

Extracellular nucleotide derivatives protect cardiomyctes against hypoxic stress

DEFF Research Database (Denmark)

Golan, O; Issan, Y; Isak, A

2011-01-01

assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. CONCLUSIONS: Extracellular tri/di-phosphates are apparently the molecule responsible...

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

OpenAIRE

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R

2008-01-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consis...

DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

Directory of Open Access Journals (Sweden)

Cheng-Yao eChen

2014-06-01

Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

Molecular recognition of nucleotides in micelles and the development and expansion of a chemistry outreach program

Science.gov (United States)

Schechinger, Linda Sue

I. To investigate the delivery of nucleotide-based drugs, we are studying molecular recognition of nucleotide derivatives in environments that are similar to cell membranes. The Nowick group previously discovered that membrane-like surfactant micelles tetradecyltrimethylammonium bromide (TTAB) micelle facilitate molecular of adenosine monophosphate (AMP) recognition. The micelles bind nucleotides by means of electrostatic interactions and hydrogen bonding. We observed binding by following 1H NMR chemical shift changes of unique hexylthymine protons upon addition of AMP. Cationic micelles are required for binding. In surfactant-free or sodium dodecylsulfate solutions, no hydrogen bonding is observed. These observations suggest that the cationic surfactant headgroups bind the nucleotide phosphate group, while the intramicellar base binds the nucleotide base. The micellar system was optimized to enhance binding and selectivity for adenosine nucleotides. The selectivity for adenosine and the number of phosphate groups attached to the adenosine were both investigated. Addition of cytidine, guanidine, or uridine monophosphates, results in no significant downfield shifting of the NH resonance. Selectivity for the phosphate is limited, since adenosine mono-, di-, and triphosphates all have similar binding constants. We successfully achieved molecular recognition of adenosine nucleotides in micellar environments. There is significant difference in the binding interactions between the adenosine nucleotides and three other natural nucleotides. II. The UCI Chemistry Outreach Program (UCICOP) addresses the declining interest of the nations youth for science. UCICOP brings fun and exciting chemistry experiments to local high schools, to remind students that science is fun and has many practical uses. Volunteer students and alumni of UCI perform the demonstrations using scripts and material provided by UCICOP. The preparation of scripts and materials is done by two coordinators

NONSPECIFIC IMMUNE RESPONSE AND RESISTANCE OF Litopenaeus vannamei FED WITH NUCLEOTIDE, β-GLUCAN, AND PROTAGEN DIETS

Directory of Open Access Journals (Sweden)

Henky Manoppo

2010-06-01

Full Text Available The objective of this research was to evaluate the nonspecific immune response and resistance of Litopenaeus vannamei fed with nucleotide, β–glucan, and protagen diets. Shrimp juveniles with an average weight of 5.39±0.56 g were reared in glass aquaria at a density of 15 shrimps/aquarium. Shrimps were fed three times a day for four weeks at a feeding rate of 3%/bw/day. Treatment diets consisted of A: basal diet (without immunostimulant, B: β–glucan, C: protagen, and D: nucleotide, each with three replicates. At the end of feeding period, the shrimps were intramuscularly injected with Vibrio harveyi 0.1 x 106 cfu.shrimp-1. Total haemocyte count (THC of shrimp fed with nucleotide-diet was significantly different compared to that of control shrimp (p=0.01, but not different compared to shrimp fed with protagen-diet. PO activity also increased significantly in shrimp fed with nucleotide-diet (p=0.02. β–glucan diet could also increase THC and PO activity, but compared to the control, the increase was not significantly different. Overall, PO activity of shrimp fed with nucleotide, β–glucan, and protagen diets was high (>0.35. Oral administration of nucleotide, β–glucan, and protagen for four consecutive weeks significantly increased resistance of shrimp to disease (<0.01 where the highest resistance rate was observed on shrimp fed with nucleotide-diet. Growth of shrimp fed with nucleotide-diet was significantly different compared to that of control shrimp (p<0.01, as well as to β–glucan, and protagen-treated shrimp. As a conclusion, supplementation of nucleotide into shrimp pellet enhanced nonspecific immune response and growth performance better than β-glucan, and protagen.

Termination of cycle rewriting

NARCIS (Netherlands)

Zantema, H.; König, B.; Bruggink, H.J.S.; Dowek, G.

2014-01-01

String rewriting can not only be applied on strings, but also on cycles and even on general graphs. In this paper we investigate termination of string rewriting applied on cycles, shortly denoted as cycle rewriting, which is a strictly stronger requirement than termination on strings. Most

78 FR 5717 - Safety Zone; Military Ocean Terminal Concord Safety Zone, Suisun Bay, Military Ocean Terminal...

Science.gov (United States)

2013-01-28

...-AA00 Safety Zone; Military Ocean Terminal Concord Safety Zone, Suisun Bay, Military Ocean Terminal... Guard is establishing a safety zone in the navigable waters of Suisun Bay near Military Ocean Terminal Concord, CA in support of military onload and offload operations. This safety zone is established to...

Nucleotide excision repair II: From yeast to mammals

NARCIS (Netherlands)

J.H.J. Hoeijmakers (Jan)

1993-01-01

textabstractAn intricate network of repair systems safeguards the integrity of genetic material, by eliminating DNA lesions induced by numerous environmental and endogenous genotoxic agents. Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. Deficiencies in this

Reverse Transcriptase Inhibitors as Potential Colorectal Microbicides▿ †

Science.gov (United States)

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J.

2009-01-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides. PMID:19258271

Resulting long-term intestinal motility in dogs following construction of a reversed jejunal segment after extensive small bowel resection.

Science.gov (United States)

Uchiyama, M; Iwafuchi, M; Ohsawa, Y; Yagi, M; Iinuma, Y; Ohtani, S

1994-10-01

To evaluate the long-term function and effective motility of a reversed jejunal segment after extensive small bowel resection, the authors continuously measured postoperative bowel motility during interdigestive and postprandial periods in conscious dogs 6 to 10 months after surgery. The long-term findings were compared with previously reported short-term results measured 2 to 4 weeks after the operation. In the long-term follow-up dogs with a 20-cm reversed jejunal segment constructed after extensive (75% to 80%) small bowel resection, the fasting duodenal migrating myoelectric (or motor) complex (MMC) was often interrupted in the jejunum above the reversed segment, and did not migrate smoothly to the reversed segment or terminal ileum. The MMCs arising from the duodenum predominantly propagated to the ileum through the inherent anatomic continuity of the bowel. In addition, brief small discordant contractions were frequent in the reversed segment and the jejunum, above the proximal anastomosis. These findings are similar to those of the MMC propagation pattern noted 2 to 4 weeks after surgery. However, the postprandial duration without duodenal MMC activity was significantly shorter in the dogs with long-term follow-up than in those with short-term follow-up (both were longer than in control dogs). Marked dilatation of the jejunum and reversed jejunal segment was noted across the proximal anastomosis. These results suggest that the transit time and passage of intestinal contents can be delayed and stagnated for at least 10 months after extensive small bowel resection with a 20-cm reversed jejunal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversibly extracellular pH controlled cellular uptake and photothermal therapy by PEGylated mixed-charge gold nanostars.

Science.gov (United States)

Wang, Shouju; Teng, Zhaogang; Huang, Peng; Liu, Dingbin; Liu, Ying; Tian, Ying; Sun, Jing; Li, Yanjun; Ju, Huangxian; Chen, Xiaoyuan; Lu, Guangming

2015-04-17

Shielding nanoparticles from nonspecific interactions with normal cells/tissues before they reach and after they leave tumors is crucial for the selective delivery of NPs into tumor cells. By utilizing the reversible protonation of weak electrolytic groups to pH changes, long-chain amine/carboxyl-terminated polyethylene glycol (PEG) decorated gold nanostars (GNSs) are designed, exhibiting reversible, significant, and sensitive response in cell affinity and therapeutic efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors. This smart nanosystem shows good dispersity and unimpaired photothermal efficacy in complex bioenvironment at pH 6.4 and 7.4 even when their surface charge is neutral. One PEGylated mixed-charge GNSs with certain surface composition, GNS-N/C 4, exhibits high cell affinity and therapeutic efficacy at pH 6.4, and low affinity and almost "zero" damage to cells at pH 7.4. Remarkably, this significant and sensitive response in cell affinity and therapeutic efficacy is reversible as local pH alternated. In vivo, GNS-N/C 4 shows higher accumulation in tumors and improved photothermal therapeutic efficacy than pH-insensitive GNSs. This newly developed smart nanosystem, whose cell affinity reversibly transforms in response to pHe gradient with unimpaired biostability, provides a novel effective means of tumor-selective therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

Directory of Open Access Journals (Sweden)

Charikleia Papadopoulou

2015-12-01

Full Text Available Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides

Directory of Open Access Journals (Sweden)

Michael E. Østergaard

2017-06-01

Full Text Available Antisense oligonucleotides (ASOs have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT and wild-type HTT (wtHTT mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.

NMR structure of the HIV-1 reverse transcriptase thumb subdomain

Energy Technology Data Exchange (ETDEWEB)

Sharaf, Naima G. [University of Pittsburgh, School of Medicine, Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions (United States); Brereton, Andrew E. [Oregon State University, Department of Biochemistry and Biophysics, 2011 Ag & Life Sciences Bldg (United States); Byeon, In-Ja L. [University of Pittsburgh, School of Medicine, Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions (United States); Andrew Karplus, P. [Oregon State University, Department of Biochemistry and Biophysics, 2011 Ag & Life Sciences Bldg (United States); Gronenborn, Angela M., E-mail: amg100@pitt.edu [University of Pittsburgh, School of Medicine, Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions (United States)

2016-12-15

The solution NMR structure of the isolated thumb subdomain of HIV-1 reverse transcriptase (RT) has been determined. A detailed comparison of the current structure with dozens of the highest resolution crystal structures of this domain in the context of the full-length enzyme reveals that the overall structures are very similar, with only two regions exhibiting local conformational differences. The C-terminal capping pattern of the αH helix is subtly different, and the loop connecting the αI and αJ helices in the p51 chain of the full-length p51/p66 heterodimeric RT differs from our NMR structure due to unique packing interactions in mature RT. Overall, our data show that the thumb subdomain folds independently and essentially the same in isolation as in its natural structural context.

46 CFR 525.2 - Terminal schedules.

Science.gov (United States)

2010-10-01

... domestic law and international conventions and agreements adopted by the United States; such terminal... their own negligence, or that impose upon others the obligation to indemnify or hold-harmless the terminals from liability for their own negligence. (2) Enforcement of terminal schedules. Any schedule that...