Structure and function of ameloblastin as an extracellular matrix protein: adhesion, calcium binding, and CD63 interaction in human and mouse.

PubMed

Zhang, Xu; Diekwisch, Thomas G H; Luan, Xianghong

2011-12-01

The functional significance of extracellular matrix proteins in the life of vertebrates is underscored by a high level of sequence variability in tandem with a substantial degree of conservation in terms of cell-cell and cell-matrix adhesion interactions. Many extracellular matrix proteins feature multiple adhesion domains for successful attachment to substrates, such as integrin, CD63, and heparin. Here we have used homology and ab initio modeling algorithms to compare mouse ameloblastin (mAMBN) and human ameloblastin (hABMN) isoforms and to analyze their potential for cell adhesion and interaction with other matrix molecules as well as calcium binding. Sequence comparison between mAMBN and hAMBN revealed a 26-amino-acid deletion in mAMBN, corresponding to a helix-loop-helix frameshift. The human AMBN domain (174Q-201G), homologous to the mAMBN 157E-178I helix-loop-helix region, formed a helix-loop motif with an extended loop, suggesting a higher degree of flexibility of hAMBN compared with mAMBN, as confirmed by molecular dynamics simulation. Heparin-binding domains, CD63-interaction domains, and calcium-binding sites in both hAMBN and mAMBN support the concept of AMBN as an extracellular matrix protein. The high level of conservation between AMBN functional domains related to adhesion and differentiation was remarkable when compared with only 61% amino acid sequence homology. © 2011 Eur J Oral Sci.

Complete Mitochondrial Genome Sequence of Acrida cinerea (Acrididae: Orthoptera) and Comparative Analysis of Mitochondrial Genomes in Orthoptera

PubMed Central

Liu, Nian; Huang, Yuan

2010-01-01

The complete 15,599-bp mitogenome of Acrida cinerea was determined and compared with that of the other 20 orthopterans. It displays characteristic gene content, genome organization, nucleotide composition, and codon usage found in other Caelifera mitogenomes. Comparison of 21 orthopteran sequences revealed that the tRNAs encoded by the H-strand appear more conserved than those by the L-stand. All tRNAs form the typical clover-leaf structure except trnS (agn), and most of the size variation among tRNAs stemmed from the length variation in the arm and loop of TΨC and the loop of DHU. The derived secondary structure models of the rrnS and rrnL from 21 orthoptera species closely resemble those from other insects on CRW except a considerably enlarged loop of helix 1399 of rrnS in Caelifera, which is a potentially autapomorphy of Caelifera. In the A+T-rich region, tandem repeats are not only conserved in the closely related mitogenome but also share some conserved motifs in the same subfamily. A stem-loop structure, 16 bp or longer, is likely to be involved in replication initiation in Caelifera and Grylloidea. A long T-stretch (>17 bp) with conserved stem-loop structure next to rrnS on the H-strand, bounded by a purine at either end, exists in the three species from Tettigoniidae. PMID:21197069

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver

PubMed Central

2018-01-01

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. PMID:29757144

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

PubMed

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

Active site CP-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

PubMed

Kamariah, Neelagandan; Eisenhaber, Birgit; Eisenhaber, Frank; Grüber, Gerhard

2018-04-01

Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (C P ) located in a conserved sequence Pxxx(T/S)xxC P motif known as C P -loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the C P -loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure. Despite many studies, little is known about the precise structural and dynamic roles of the C P -loop on Prxs functions. Herein, the comprehensive biochemical and biophysical studies on Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) and the C P -loop mutants, EcAhpC-F45A and EcAhpC-F45P reveal that the reduced form of the C P -loop adopts conformational dynamics, which is essential for effective peroxide reduction. Furthermore, the point mutants alter the structure and dynamics of the reduced form of the C P -loop and, thereby, affect substrate binding, catalysis, oligomerization, stability and overoxidiation. In the oxidized form, due to restricted C P -loop dynamics, the EcAhpC-F45P mutant favours a decamer formation, which enhances the effective recycling by physiological reductases compared to wild-type EcAhpC. In addition, the study reveals that residue F45 increases the specificity of Prxs-reductase interactions. Based on these studies, we propose an evolution of the C P -loop with confined sequence conservation within Prxs subfamilies that might optimize the functional adaptation of Prxs into various physiological roles. Copyright © 2018 Elsevier Inc. All rights reserved.

The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis

PubMed Central

Yunus, Muhammad Amir; Lin, Xiaoyan; Bailey, Dalan; Karakasiliotis, Ioannis; Chaudhry, Yasmin; Vashist, Surender; Zhang, Guo; Thorne, Lucy; Kao, C. Cheng

2014-01-01

ABSTRACT All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3′ of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. IMPORTANCE Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. PMID:25392209

The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

PubMed

Yunus, Muhammad Amir; Lin, Xiaoyan; Bailey, Dalan; Karakasiliotis, Ioannis; Chaudhry, Yasmin; Vashist, Surender; Zhang, Guo; Thorne, Lucy; Kao, C Cheng; Goodfellow, Ian

2015-01-15

All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

PubMed Central

Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

2015-01-01

Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus. PMID:26447800

The structural coupling between ATPase activation and recovery stroke in the myosin II motor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koppole, Sampath; Smith, Jeremy C; Fischer, S.

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transitionmore » at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.« less

Short communication: evidence of HIV type 1 clade C env clones containing low V3 loop charge obtained from an AIDS patient in India that uses CXCR6 and CCR8 for entry in addition to CCR5.

PubMed

Gharu, Lavina; Ringe, Rajesh; Satyakumar, Anupindi; Patil, Ajit; Bhattacharya, Jayanta

2011-02-01

Abstract HIV-1 clade C is the major subtype circulating in India and preferentially uses CCR5 during the entire disease course. We have recently shown that env clones from an Indian patient; NARI-VB105 uses multiple coreceptors for entry and was presented with an unusual V3 loop sequence giving rise to high net V3 loop positive charges. Here we show that env clones belonging to subtype C obtained from an AIDS patient, NARI-VB52, use CXCR6 and CCR8 in addition to CCR5 for entry. However, unlike the NARI-105 patient, the env clones contained a low V3 loop net charge of +3 with a conserved GPGQ motif typical of CCR5 using subtype C strains, indicating that residues outside the V3 loop contributed to extended coreceptor use in this particular patient.

The structural coupling between ATPase activation and recovery stroke in the myosin II motor.

PubMed

Koppole, Sampath; Smith, Jeremy C; Fischer, Stefan

2007-07-01

Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates approximately 65 degrees . Simultaneous with this "recovery stroke," myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 A-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity.

PubMed

Faroux-Corlay, B; Clary, L; Gadras, C; Hammache, D; Greiner, J; Santaella, C; Aubertin, A M; Vierling, P; Fantini, J

2000-07-24

Galactosylceramide (GalCer) is an alternative receptor allowing HIV-1 entry into CD4(-)/GalCer(+) cells. This glycosphingolipid recognizes the V3 loop of HIV gp120, which plays a key role in the fusion of the HIV envelope and cellular membrane. To inhibit HIV uptake and infection, we designed and synthesized analogs of GalCer. These amphiphiles and bolaamphiphiles consist of single and double hydrocarbon and/or fluorocarbon chain beta-linked to galactose and galactosamine. They derive from serine (GalSer), cysteine (GalCys), and ethanolamine (GalAE). The anti-HIV activity and cytotoxicity of these galactolipids were evaluated in vitro on CEM-SS (a CD4(+) cell line), HT-29, a CD4(-) cell line expressing high levels of GalCer receptor, and/or HT29 genetically modified to express CD4. GalSer and GalAE derivatives, tested in aqueous medium or as part of liposome preparation, showed moderate anti-HIV-1 activities (IC50 in the 20-220 microM range), whereas none of the GalCys derivatives was found to be active. Moreover, only some of these anti-HIV active analogs inhibited the binding of [3H]suramin (a polysulfonyl compound which displays a high affinity for the V3 loop) to SPC3, a synthetic peptide which contains the conserved GPGRAF region of the V3 loop. Our results most likely indicate that the neutralization of the virion through masking of this conserved V3 loop region is not the only mechanism involved in the HIV-1 antiviral activity of our GalCer analogs.

Extended molecular dynamics of a c-kit promoter quadruplex

PubMed Central

Islam, Barira; Stadlbauer, Petr; Krepl, Miroslav; Koca, Jaroslav; Neidle, Stephen; Haider, Shozeb; Sponer, Jiri

2015-01-01

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 μs long, ∼50 μs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal μs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex. PMID:26245347

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

PubMed

Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

1997-04-15

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Selection of the simplest RNA that binds isoleucine

PubMed Central

LOZUPONE, CATHERINE; CHANGAYIL, SHANKAR; MAJERFELD, IRENE; YARUS, MICHAEL

2003-01-01

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA–amino acid binding structures. PMID:14561881

Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGF-beta signaling.

PubMed

Wu, Jia Wei; Krawitz, Ariel R; Chai, Jijie; Li, Wenyu; Zhang, Fangjiu; Luo, Kunxin; Shi, Yigong

2002-11-01

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.

Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

PubMed Central

Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

2016-01-01

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

PubMed

Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

2015-09-09

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to "W3:34 and an top-loop", and "solely W2:41", accounting for 82% of published RGD-integrin-mAbs.

Genome-wide identification of conserved microRNA and their response to drought stress in Dongxiang wild rice (Oryza rufipogon Griff.).

PubMed

Zhang, Fantao; Luo, Xiangdong; Zhou, Yi; Xie, Jiankun

2016-04-01

To identify drought stress-responsive conserved microRNA (miRNA) from Dongxiang wild rice (Oryza rufipogon Griff., DXWR) on a genome-wide scale, high-throughput sequencing technology was used to sequence libraries of DXWR samples, treated with and without drought stress. 505 conserved miRNAs corresponding to 215 families were identified. 17 were significantly down-regulated and 16 were up-regulated under drought stress. Stem-loop qRT-PCR revealed the same expression patterns as high-throughput sequencing, suggesting the accuracy of the sequencing result was high. Potential target genes of the drought-responsive miRNA were predicted to be involved in diverse biological processes. Furthermore, 16 miRNA families were first identified to be involved in drought stress response from plants. These results present a comprehensive view of the conserved miRNA and their expression patterns under drought stress for DXWR, which will provide valuable information and sequence resources for future basis studies.

The highly conserved codon following the slippery sequence supports -1 frameshift efficiency at the HIV-1 frameshift site.

PubMed

Mathew, Suneeth F; Crowe-McAuliffe, Caillan; Graves, Ryan; Cardno, Tony S; McKinney, Cushla; Poole, Elizabeth S; Tate, Warren P

2015-01-01

HIV-1 utilises -1 programmed ribosomal frameshifting to translate structural and enzymatic domains in a defined proportion required for replication. A slippery sequence, U UUU UUA, and a stem-loop are well-defined RNA features modulating -1 frameshifting in HIV-1. The GGG glycine codon immediately following the slippery sequence (the 'intercodon') contributes structurally to the start of the stem-loop but has no defined role in current models of the frameshift mechanism, as slippage is inferred to occur before the intercodon has reached the ribosomal decoding site. This GGG codon is highly conserved in natural isolates of HIV. When the natural intercodon was replaced with a stop codon two different decoding molecules-eRF1 protein or a cognate suppressor tRNA-were able to access and decode the intercodon prior to -1 frameshifting. This implies significant slippage occurs when the intercodon is in the (perhaps distorted) ribosomal A site. We accommodate the influence of the intercodon in a model of frame maintenance versus frameshifting in HIV-1.

Structurally conserved water molecules in ribonuclease T1.

PubMed

Malin, R; Zielenkiewicz, P; Saenger, W

1991-03-15

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

PubMed

Lai, Chih-Yun; Tsai, Wen-Yang; Lin, Su-Ru; Kao, Chuan-Liang; Hu, Hsien-Ping; King, Chwan-Chuen; Wu, Han-Chung; Chang, Gwong-Jen; Wang, Wei-Kung

2008-07-01

The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

PubMed

Korkosh, V S; Zhorov, B S; Tikhonov, D B

2016-05-01

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue-residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

PubMed Central

Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

2008-01-01

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

GalaxyGPCRloop: Template-Based and Ab Initio Structure Sampling of the Extracellular Loops of G-Protein-Coupled Receptors.

PubMed

Won, Jonghun; Lee, Gyu Rie; Park, Hahnbeom; Seok, Chaok

2018-06-07

The second extracellular loops (ECL2s) of G-protein-coupled receptors (GPCRs) are often involved in GPCR functions, and their structures have important implications in drug discovery. However, structure prediction of ECL2 is difficult because of its long length and the structural diversity among different GPCRs. In this study, a new ECL2 conformational sampling method involving both template-based and ab initio sampling was developed. Inspired by the observation of similar ECL2 structures of closely related GPCRs, a template-based sampling method employing loop structure templates selected from the structure database was developed. A new metric for evaluating similarity of the target loop to templates was introduced for template selection. An ab initio loop sampling method was also developed to treat cases without highly similar templates. The ab initio method is based on the previously developed fragment assembly and loop closure method. A new sampling component that takes advantage of secondary structure prediction was added. In addition, a conserved disulfide bridge restraining ECL2 conformation was predicted and analytically incorporated into sampling, reducing the effective dimension of the conformational search space. The sampling method was combined with an existing energy function for comparison with previously reported loop structure prediction methods, and the benchmark test demonstrated outstanding performance.

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits

PubMed Central

Nishimichi, Norihisa; Kawashima, Nagako; Yokosaki, Yasuyuki

2015-01-01

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs. PMID:26349930

Stem loop recognition by DDX17 facilitates miRNA processing and antiviral defense

PubMed Central

Moy, Ryan H.; Cole, Brian S.; Yasunaga, Ari; Gold, Beth; Shankarling, Ganesh; Varble, Andrew; Molleston, Jerome M.; tenOever, Benjamin R.; Lynch, Kristen W.; Cherry, Sara

2014-01-01

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using cross-linking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing, and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous miRNA biogenesis and in the cytoplasm for surveillance against structured non-self elements. PMID:25126784

Identification and Functional Characterization of a Novel Acetylcholine-binding Protein from the Marine Annelid Capitella teleta

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCormack, T.; Petrovich,; Mercier, K

2010-01-01

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) {alpha} subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has beenmore » implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and {alpha}-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 {micro}M, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K{sub D} = 163 {micro}M). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.« less

Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

NASA Astrophysics Data System (ADS)

Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

2017-03-01

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

Conservation of ζ with radiative corrections from heavy field

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanaka, Takahiro; Yukawa Institute for Theoretical Physics, Kyoto University,Kyoto, 606-8502; Urakawa, Yuko

2016-06-08

In this paper, we address a possible impact of radiative corrections from a heavy scalar field χ on the curvature perturbation ζ. Integrating out χ, we derive the effective action for ζ, which includes the loop corrections of the heavy field χ. When the mass of χ is much larger than the Hubble scale H, the loop corrections of χ only yield a local contribution to the effective action and hence the effective action simply gives an action for ζ in a single field model, where, as is widely known, ζ is conserved in time after the Hubble crossing time.more » Meanwhile, when the mass of χ is comparable to H, the loop corrections of χ can give a non-local contribution to the effective action. Because of the non-local contribution from χ, in general, ζ may not be conserved, even if the classical background trajectory is determined only by the evolution of the inflaton. In this paper, we derive the condition that ζ is conserved in time in the presence of the radiative corrections from χ. Namely, we show that when the dilatation invariance, which is a part of the diffeomorphism invariance, is preserved at the quantum level, the loop corrections of the massive field χ do not disturb the constant evolution of ζ at super Hubble scales. In this discussion, we show the Ward-Takahashi identity for the dilatation invariance, which yields a consistency relation for the correlation functions of the massive field χ.« less

A conserved loop-wedge motif moderates reaction site search and recognition by FEN1.

PubMed

Thompson, Mark J; Gotham, Victoria J B; Ciani, Barbara; Grasby, Jane A

2018-06-07

DNA replication and repair frequently involve intermediate two-way junction structures with overhangs, or flaps, that must be promptly removed; a task performed by the essential enzyme flap endonuclease 1 (FEN1). We demonstrate a functional relationship between two intrinsically disordered regions of the FEN1 protein, which recognize opposing sides of the junction and order in response to the requisite substrate. Our results inform a model in which short-range translocation of FEN1 on DNA facilitates search for the annealed 3'-terminus of a primer strand, which is recognized by breaking the terminal base pair to generate a substrate with a single nucleotide 3'-flap. This recognition event allosterically signals hydrolytic removal of the 5'-flap through reaction in the opposing junction duplex, by controlling access of the scissile phosphate diester to the active site. The recognition process relies on a highly-conserved 'wedge' residue located on a mobile loop that orders to bind the newly-unpaired base. The unanticipated 'loop-wedge' mechanism exerts control over substrate selection, rate of reaction and reaction site precision, and shares features with other enzymes that recognize irregular DNA structures. These new findings reveal how FEN1 precisely couples 3'-flap verification to function.

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

PubMed

D'Souza, V; Melamed, J; Habib, D; Pullen, K; Wallace, K; Summers, M F

2001-11-23

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops. Copyright 2001 Academic Press.

Classification and evolutionary analysis of the basic helix-loop-helix gene family in the green anole lizard, Anolis carolinensis.

PubMed

Liu, Ake; Wang, Yong; Zhang, Debao; Wang, Xuhua; Song, Huifang; Dang, Chunwang; Yao, Qin; Chen, Keping

2013-08-01

Helix-loop-helix (bHLH) proteins play essential regulatory roles in a variety of biological processes. These highly conserved proteins form a large transcription factor superfamily, and are commonly identified in large numbers within animal, plant, and fungal genomes. The bHLH domain has been well studied in many animal species, but has not yet been characterized in non-avian reptiles. In this study, we identified 102 putative bHLH genes in the genome of the green anole lizard, Anolis carolinensis. Based on phylogenetic analysis, these genes were classified into 43 families, with 43, 24, 16, 3, 10, and 3 members assigned into groups A, B, C, D, E, and F, respectively, and 3 members categorized as "orphans". Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with highly conserved patterns observed for introns and additional domains. Results from phylogenetic analysis of the H/E(spl) family suggest that genome and tandem gene duplications have contributed to this family's expansion. Our classification and evolutionary analysis has provided insights into the evolutionary diversification of animal bHLH genes, and should aid future studies on bHLH protein regulation of key growth and developmental processes.

Temperature impact on the micro structure of tungsten exposed to He irradiation in LHD

NASA Astrophysics Data System (ADS)

Bernard, Elodie; Sakamoto, Ryuichi; Tokitani, Masayuki; Masuzaki, Suguru; Hayashi, Hiromi; Yamada, Hiroshi; Yoshida, Naoaki

2017-02-01

A new temperature controlled material probe was designed for the exposure of tungsten samples to helium plasma in the LHD. Samples were exposed to estimated fluences of ∼1023 m-2 and temperatures ranging from 65 to 600 °C. Transmission Electron Microscopy analysis allowed the study of the impact of He irradiation under high temperatures on tungsten micro structure for the first time in real-plasma exposure conditions. Both dislocation loops and bubbles appeared from low to medium temperatures and saw an impressive increase of size (factor 4 to 6) most probably by coalescence as the temperature reaches 600 °C, with 500 °C appearing as a threshold for bubble growth. Annealing of the samples up to 800 C highlighted the stability of the dislocation damages formed by helium irradiation at high surface temperature, as bubbles and dislocation loops seem to conserve their characteristics. Additional studies on cross-sections showed that bubbles were formed much deeper (70-100 nm) than the heavily damaged surface layer (10-20 nm), raising concern about the impact on the material mechanical properties conservation and potential additional trapping of hydrogen isotopes.

Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants.

PubMed

Mohanta, Tapan Kumar; Arora, Pankaj Kumar; Mohanta, Nibedita; Parida, Pratap; Bae, Hanhong

2015-02-06

Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.

The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

PubMed

Leontis, N B; Westhof, E

1998-09-01

A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings. Thus, each non-Watson-Crick pair could be characterized by a phylogenetic signature of variations between isosteric-like pairings. In addition to the conservative changes, which form a dictionary of pairings isosterically compatible with those observed in the crystal structure, concerted changes involving several base pairs also occur. The latter covariations may indicate transitions between related but distinctive motifs within the loop E of 5S ribosomal RNA.

Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

PubMed

Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

2017-08-01

The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses. Copyright © 2017 American Society for Microbiology.

High resolution structure of cleaved Serpin 42 Da from Drosophila melanogaster.

PubMed

Ellisdon, Andrew M; Zhang, Qingwei; Henstridge, Michelle A; Johnson, Travis K; Warr, Coral G; Law, Ruby Hp; Whisstock, James C

2014-04-24

The Drosophila melanogaster Serpin 42 Da gene (previously Serpin 4) encodes a serine protease inhibitor that is capable of remarkable functional diversity through the alternative splicing of four different reactive centre loop exons. Eight protein isoforms of Serpin 42 Da have been identified to date, targeting the protease inhibitor to both different proteases and cellular locations. Biochemical and genetic studies suggest that Serpin 42 Da inhibits target proteases through the classical serpin 'suicide' inhibition mechanism, however the crystal structure of a representative Serpin 42 Da isoform remains to be determined. We report two high-resolution crystal structures of Serpin 42 Da representing the A/B isoforms in the cleaved conformation, belonging to two different space-groups and diffracting to 1.7 Å and 1.8 Å. Structural analysis reveals the archetypal serpin fold, with the major elements of secondary structure displaying significant homology to the vertebrate serpin, neuroserpin. Key residues known to have central roles in the serpin inhibitory mechanism are conserved in both the hinge and shutter regions of Serpin 42 Da. Furthermore, these structures identify important conserved interactions that appear to be of crucial importance in allowing the Serpin 42 Da fold to act as a versatile template for multiple reactive centre loops that have different sequences and protease specificities. In combination with previous biochemical and genetic studies, these structures confirm for the first time that the Serpin 42 Da isoforms are typical inhibitory serpin family members with the conserved serpin fold and inhibitory mechanism. Additionally, these data reveal the remarkable structural plasticity of serpins, whereby the basic fold is harnessed as a template for inhibition of a large spectrum of proteases by reactive centre loop exon 'switching'. This is the first structure of a Drosophila serpin reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of each of the Serpin 42 Da isoforms.

Closing the Loop Sampler.

ERIC Educational Resources Information Center

California Integrated Waste Management Board, Sacramento.

Closing the Loop (CTL) is a science curriculum designed to introduce students to integrated waste management through awareness. This document presents five lesson plans focusing on developing an understanding of natural resources, solid wastes, conservation, and the life of landfills. Contents include: (1) "What Are Natural Resources?"; (2)…

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation.

PubMed

Ruff, Emily F; Muretta, Joseph M; Thompson, Andrew R; Lake, Eric W; Cyphers, Soreen; Albanese, Steven K; Hanson, Sonya M; Behr, Julie M; Thomas, David D; Chodera, John D; Levinson, Nicholas M

2018-02-21

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases. © 2018, Ruff et al.

Solution structure of conserved AGNN tetraloops: insights into Rnt1p RNA processing

PubMed Central

Lebars, Isabelle; Lamontagne, Bruno; Yoshizawa, Satoko; Abou Elela, Sherif; Fourmy, Dominique

2001-01-01

Rnt1p, the yeast orthologue of RNase III, cleaves rRNAs, snRNAs and snoRNAs at a stem capped with conserved AGNN tetraloop. Here we show that 9 bp long stems ending with AGAA or AGUC tetraloops bind to Rnt1p and direct specific but sequence-independent RNA cleavage when provided with stems longer than 13 bp. The solution structures of these two tetraloops reveal a common fold for the terminal loop stabilized by non-canonical A–A or A–C pairs and extensive base stacking. The conserved nucleotides are stacked at the 5′ side of the loop, exposing their Watson–Crick and Hoogsteen faces for recognition by Rnt1p. These results indicate that yeast RNase III recognizes the fold of a conserved single-stranded tetraloop to direct specific dsRNA cleavage. PMID:11743001

Mitochondrial DNA variation in natural populations of endangered Indian feather-back fish, Chitala chitala.

PubMed

Mandal, Anup; Mohindra, Vindhya; Singh, Rajeev Kumar; Punia, Peyush; Singh, Ajay Kumar; Lal, Kuldeep Kumar

2012-02-01

Genetic variation at mitochondrial cytochrome b (cyt b) and D-loop region reveals the evidence of population sub-structuring in Indian populations of highly endangered primitive feather-back fish Chitala chitala. Samples collected through commercial catches from eight riverine populations from different geographical locations of India were analyzed for cyt b region (307 bp) and D-loop region (636-716 bp). The sequences of the both the mitochondrial regions revealed high haplotype diversity and low nucleotide diversity. The patterns of genetic diversity, haplotypes networks clearly indicated two distinct mitochondrial lineages and mismatch distribution strongly suggest a historical influence on the genetic structure of C. chitala populations. The baseline information on genetic variation and the evidence of population sub-structuring generated from this study would be useful for planning effective strategies for conservation and rehabilitation of this highly endangered species.

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

PubMed

Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

2012-09-01

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

PubMed Central

Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

2015-01-01

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Ning; Shen, Tielong; Kurtz, Richard

The properties of nano-scale interstitial dislocation loops under the coupling effect of stress and temperature are studied using atomistic simulation methods and experiments. The decomposition of a loop by the emission of smaller loops is identified as one of the major mechanisms to release the localized stress induced by the coupling effect, which is validated by the TEM observations. The classical conservation law of Burgers vector cannot be applied during such decomposition process. The dislocation network is formed from the decomposed loops, which may initiate the irradiation creep much earlier than expected through the mechanism of climb-controlled glide of dislocations.

Solution structure of an ATP-binding RNA aptamer reveals a novel fold.

PubMed Central

Dieckmann, T; Suzuki, E; Nakamura, G K; Feigon, J

1996-01-01

In vitro selection has been used to isolate several RNA aptamers that bind specifically to biological cofactors. A well-characterized example in the ATP-binding RNA aptamer family, which contains a conserved 11-base loop opposite a bulged G and flanked by regions of double-stranded RNA. The nucleotides in the consensus sequence provide a binding pocket for ATP (or AMP), which binds with a Kd in the micromolar range. Here we present the three-dimensional solution structure of a 36-nucleotide ATP-binding RNA aptamer complexed with AMP, determined from NMR-derived distance and dihedral angle restraints. The conserved loop and bulged G form a novel compact, folded structure around the AMP. The backbone tracing of the loop nucleotides can be described by a Greek zeta (zeta). Consecutive loop nucleotides G, A, A form a U-turn at the bottom of the zeta, and interact with the AMP to form a structure similar to a GNRA tetraloop, with AMP standing in for the final A. Two asymmetric G. G base pairs close the stems flanking the internal loop. Mutated aptamers support the existence of the tertiary interactions within the consensus nucleotides and with the AMP found in the calculated structures. PMID:8756406

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Comparative evolutionary genomics of the HADH2 gene encoding Aβ-binding alcohol dehydrogenase/17β-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10)

PubMed Central

Marques, Alexandra T; Antunes, Agostinho; Fernandes, Pedro A; Ramos, Maria J

2006-01-01

Background The Aβ-binding alcohol dehydrogenase/17β-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. Results Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand βF. This strand is one of the seven strands that compose the core β-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Aβ-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). Conclusion Our study revealed that HADH2 genes maintained a reasonable conserved organization across a large evolutionary distance. The conserved noncoding regions identified among mammals and between pufferfishes, the evidence of an alternative splicing variant conserved between human and dog, and the detection of positive selection across eutherian mammals, may be of importance for further research on ABAD/HSD10 function and its implication in the Alzheimer's disease. PMID:16899120

RECQ-like helicases Sgs1 and BLM regulate R-loop–associated genome instability

PubMed Central

Chang, Emily Yun-Chia; Novoa, Carolina A.; Aristizabal, Maria J.; Coulombe, Yan; Segovia, Romulo; Shen, Yaoqing; Keong, Christelle; Tam, Annie S.; Jones, Steven J.M.; Masson, Jean-Yves; Kobor, Michael S.

2017-01-01

Sgs1, the orthologue of human Bloom’s syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription–replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1Δ reveals copy number changes flanked by repetitive regions with high R-loop–forming potential. Analysis of BLM in Bloom’s syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop–associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop–mediated genome instability. PMID:29042409

Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization.

PubMed

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De; Qin, Cheng-Feng

2013-06-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

PubMed Central

Liu, Zhong-Yu; Li, Xiao-Feng; Jiang, Tao; Deng, Yong-Qiang; Zhao, Hui; Wang, Hong-Jiang; Ye, Qing; Zhu, Shun-Ya; Qiu, Yang; Zhou, Xi; Qin, E-De

2013-01-01

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5′ cyclization sequence (5′CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5′CS, and the presence of DCS-PK facilitates the formation of 5′-3′ RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution. PMID:23576500

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

PubMed

Nandy, Suman Kumar; Seal, Alpana

2016-01-01

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

Mining protein loops using a structural alphabet and statistical exceptionality

PubMed Central

2010-01-01

Background Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. Results We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 Å). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/. PMID:20132552

Mining protein loops using a structural alphabet and statistical exceptionality.

PubMed

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

Protein loops encompass 50% of protein residues in available three-dimensional structures. These regions are often involved in protein functions, e.g. binding site, catalytic pocket... However, the description of protein loops with conventional tools is an uneasy task. Regular secondary structures, helices and strands, have been widely studied whereas loops, because they are highly variable in terms of sequence and structure, are difficult to analyze. Due to data sparsity, long loops have rarely been systematically studied. We developed a simple and accurate method that allows the description and analysis of the structures of short and long loops using structural motifs without restriction on loop length. This method is based on the structural alphabet HMM-SA. HMM-SA allows the simplification of a three-dimensional protein structure into a one-dimensional string of states, where each state is a four-residue prototype fragment, called structural letter. The difficult task of the structural grouping of huge data sets is thus easily accomplished by handling structural letter strings as in conventional protein sequence analysis. We systematically extracted all seven-residue fragments in a bank of 93000 protein loops and grouped them according to the structural-letter sequence, named structural word. This approach permits a systematic analysis of loops of all sizes since we consider the structural motifs of seven residues rather than complete loops. We focused the analysis on highly recurrent words of loops (observed more than 30 times). Our study reveals that 73% of loop-lengths are covered by only 3310 highly recurrent structural words out of 28274 observed words). These structural words have low structural variability (mean RMSd of 0.85 A). As expected, half of these motifs display a flanking-region preference but interestingly, two thirds are shared by short (less than 12 residues) and long loops. Moreover, half of recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Cross-cohort analysis identifies a TEAD4 ↔ MYCN positive-feedback loop as the core regulatory element of high-risk neuroblastoma. | Office of Cancer Genomics

Cancer.gov

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator (MR) proteins that were conserved across independent cohorts.

High diversification of CD94 by alternative splicing in New World primates.

PubMed

Galindo, John A; Cadavid, Luis F

2013-04-01

CD94 forms heterodimers with NKG2A, -C, or -E to constitute lectin-like natural killer cell receptors for MHC-E. Its structure differs from other C-type lectins in that the second α-helix is replaced by a loop that forms the interacting interface with the NKG2 molecules. Although CD94 has remained highly conserved mammals, several alternative splicing variants have been detected in some species. To evaluate the prevalence and significance of this phenomenon, we have cloned and sequenced CD94 cDNAs in six species of New World primates from the Cebidae and Atelidae families. Full-length sequences had a mean similarity of 96 % amongst New World primates and of 90 % to the human orthologue, with little variation in the residues interacting with NKG2 or MHC-E molecules. Despite this high conservation, a total of 14 different splice variants were identified, half of which were shared by two or more primate species. Homology-based modeling of the C-type lectin domain showed that most isoforms folded stably, although they had modifications that prevented its interaction with NKG2 and MHC-E. Two isoforms were predicted to replace the typical CD94 loop by a second α-helix, evidencing a domain fold transition from a CD94 structure to a canonical C-type lectin. These two structures were more similar to members of the CLEC lectin family than to the native CD94. Thus, CD94 has remained conserved in primates to maintain functional interactions with NKG2 and MHC-E, while at the same time has diversified by alternative splicing potentially providing additional functional scenarios.

Molecular Characterization of the Skate Peripherin/rds Gene: Relationship to Its Orthologues and Paralogues

PubMed Central

Li, Chibo; Ding, Xi-Qin; O’Brien, John; Al-Ubaidi, Muayyad R.

2010-01-01

PURPOSE A great deal of information about functionally significant domains of a protein may be obtained by comparison of primary sequences of gene homologues over a broad phylogenetic base. This study was designed to identify evolutionarily conserved domains of the photoreceptor disc membrane protein peripherin/rds by analysis of the homologue in a primitive vertebrate, the skate. METHODS A skate retinal cDNA library was screened using a mouse peripherin/rds clone. The 5′ and 3′ untranslated regions of the skate peripherin/rds (srds) cDNA were isolated by the rapid amplification of cDNA ends (RACE) approach. The gene structure was characterized by PCR amplification and sequencing of genomic fragments. Northern and Western blot analyses were used to identify srds transcript and protein, respectively. RESULTS A new homologue of peripherin/rds was identified from the skate retinal cDNA library. SRDS is a glycoprotein with a predicted molecular mass of 40.2 kDa. The srds gene consists of two exons and one small intron and transcribes into a single 6-kb message. Phylogenetic analysis places SRDS at the base of peripherin/rds family and near the division of that group and the branch leading to rds-like and rom-1 genes. SRDS protein is 54.5% identical with peripherin/rds across species. Identity is significantly higher (73%) in the intradiscal domains. Sequence comparison revealed the conservation of all residues that have been shown, on mutation, to associate with retinitis pigmentosa and showed conservation of most residues associated with macular dystrophies. Comparison with ROM-1 and other rds-like proteins revealed the presence of a highly conserved domain in the large intradiscal loop. CONCLUSIONS Srds represents the skate orthologue of mammalian peripherin/rds genes. Conservation of most of the residues associated with human retinal diseases indicates that these residues serve important functional roles. The high degree of conservation of a short stretch within the large intradiscal loop also suggests an important function for this domain. PMID:12766040

Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems.

PubMed

Okuda, Ken-ichi; Yanagihara, Sae; Sugayama, Tomomichi; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-06-01

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.

Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

2013-11-20

Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures ofmore » IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.« less

Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and Tendon Cell Differentiation in Drosophila

PubMed Central

Cattenoz, Pierre B.; Popkova, Anna; Southall, Tony D.; Aiello, Giuseppe; Brand, Andrea H.; Giangrande, Angela

2016-01-01

High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades. PMID:26567182

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

PubMed

Venkatesan, G; Balamurugan, V; Bhanuprakash, V; Singh, R K; Pandey, A B

2016-06-01

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories. Copyright © 2016 Elsevier Ltd. All rights reserved.

Note: A dual temperature closed loop batch reactor for determining the partitioning of trace gases within CO2-water systems.

PubMed

Warr, Oliver; Rochelle, Christopher A; Masters, Andrew J; Ballentine, Christopher J

2016-01-01

An experimental approach is presented which can be used to determine partitioning of trace gases within CO2-water systems. The key advantages of this system are (1) The system can be isolated with no external exchange, making it ideal for experiments with conservative tracers. (2) Both phases can be sampled concurrently to give an accurate composition at each phase at any given time. (3) Use of a lower temperature flow loop outside of the reactor removes contamination and facilitates sampling. (4) Rapid equilibration at given pressure/temperature conditions is significantly aided by stirring and circulating the water phase using a magnetic stirrer and high-pressure liquid chromatography pump, respectively.

Replacing Arginine 33 for Alanine in the Hemophore HasA from Pseudomonas aeruginosa Causes Closure of the H32 Loop in the Apo-Protein

PubMed Central

Kumar, Ritesh; Qi, Yifei; Matsumura, Hirotoshi; Lovell, Scott; Yao, Huili; Battaile, Kevin P.; Im, Wonpil; Moënne-Loccoz, Pierre; Rivera, Mario

2017-01-01

Previous characterization of hemophores from Serratia marcescens (HasAs), Pseudomonas aeruginosa (HasAp) and Yersinia pestis (HasAyp) showed that hemin binds between two loops, where it is axially coordinated by H32 and Y75. The Y75 loop is structurally conserved in all three hemophores and harbors conserved ligand Y75. The other loop contains H32 in HasAs and HasAp, but a noncoordinating Q32 in HasAyp. The H32 loop in apo-HasAs and apo-HasAp is in an open conformation, which places H32 about 30 Å from the hemin-binding site. Hence, hemin binding onto the Y75 loop of HasAs or HasAp triggers a large relocation of the H32 loop from an open- to a closed-loop conformation and enables coordination of the hemin-iron by H32. In comparison, the Q32 loop in apo-HasAyp is in the closed conformation and hemin binding occurs with minimal reorganization and without coordinative interactions with the Q32 loop. Studies in crystallo and in solution have established that the open H32 loop in apo-HasAp and apo-HasAs is well structured and minimally affected by conformational dynamics. In this study we address the intriguing issue of the stability of the H32 loop in apo-HasAp and how hemin binding triggers its relocation. We address this question with a combination of NMR spectroscopy, X-ray crystallography, and molecular dynamics simulations and find that R33 is critical to the stability of the open H32 loop. Replacing R33 with A causes the H32 loop in R33A apo-HasAp to adopt a conformation similar to that of holo-HasAp. Finally, stopped-flow absorption and resonance Raman analyses of hemin binding to apo-R33A HasAp indicates that the closed H32 loop slows down the insertion of the heme inside the binding pocket, presumably as it obstructs access to the hydrophobic platform on the Y75 loop, but accelerate the completion of the heme iron coordination. PMID:27074415

The Potential for Double-Loop Learning to Enable Landscape Conservation Efforts

NASA Astrophysics Data System (ADS)

Petersen, Brian; Montambault, Jensen; Koopman, Marni

2014-10-01

As conservation increases its emphasis on implementing change at landscape-level scales, multi-agency, cross-boundary, and multi-stakeholder networks become more important. These elements complicate traditional notions of learning. To investigate this further, we examined structures of learning in the Landscape Conservation Cooperatives (LCCs), which include the entire US and its territories, as well as parts of Canada, Mexico, and Caribbean and Pacific island states. We used semi-structured interviews, transcribed and analyzed using NVivo, as well as a charrette-style workshop to understand the difference between the original stated goals of individual LCCs and the values and purposes expressed as the collaboration matured. We suggest double-loop learning as a theoretical framework appropriate to landscape-scale conservation, recognizing that concerns about accountability are among the valid points of view that must be considered in multi-stakeholder collaborations. Methods from the social sciences and public health sectors provide insights on how such learning might be actualized.

Length variation and sequence divergence in mitochondrial control region of Schizothoracine (Teleostei: Cyperinidae) species.

PubMed

Syed, Mudasir Ahmad; Bhat, Farooz Ahmad; Balkhi, Masood-ul Hassan; Bhat, Bilal Ahmad

2016-01-01

Schizothoracine fish commonly called snow trouts inhibit the entire network of snow and spring fed cool waters of Kashmir, India. Over 10 species reported earlier, only five species have been found, these include Schizothorax niger, Schizothorax esocinus, Schizothorax plagiostomus, Schizothorax curvifrons and Schizothorax labiatus. The relationship between these species is contradicting. To understand the evolutionary relation of these species, we examined the sequence information of mitochondrial D-loop of 25 individuals representing five species. Sequence alignment showed D-loop region highly variable and length variation was observed in di-nucleotide (TA)n microsatellite between and within species. Interestingly, all these species have (TA)n microsatellite not associated with longer tandem repeats at the 3' end of the mitochondrial control region and do not show heteroplasmy. Our analysis also indicates the presence of four conserved sequence blocks (CSB), CSB-D, CSB-1, CSB-II and CSB-III, four (Termination Associated Sequence) TAS motifs and 15bp pyrimidine block within the mitochondrial control region, that are highly conserved within genus Schizothorax when compared with other species. The phylogenetic analysis carried by Maximum likelihood (ML), Neighbor Joining (NJ) and Bayesian inference (BI) generated almost identical results. The resultant BI tree showed a close genetic relationship of all the five species and supports two distinct grouping of S. esocinus species. Besides the species relation, the presence of length variation in tandem repeats is attributed to differences in predicting the stability of secondary structures. The role of CSBs and TASs, reported so far as main regulatory signals, would explain the conservation of these elements in evolution.

Identification of microRNAs and their targets in Finger millet by high throughput sequencing.

PubMed

Usha, S; Jyothi, M N; Sharadamma, N; Dixit, Rekha; Devaraj, V R; Nagesh Babu, R

2015-12-15

MicroRNAs are short non-coding RNAs which play an important role in regulating gene expression by mRNA cleavage or by translational repression. The majority of identified miRNAs were evolutionarily conserved; however, others expressed in a species-specific manner. Finger millet is an important cereal crop; nonetheless, no practical information is available on microRNAs to date. In this study, we have identified 95 conserved microRNAs belonging to 39 families and 3 novel microRNAs by high throughput sequencing. For the identified conserved and novel miRNAs a total of 507 targets were predicted. 11 miRNAs were validated and tissue specificity was determined by stem loop RT-qPCR, Northern blot. GO analyses revealed targets of miRNA were involved in wide range of regulatory functions. This study implies large number of known and novel miRNAs found in Finger millet which may play important role in growth and development. Copyright © 2015 Elsevier B.V. All rights reserved.

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap)

PubMed Central

Wang, Aibing; Zhang, Lijie; Khayat, Reza

2016-01-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis. PMID:27902320

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap).

PubMed

Wang, Naidong; Zhan, Yang; Wang, Aibing; Zhang, Lijie; Khayat, Reza; Yang, Yi

2016-12-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites

PubMed Central

Piazza, Aurèle; Adrian, Michael; Samazan, Frédéric; Heddi, Brahim; Hamon, Florian; Serero, Alexandre; Lopes, Judith; Teulade-Fichou, Marie-Paule; Phan, Anh Tuân; Nicolas, Alain

2015-01-01

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or functionin vivo are unknown. InS. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with thein vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved inC. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequencesin vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to formin vivo. PMID:25956747

Landau-Zener-Stückelberg-Majorana Interferometry of a Single Hole

NASA Astrophysics Data System (ADS)

Bogan, Alex; Studenikin, Sergei; Korkusinski, Marek; Gaudreau, Louis; Zawadzki, Piotr; Sachrajda, Andy S.; Tracy, Lisa; Reno, John; Hargett, Terry

2018-05-01

We perform Landau-Zener-Stückelberg-Majorana (LZSM) spectroscopy on a system with strong spin-orbit interaction (SOI), realized as a single hole confined in a gated double quantum dot. Analogous to electron systems, at a magnetic field B =0 and high modulation frequencies, we observe photon-assisted tunneling between dots, which smoothly evolves into the typical LZSM funnel-shaped interference pattern as the frequency is decreased. In contrast to electrons, the SOI enables an additional, efficient spin-flip interdot tunneling channel, introducing a distinct interference pattern at finite B . Magnetotransport spectra at low-frequency LZSM driving show the two channels to be equally coherent. High-frequency LZSM driving reveals complex photon-assisted tunneling pathways, both spin conserving and spin flip, which form closed loops at critical magnetic fields. In one such loop, an arbitrary hole spin state is inverted, opening the way toward its all-electrical manipulation.

A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

PubMed

Kudalkar, Shalley N; Njuma, Olive J; Li, Yongjiang; Muldowney, Michelle; Fuanta, N Rene; Goodwin, Douglas C

2015-03-03

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

Robustness of the Rotary Catalysis Mechanism of F1-ATPase*

PubMed Central

Watanabe, Rikiya; Matsukage, Yuki; Yukawa, Ayako; Tabata, Kazuhito V.; Noji, Hiroyuki

2014-01-01

F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought. PMID:24876384

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

PubMed Central

Sundar, Shankar; Baker, Tania A; Sauer, Robert T

2012-01-01

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase KM and decrease Vmax for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine. PMID:22102327

One-step reverse transcription loop mediated isothermal amplification assay for detection of Apple chlorotic leaf spot virus

USDA-ARS?s Scientific Manuscript database

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Apple chlorotic leaf spot virus (ACLSV) was developed. In this method, a set of four primers was designed based on the conserved regions in the coat protein gene of ACLSV, and was synthesized for the ...

Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes

PubMed Central

Nandy, Suman Kumar; Seal, Alpana

2016-01-01

Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases. PMID:27764212

G-quadruplex prediction in E. coli genome reveals a conserved putative G-quadruplex-Hairpin-Duplex switch.

PubMed

Kaplan, Oktay I; Berber, Burak; Hekim, Nezih; Doluca, Osman

2016-11-02

Many studies show that short non-coding sequences are widely conserved among regulatory elements. More and more conserved sequences are being discovered since the development of next generation sequencing technology. A common approach to identify conserved sequences with regulatory roles relies on topological changes such as hairpin formation at the DNA or RNA level. G-quadruplexes, non-canonical nucleic acid topologies with little established biological roles, are increasingly considered for conserved regulatory element discovery. Since the tertiary structure of G-quadruplexes is strongly dependent on the loop sequence which is disregarded by the generally accepted algorithm, we hypothesized that G-quadruplexes with similar topology and, indirectly, similar interaction patterns, can be determined using phylogenetic clustering based on differences in the loop sequences. Phylogenetic analysis of 52 G-quadruplex forming sequences in the Escherichia coli genome revealed two conserved G-quadruplex motifs with a potential regulatory role. Further analysis revealed that both motifs tend to form hairpins and G quadruplexes, as supported by circular dichroism studies. The phylogenetic analysis as described in this work can greatly improve the discovery of functional G-quadruplex structures and may explain unknown regulatory patterns. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Note: A dual temperature closed loop batch reactor for determining the partitioning of trace gases within CO{sub 2}-water systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warr, Oliver, E-mail: oliver.warr@earth.ox.ac.uk; Ballentine, Christopher J.; Rochelle, Christopher A.

An experimental approach is presented which can be used to determine partitioning of trace gases within CO{sub 2}-water systems. The key advantages of this system are (1) The system can be isolated with no external exchange, making it ideal for experiments with conservative tracers. (2) Both phases can be sampled concurrently to give an accurate composition at each phase at any given time. (3) Use of a lower temperature flow loop outside of the reactor removes contamination and facilitates sampling. (4) Rapid equilibration at given pressure/temperature conditions is significantly aided by stirring and circulating the water phase using a magneticmore » stirrer and high-pressure liquid chromatography pump, respectively.« less

Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.

PubMed

Stevenson, Andrew; Macdonald, Julie; Roberts, Mark

2003-03-20

Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs.

PubMed

Ji, Yongchang; Buel, Sharleen M; Amack, Jeffrey D

2016-08-01

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2(-/-) knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFβ) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling-independent of Pitx2-controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lentz, T.L.

1991-11-12

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similarmore » segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.« less

Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of Sugarcane mosaic virus and Sorghum mosaic virus in sugarcane

USDA-ARS?s Scientific Manuscript database

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) in sugarcane. Six sets of four primers corresponding to the conserved coat protein gene were designed for each virus and their succ...

Performance of the Extravehicular Mobility Unit (EMU): Airlock Coolant Loop Recovery (A/L CLR) Hardware - Phase II

NASA Technical Reports Server (NTRS)

Steele, John; Rector, tony; Gazda, Daniel; Lewis, John

2009-01-01

An EMU water processing kit (Airlock Coolant Loop Recovery A/L CLR) was developed as a corrective action to Extravehicular Mobility Unit (EMU) coolant flow disruptions experienced on the International Space Station (ISS) in May of 2004 and thereafter. Conservative schedules for A/L CLR use and component life were initially developed and implemented based on prior analysis results and analytical modeling. The examination of postflight samples and EMU hardware in November of 2006 indicated that the A/L CLR kits were functioning well and had excess capacity that would allow a relaxation of the initially conservative schedules of use and component life. A relaxed use schedule and list of component lives was implemented thereafter. Since the adoption of the relaxed A/L CLR schedules of use and component lives, several A/L CLR kit components, transport loop water samples and sensitive EMU transport loop components have been examined to gage the impact of the relaxed requirements. The intent of this paper is to summarize the findings of that evaluation, and to outline updated schedules for A/L CLR use and component life.

Molecular dynamics simulations reveal a new role for a conserved active site asparagine in a ubiquitin-conjugating enzyme.

PubMed

Wilson, R Hunter; Zamfir, Serban; Sumner, Isaiah

2017-09-01

The role of a highly conserved active site asparagine (N79) in the ubiquitin conjugating enzyme, Ubc13, is probed using molecular dynamics simulations. Both wild type and mutant enzymes (N79A and N79D) are studied. Contrary to a popular hypothesis, we show that it is unlikely that N79 stabilizes a reaction intermediate, but instead preferentially hydrogen bonds to a loop near the active site. This keeps the sidechain carboxylate of an aspartate in the loop (D119) near the sidechain amine of the substrate lysine. Our simulations show that this distance increases in the mutants. D119 has been hypothesized to play a variety of roles in the enzyme, including deprotonating the substrate lysine, so changing this distance can have an effect on the enzyme's efficiency. Finally, we show that it is possible for the aspartate to deprotonate the substrate even across long distances if short water wires form that connect the proton donor and acceptor. Short water wires form with greater probability in the wild type than in mutant enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

Overcoming Antigenic Diversity by Enhancing the Immunogenicity of Conserved Epitopes on the Malaria Vaccine Candidate Apical Membrane Antigen-1

PubMed Central

Dutta, Sheetij; Dlugosz, Lisa S.; Drew, Damien R.; Ge, Xiopeng; Ababacar, Diouf; Rovira, Yazmin I.; Moch, J. Kathleen; Shi, Meng; Long, Carole A.; Foley, Michael; Beeson, James G.; Anders, Robin F.; Miura, Kazutoyo; Haynes, J. David; Batchelor, Adrian H.

2013-01-01

Malaria vaccine candidate Apical Membrane Antigen-1 (AMA1) induces protection, but only against parasite strains that are closely related to the vaccine. Overcoming the AMA1 diversity problem will require an understanding of the structural basis of cross-strain invasion inhibition. A vaccine containing four diverse allelic proteins 3D7, FVO, HB3 and W2mef (AMA1 Quadvax or QV) elicited polyclonal rabbit antibodies that similarly inhibited the invasion of four vaccine and 22 non-vaccine strains of P. falciparum. Comparing polyclonal anti-QV with antibodies against a strain-specific, monovalent, 3D7 AMA1 vaccine revealed that QV induced higher levels of broadly inhibitory antibodies which were associated with increased conserved face and domain-3 responses and reduced domain-2 response. Inhibitory monoclonal antibodies (mAb) raised against the QV reacted with a novel cross-reactive epitope at the rim of the hydrophobic trough on domain-1; this epitope mapped to the conserved face of AMA1 and it encompassed the 1e-loop. MAbs binding to the 1e-loop region (1B10, 4E8 and 4E11) were ∼10-fold more potent than previously characterized AMA1-inhibitory mAbs and a mode of action of these 1e-loop mAbs was the inhibition of AMA1 binding to its ligand RON2. Unlike the epitope of a previously characterized 3D7-specific mAb, 1F9, the 1e-loop inhibitory epitope was partially conserved across strains. Another novel mAb, 1E10, which bound to domain-3, was broadly inhibitory and it blocked the proteolytic processing of AMA1. By itself mAb 1E10 was weakly inhibitory but it synergized with a previously characterized, strain-transcending mAb, 4G2, which binds close to the hydrophobic trough on the conserved face and inhibits RON2 binding to AMA1. Novel inhibition susceptible regions and epitopes, identified here, can form the basis for improving the antigenic breadth and inhibitory response of AMA1 vaccines. Vaccination with a few diverse antigenic proteins could provide universal coverage by redirecting the immune response towards conserved epitopes. PMID:24385910

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

A Redox 2-Cys Mechanism Regulates the Catalytic Activity of Divergent Cyclophilins1[W

PubMed Central

Campos, Bruna Medéia; Sforça, Mauricio Luis; Ambrosio, Andre Luis Berteli; Domingues, Mariane Noronha; Brasil de Souza, Tatiana de Arruda Campos; Barbosa, João Alexandre Ribeiro Gonçalvez; Leme, Adriana Franco Paes; Perez, Carlos Alberto; Whittaker, Sara Britt-Marie; Murakami, Mario Tyago; Zeri, Ana Carolina de Matos; Benedetti, Celso Eduardo

2013-01-01

The citrus (Citrus sinensis) cyclophilin CsCyp is a target of the Xanthomonas citri transcription activator-like effector PthA, required to elicit cankers on citrus. CsCyp binds the citrus thioredoxin CsTdx and the carboxyl-terminal domain of RNA polymerase II and is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteine (Cys) residues Cys-40 and Cys-168, and the conserved glutamate (Glu) Glu-83. Despite the suggested roles in ATP and metal binding, the functions of these unique structural elements remain unknown. Here, we show that the conserved Cys residues form a disulfide bond that inactivates the enzyme, whereas Glu-83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that Cys-40 and Cys-168 are required for the interaction with CsTdx and that CsCyp binds the citrus carboxyl-terminal domain of RNA polymerase II YSPSAP repeat. Our data support a model where formation of the Cys-40-Cys-168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via Glu-83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop-displacement mechanism. PMID:23709667

Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.

PubMed

Jacob, F; Joris, B; Lepage, S; Dusart, J; Frère, J M

1990-10-15

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

PubMed Central

Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

2014-01-01

The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5′-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5′-CCCCGGCAAGGAGGGG-3′. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication. PMID:25175101

Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

PubMed

Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

2010-01-01

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

Structural maintenance of chromosome complexes differentially compact mitotic chromosomes according to genomic context

PubMed Central

Schalbetter, S. A.; Goloborodko, A.; Fudenberg, G.; Belton, J.-M.; Miles, C.; Yu, M.; Dekker, J.; Mirny, L.; Baxter, J.

2017-01-01

Structural Maintenance of Chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modeling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids whilst condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes. PMID:28825700

Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine

PubMed Central

Iosefson, Ohad; Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.

2014-01-01

Hexameric AAA+ unfoldases of ATP-dependent proteases and protein-remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the E. coli ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, with the number of wild-type loops required for efficient degradation depending upon the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate. PMID:25599533

Virtual solar field - An opportunity to optimize transient processes in line-focus CSP power plants

NASA Astrophysics Data System (ADS)

Noureldin, Kareem; Hirsch, Tobias; Pitz-Paal, Robert

2017-06-01

Optimizing solar field operation and control is a key factor to improve the competitiveness of line-focus solar thermal power plants. However, the risks of assessing new and innovative control strategies on operational power plants hinder such optimizations and result in applying more conservative control schemes. In this paper, we describe some applications for a whole solar field transient in-house simulation tool developed at the German Aerospace Centre (DLR), the Virtual Solar Field (VSF). The tool offers a virtual platform to simulate real solar fields while coupling the thermal and hydraulic conditions of the field with high computational efficiency. Using the tool, developers and operator can probe their control strategies and assess the potential benefits while avoiding the high risks and costs. In this paper, we study the benefits gained from controlling the loop valves and of using direct normal irradiance maps and forecasts for the field control. Loop valve control is interesting for many solar field operators since it provides a high degree of flexibility to the control of the solar field through regulating the flow rate in each loop. This improves the reaction to transient condition, such as passing clouds and field start-up in the morning. Nevertheless, due to the large number of loops and the sensitivity of the field control to the valve settings, this process needs to be automated and the effect of changing the setting of each valve on the whole field control needs to be taken into account. We used VSF to implement simple control algorithms to control the loop valves and to study the benefits that could be gained from using active loop valve control during transient conditions. Secondly, we study how using short-term highly spatially-resolved DNI forecasts provided by cloud cameras could improve the plant energy yield. Both cases show an improvement in the plant efficiency and outlet temperature stability. This paves the road for further investigations of new control strategies or for optimizations of the currently implemented ones.

Genetic analysis of the Hungarian draft horse population using partial mitochondrial DNA D-loop sequencing.

PubMed

Csizmár, Nikolett; Mihók, Sándor; Jávor, András; Kusza, Szilvia

2018-01-01

The Hungarian draft is a horse breed with a recent mixed ancestry created in the 1920s by crossing local mares with draught horses imported from France and Belgium. The interest in its conservation and characterization has increased over the last few years. The aim of this work is to contribute to the characterization of the endangered Hungarian heavy draft horse populations in order to obtain useful information to implement conservation strategies for these genetic stocks. To genetically characterize the breed and to set up the basis for a conservation program, in the present study a hypervariable region of the mitochrondial DNA (D-loop) was used to assess genetic diversity in Hungarian draft horses. Two hundred and eighty five sequences obtained in our laboratory and 419 downloaded sequences available from Genbank were analyzed. One hundred and sixty-four haplotypes and thirty-six polymorphic sites were observed. High haplotype and nucleotide diversity values ( H d  = 0.954 ± 0.004; π  = 0.028 ± 0.0004) were identified in Hungarian population, although they were higher within than among the different populations ( H d  = 0.972 ± 0.002; π  = 0.03097 ± 0.002). Fourteen of the previously observed seventeen haplogroups were detected. Our samples showed a large intra- and interbreed variation. There was no clear clustering on the median joining network figure. The overall information collected in this work led us to consider that the genetic scenario observed for Hungarian draft breed is more likely the result of contributions from 'ancestrally' different genetic backgrounds. This study could contribute to the development of a breeding plan for Hungarian draft horses and help to formulate a genetic conservation plan, avoiding inbreeding while.

Automated classification of RNA 3D motifs and the RNA 3D Motif Atlas

PubMed Central

Petrov, Anton I.; Zirbel, Craig L.; Leontis, Neocles B.

2013-01-01

The analysis of atomic-resolution RNA three-dimensional (3D) structures reveals that many internal and hairpin loops are modular, recurrent, and structured by conserved non-Watson–Crick base pairs. Structurally similar loops define RNA 3D motifs that are conserved in homologous RNA molecules, but can also occur at nonhomologous sites in diverse RNAs, and which often vary in sequence. To further our understanding of RNA motif structure and sequence variability and to provide a useful resource for structure modeling and prediction, we present a new method for automated classification of internal and hairpin loop RNA 3D motifs and a new online database called the RNA 3D Motif Atlas. To classify the motif instances, a representative set of internal and hairpin loops is automatically extracted from a nonredundant list of RNA-containing PDB files. Their structures are compared geometrically, all-against-all, using the FR3D program suite. The loops are clustered into motif groups, taking into account geometric similarity and structural annotations and making allowance for a variable number of bulged bases. The automated procedure that we have implemented identifies all hairpin and internal loop motifs previously described in the literature. All motif instances and motif groups are assigned unique and stable identifiers and are made available in the RNA 3D Motif Atlas (http://rna.bgsu.edu/motifs), which is automatically updated every four weeks. The RNA 3D Motif Atlas provides an interactive user interface for exploring motif diversity and tools for programmatic data access. PMID:23970545

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

PubMed

Lim, Chun Shen; Brown, Chris M

2016-09-01

Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

PubMed Central

Lim, Chun Shen; Brown, Chris M.

2016-01-01

ABSTRACT Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

Mechanisms of zinc binding to the solute-binding protein AztC and transfer from the metallochaperone AztD.

PubMed

Neupane, Durga P; Avalos, Dante; Fullam, Stephanie; Roychowdhury, Hridindu; Yukl, Erik T

2017-10-20

Bacteria can acquire the essential metal zinc from extremely zinc-limited environments by using ATP-binding cassette (ABC) transporters. These transporters are critical virulence factors, relying on specific and high-affinity binding of zinc by a periplasmic solute-binding protein (SBP). As such, the mechanisms of zinc binding and release among bacterial SBPs are of considerable interest as antibacterial drug targets. Zinc SBPs are characterized by a flexible loop near the high-affinity zinc-binding site. The function of this structure is not always clear, and its flexibility has thus far prevented structural characterization by X-ray crystallography. Here, we present intact structures for the zinc-specific SBP AztC from the bacterium Paracoccus denitrificans in the zinc-bound and apo-states. A comparison of these structures revealed that zinc loss prompts significant structural rearrangements, mediated by the formation of a sodium-binding site in the apo-structure. We further show that the AztC flexible loop has no impact on zinc-binding affinity, stoichiometry, or protein structure, yet is essential for zinc transfer from the metallochaperone AztD. We also found that 3 His residues in the loop appear to temporarily coordinate zinc and then convey it to the high-affinity binding site. Thus, mutation of any of these residues to Ala abrogated zinc transfer from AztD. Our structural and mechanistic findings conclusively identify a role for the AztC flexible loop in zinc acquisition from the metallochaperone AztD, yielding critical insights into metal binding by AztC from both solution and AztD. These proteins are highly conserved in human pathogens, making this work potentially useful for the development of novel antibiotics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial genome of the tomato clownfish Amphiprion frenatus (Pomacentridae, Amphiprioninae).

PubMed

Ye, Le; Hu, Jing; Wu, Kaichang; Wang, Yu; Li, Jianlong

2016-01-01

The complete mitochondrial (mt) genome of the tomato clownfish Amphiprion frenatus was obtained in this study. The circular mtDNA molecule was 16,774 bp in size and the overall nucleotide composition of the H-strand was 29.72% A, 25.81% T, 15.38% G and 29.09% C, with an A + T bias. The complete mitogenome encoded 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a control region (D-loop), with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. The D-loop included termination associated sequence (TAS), central conserved domain (CCD) and conserved sequence block (CSB), and was composed of 6 complete continuity tandem repeat units and an imperfect tandem repeat unit.

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection

PubMed Central

Maciag, Joseph J.; Mackenzie, Sarah H.; Tucker, Matthew B.; Schipper, Joshua L.; Swartz, Paul; Clark, A. Clay

2016-01-01

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection. PMID:27681633

Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection.

PubMed

Maciag, Joseph J; Mackenzie, Sarah H; Tucker, Matthew B; Schipper, Joshua L; Swartz, Paul; Clark, A Clay

2016-10-11

The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.

Nicotiana alata Defensin Chimeras Reveal Differences in the Mechanism of Fungal and Tumor Cell Killing and an Enhanced Antifungal Variant

PubMed Central

Payne, Jennifer A. E.; Hayes, Brigitte M. E.; Durek, Thomas; Craik, David J.; Shafee, Thomas M. A.; Poon, Ivan K. H.; Hulett, Mark D.; van der Weerden, Nicole L.

2016-01-01

The plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2 binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants. PMID:27503651

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

PubMed Central

Buchko, Garry W.; Berg, Howard R.; Kaur, Jagdeep; Pandurangi, Raghu S.; Smith, Thomas J.; Shah, Dilip M.

2013-01-01

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA. PMID:24324798

Minimally doubled fermions at one loop

NASA Astrophysics Data System (ADS)

Capitani, Stefano; Weber, Johannes; Wittig, Hartmut

2009-10-01

Minimally doubled fermions have been proposed as a cost-effective realization of chiral symmetry at non-zero lattice spacing. Using lattice perturbation theory at one loop, we study their renormalization properties. Specifically, we investigate the consequences of the breaking of hyper-cubic symmetry, which is a typical feature of this class of fermionic discretizations. Our results for the quark self-energy indicate that the four-momentum undergoes a renormalization which is linearly divergent. We also compute renormalization factors for quark bilinears, construct the conserved vector and axial-vector currents and verify that at one loop the renormalization factors of the latter are equal to one.

General calculation of the cross section for dark matter annihilations into two photons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Cely, Camilo; Rivera, Andres, E-mail: Camilo.Alfredo.Garcia.Cely@ulb.ac.be, E-mail: afelipe.rivera@udea.edu.co

2017-03-01

Assuming that the underlying model satisfies some general requirements such as renormalizability and CP conservation, we calculate the non-relativistic one-loop cross section for any self-conjugate dark matter particle annihilating into two photons. We accomplish this by carefully classifying all possible one-loop diagrams and, from them, reading off the dark matter interactions with the particles running in the loop. Our approach is general and leads to the same results found in the literature for popular dark matter candidates such as the neutralinos of the MSSM, minimal dark matter, inert Higgs and Kaluza-Klein dark matter.

3D Studies of the Solar Corona and its Evolution with SOHO/EIT

NASA Astrophysics Data System (ADS)

Portier-Fozzani, F.

This thesis deals with 3D evolution of coronal structures based upon the ultraviolet telescope of SOHO : EIT. Anaglyphs and incertainties on a complete stereovision reconstruction are described. Stereoscopic methods for loop reconstruction were successfully made to find 3D parameters. With dynamical stereoscopy, physical conditions were derived for 30 loops of temperature around 1MK. A method which is able to derive twist variation were also built. Emerging loops were found highly twisted and they detwist as they grow. According to helicity conservation, this correspond to a transfert of twist into expansion. Long time twist evolution of magnetic flux tubes are followed in relation with flares as relaxation. Interaction between magnetic field lines were analysed. An example of reconnection between open and closed field line were observed. Other interactions were found with multi-wavelength observations : coronal holes borders (and thus CH) are better defined when an active region nearby is growing. Other imaging techniques were used to better take profit as possible of SOHO/EIT. A multiscale vision model (MVM) was applied with success to show small coronal structures evolutions hidden by the noise level.

Molecular characterization and application of lipase from Bacillus sp. PU1 and investigation of structural changes based on pH and temperature using MD simulation.

PubMed

Esakkiraj, Palanichamy; Antonyraj, Christian Bharathi; Meleppat, Balraj; Ankaiah, Dasari; Ayyanna, Repally; Ahamed, Syed Ibrahim Basheer; Arul, Venkatesan

2017-10-01

A gene coding lipase from Bacillus sp. PU1 was cloned and expressed in E. coli BL21(DE3) pLysS. The purified lipase has a molecular weight of 23kDa, is highly alkaline (pH range 8-10) and mesophilic (20-50°C). Three dimensional structure of the lipase was modeled by comparative homology and identified as a typical serine lipase by the presence of conserved Ser77, Asp133, His156. The molecular stability and behavior of the lipase was carried out using MD simulation studies at different pH and temperature was performed in comparison with biochemical analysis. Structural modifications of the lipase under these conditions were trapped by dihedral based FEL analysis and the functional loops (loop-H5/B4 and loop-H6/B5 of lipase) are identified which would cause the catalytic behavior of the lipase by high flexibility. Further characteristic feature of lipase are observed as follows; SDS completely inhibits the lipase activity and enzyme activity is enhanced with non-ionic surfactants. The lipase was highly stable in different organic solvents and also it could tolerate NaCl (0.4-0.8M). This enzyme was found to disrupt the biofilm of tested pathogenic bacterial strains. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Qiaozhen; Krug, Robert M.; Tao, Yizhi Jane

Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 Angstrom crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighboring molecule. Thismore » flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.« less

Genome Organization Drives Chromosome Fragility.

PubMed

Canela, Andres; Maman, Yaakov; Jung, Seolkyoung; Wong, Nancy; Callen, Elsa; Day, Amanda; Kieffer-Kwon, Kyong-Rim; Pekowska, Aleksandra; Zhang, Hongliang; Rao, Suhas S P; Huang, Su-Chen; Mckinnon, Peter J; Aplan, Peter D; Pommier, Yves; Aiden, Erez Lieberman; Casellas, Rafael; Nussenzweig, André

2017-07-27

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT. Published by Elsevier Inc.

Adenocarcinoma in situ of the uterine cervix--a systematic review.

PubMed

Baalbergen, Astrid; Helmerhorst, Theo J M

2014-11-01

This study aimed to review literature if therapeutic strategies in adenocarcinoma in situ of the cervix could lead to a more conservative approach. A review of the literature was conducted using a Medline search for articles published between 1966 and 2013. Thirty-five studies showed that after a radical cone, 16.5% residual disease in the re-cone or uterus was found. After cone with positive margins, residual abnormalities were found in 49.3%. Thirty-seven studies showed 5% recurrence rate after conservative therapy (large loop excision transformation zone-cold knife conization. After conization with negative margins, the risk of recurrence was 3%. Adenocarcinoma in situ is a relatively rare premalignant but increasingly frequent lesion of the cervix. Although there is a risk of relapse (3%) with a chance of malignancy (<1%), this risk is so small that conservative treatment with negative margins by large loop excision transformation zone or cold knife conization is justified and justifiable not only for women to have children.

Passivity-based control of linear time-invariant systems modelled by bond graph

NASA Astrophysics Data System (ADS)

Galindo, R.; Ngwompo, R. F.

2018-02-01

Closed-loop control systems are designed for linear time-invariant (LTI) controllable and observable systems modelled by bond graph (BG). Cascade and feedback interconnections of BG models are realised through active bonds with no loading effect. The use of active bonds may lead to non-conservation of energy and the overall system is modelled by proposed pseudo-junction structures. These structures are build by adding parasitic elements to the BG models and the overall system may become singularly perturbed. The structures for these interconnections can be seen as consisting of inner structures that satisfy energy conservation properties and outer structures including multiport-coupled dissipative fields. These fields highlight energy properties like passivity that are useful for control design. In both interconnections, junction structures and dissipative fields for the controllers are proposed, and passivity is guaranteed for the closed-loop systems assuring robust stability. The cascade interconnection is applied to the structural representation of closed-loop transfer functions, when a stabilising controller is applied to a given nominal plant. Applications are given when the plant and the controller are described by state-space realisations. The feedback interconnection is used getting necessary and sufficient stability conditions based on the closed-loop characteristic polynomial, solving a pole-placement problem and achieving zero-stationary state error.

Effects of ligand binding on the dynamics of rice nonspecific lipid transfer protein 1: a model from molecular simulations.

PubMed

Lai, Yen-Ting; Cheng, Chao-Sheng; Liu, Yu-Nan; Liu, Yaw-Jen; Lyu, Ping-Chiang

2008-09-01

Plant nonspecific lipid transfer proteins (nsLTPs) are small, basic proteins constituted mainly of alpha-helices and stabilized by four conserved disulfide bridges. They are characterized by the presence of a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers in vitro. In this study, molecular dynamics (MD) simulations were performed at room temperature to investigate the effects of lipid binding on the dynamic properties of rice nsLTP1. Rice nsLTP1, either in the free form or complexed with one or two lipids was subjected to MD simulations. The C-terminal loop was very flexible both before and after lipid binding, as revealed by calculating the root-mean-square fluctuation. After lipid binding, the flexibility of some residues that were not in direct contact with lipid molecules increased significantly, indicating an increase of entropy in the region distal from the binding site. Essential dynamics analysis revealed clear differences in motion between unliganded and liganded rice nsLTP1s. In the free form of rice nsLTP1, loop1 exhibited the largest directional motion. This specific essential motion mode diminished after binding one or two lipid molecules. To verify the origin of the essential motion observed in the free form of rice nsLTP1, we performed multiple sequence alignments to probe the intrinsic motion encoded in the primary sequence. We found that the amino acid sequence of loop1 is highly conserved among plant nsLTP1s, thus revealing its functional importance during evolution. Furthermore, the sequence of loop1 is composed mainly of amino acids with short side chains. In this study, we show that MD simulations, together with essential dynamics analysis, can be used to determine structural and dynamic differences of rice nsLTP1 upon lipid binding. 2008 Wiley-Liss, Inc.

Distinguishing West Nile virus infection using a recombinant envelope protein with mutations in the conserved fusion-loop.

PubMed

Chabierski, Stefan; Barzon, Luisa; Papa, Anna; Niedrig, Matthias; Bramson, Jonathan L; Richner, Justin M; Palù, Giorgio; Diamond, Michael S; Ulbert, Sebastian

2014-05-09

West Nile Virus (WNV) is an emerging mosquito-transmitted flavivirus that continues to spread and cause disease throughout several parts of the world, including Europe and the Americas. Specific diagnosis of WNV infections using current serological testing is complicated by the high degree of cross-reactivity between antibodies against other clinically relevant flaviviruses, including dengue, tick-borne encephalitis (TBEV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. Cross-reactivity is particularly problematic in areas where different flaviviruses co-circulate or in populations that have been immunized with vaccines against TBEV, JEV, or YFV. The majority of cross-reactive antibodies against the immunodominant flavivirus envelope (E) protein target a conserved epitope in the fusion loop at the distal end of domain II. We tested a loss-of-function bacterially expressed recombinant WNV E protein containing mutations in the fusion loop and an adjacent loop domain as a possible diagnostic reagent. By comparing the binding of sera from humans infected with WNV or other flaviviruses to the wild type and the mutant E proteins, we analyzed the potential of this technology to specifically detect WNV antibodies. Using this system, we could reliably determine WNV infections. Antibodies from WNV-infected individuals bound equally well to the wild type and the mutant protein. In contrast, sera from persons infected with other flaviviruses showed significantly decreased binding to the mutant protein. By calculating the mean differences between antibody signals detected using the wild type and the mutant proteins, a value could be assigned for each of the flaviviruses, which distinguished their pattern of reactivity. Recombinant mutant E proteins can be used to discriminate infections with WNV from those with other flaviviruses. The data have important implications for the development of improved, specific serological assays for the detection of WNV antibodies in regions where other flaviviruses co-circulate or in populations that are immunized with other flavivirus vaccines.

Characterization of a native hammerhead ribozyme derived from schistosomes

PubMed Central

OSBORNE, EDITH M.; SCHAAK, JANELL E.; DEROSE, VICTORIA J.

2005-01-01

A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop–loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop–loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop–loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of Rp-phosphorothioate substitutions at the cleavage site and 5′ to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5′ to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence. PMID:15659358

γδ T cell receptors recognize the non-classical major histocompatibility complex (MHC) molecule T22 via conserved anchor residues in a MHC peptide-like fashion.

PubMed

Sandstrom, Andrew; Scharf, Louise; McRae, Gabrielle; Hawk, Andrew J; Meredith, Stephen C; Adams, Erin J

2012-02-17

The molecular mechanisms by which γδ T cells recognize ligand remain a mystery. The non-classical MHC molecule T22 represents the best characterized ligand for murine γδ T cells, with a motif (W … EGYEL) present in the γδ T cell receptor complementary-determining region 3δ (CDR3δ) loop mediating γδ T cell recognition of this molecule. Produced through V(D)J recombination, this loop is quite diverse, with different numbers and chemical types of amino acids between Trp and EGYEL, which have unknown functional consequences for T22 recognition. We have investigated the biophysical and structural effects of CDR3δ loop diversity, revealing a range of affinities for T22 but a common thermodynamic pattern. Mutagenesis of these CDR3δ loops defines the key anchor residues involved in T22 recognition as W … EGYEL, similar to those found for the G8 CDR3δ loop, and demonstrates that spacer residues modulate but are not required for T22 recognition. Comparison of the location of these residues in the T22 interface reveals a striking similarity to peptide anchor residues in classically presented MHC peptides, with the key Trp residue of the CDR3δ motif completing the deficient peptide-binding groove of T22. This suggests that γδ T cell recognition of T22 utilizes the conserved ligand-presenting nature of the MHC fold.

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

PubMed

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

New understanding of nano-scale interstitial dislocation loops in BCC iron

NASA Astrophysics Data System (ADS)

Gao, N.; Chen, J.; Kurtz, R. J.; Wang, Z. G.; Zhang, R. F.; Gao, F.

2017-11-01

Complex states of nanoscale interstitial dislocation loop can be described by its habit plane and Burgers vector. Using atomistic simulations, we provide direct evidences on the change of the habit plane of a 1/2〈1 1 1〉 loop from {1 1 1} to {1 1 0} and {2 1 1}, in agreement with TEM observations. A new {1 0 0} habit plane of this loop is also predicted by simulations. The non-conservation of the Burgers vector is approved theoretically for: (1) dislocation reactions between loops with different Burgers vectors and (2) the transition between 〈1 0 0〉 loops and 1/2〈1 1 1〉 loops. The rotation from a 1/2〈1 1 1〉 to a 〈1 0 0〉 loop has also been explored, which occurs at 570 K for time on the order of 10 s. The dislocation-precipitate phase duality and change of habit plane are then proposed as new features for nano-scale dislocation loops.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Cannabis sativa.

PubMed

Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

2016-07-01

In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66°C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.

Alteration of the mode of antibacterial action of a defensin by the amino-terminal loop substitution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Bin; Zhu, Shunyi, E-mail: Zhusy@ioz.ac.cn

Highlights: Black-Right-Pointing-Pointer Al-M is an engineered fungal defensin with the n-loop of an insect defensin. Black-Right-Pointing-Pointer Al-M adopts a native defensin-like structure with high antibacterial potency. Black-Right-Pointing-Pointer Al-M kills bacteria through a membrane disruptive mechanism. Black-Right-Pointing-Pointer This work sheds light on the functional evolution of CS{alpha}{beta}-type defensins. -- Abstract: Ancient invertebrate-type and classical insect-type defensins (AITDs and CITDs) are two groups of evolutionarily related antimicrobial peptides (AMPs) that adopt a conserved cysteine-stabilized {alpha}-helical and {beta}-sheet (CS{alpha}{beta}) fold with a different amino-terminal loop (n-loop) size and diverse modes of antibacterial action. Although they both are identified as inhibitors of cell wallmore » biosynthesis, only CITDs evolved membrane disruptive ability by peptide oligomerization to form pores. To understand how this occurred, we modified micasin, a fungus-derived AITDs with a non-membrane disruptive mechanism, by substituting its n-loop with that of an insect-derived CITDs. After air oxidization, the synthetic hybrid defensin (termed Al-M) was structurally identified by circular dichroism (CD) and functionally evaluated by antibacterial and membrane permeability assays and electronic microscopic observation. Results showed that Al-M folded into a native-like defensin structure, as determined by its CD spectrum that is similar to that of micasin. Al-M was highly efficacious against the Gram-positive bacterium Bacillus megaterium with a lethal concentration of 1.76 {mu}M. As expected, in contrast to micasin, Al-M killed the bacteria through a membrane disruptive mechanism of action. The alteration in modes of action supports a key role of the n-loop extension in assembling functional surface of CITDs for membrane disruption. Our work provides mechanical evidence for evolutionary relationship between AITDs and CITDs.« less

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

PubMed Central

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

2011-01-01

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. PMID:21622960

Enthalpy-Based Thermal Evolution of Loops: III. Comparison of Zero-Dimensional Models

NASA Technical Reports Server (NTRS)

Cargill, P. J.; Bradshaw, Stephen J.; Klimchuk, James A.

2012-01-01

Zero dimensional (0D) hydrodynamic models, provide a simple and quick way to study the thermal evolution of coronal loops subjected to time-dependent heating. This paper presents a comparison of a number of 0D models that have been published in the past and is intended to provide a guide for those interested in either using the old models or developing new ones. The principal difference between the models is the way the exchange of mass and energy between corona, transition region and chromosphere is treated, as plasma cycles into and out of a loop during a heating-cooling cycle. It is shown that models based on the principles of mass and energy conservation can give satisfactory results at some, or, in the case of the Enthalpy Based Thermal Evolution of Loops (EBTEL) model, all stages of the loop evolution. Empirical models can lead to low coronal densities, spurious delays between the peak density and temperature, and, for short heating pulses, overly short loop lifetimes.

Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability.

PubMed

Bonnet, Amandine; Grosso, Ana R; Elkaoutari, Abdessamad; Coleno, Emeline; Presle, Adrien; Sridhara, Sreerama C; Janbon, Guilhem; Géli, Vincent; de Almeida, Sérgio F; Palancade, Benoit

2017-08-17

Transcription is a source of genetic instability that can notably result from the formation of genotoxic DNA:RNA hybrids, or R-loops, between the nascent mRNA and its template. Here we report an unexpected function for introns in counteracting R-loop accumulation in eukaryotic genomes. Deletion of endogenous introns increases R-loop formation, while insertion of an intron into an intronless gene suppresses R-loop accumulation and its deleterious impact on transcription and recombination in yeast. Recruitment of the spliceosome onto the mRNA, but not splicing per se, is shown to be critical to attenuate R-loop formation and transcription-associated genetic instability. Genome-wide analyses in a number of distant species differing in their intron content, including human, further revealed that intron-containing genes and the intron-richest genomes are best protected against R-loop accumulation and subsequent genetic instability. Our results thereby provide a possible rationale for the conservation of introns throughout the eukaryotic lineage. Copyright © 2017 Elsevier Inc. All rights reserved.

Method for spinning up a three-axis controlled spacecraft

NASA Technical Reports Server (NTRS)

Vorlicek, Preston L. (Inventor)

1988-01-01

A three-axis controlled spacecraft (1), typically a satellite, is spun up about its roll axis (20) prior to firing a motor (2), i.e., a perigee kick motor, to achieve the requisite degree of angular momentum stiffness. Thrusters (21) for imparting rotation about the roll axis (20) are activated in open-loop fashion, typically at less than full duty cycle. Cross-axis torques induced by this rotational motion are compensated for by means of closed control loops for each of the pitch and yaw axes (30, 40, respectively). Each closed control loop combines a prebias torque (72) with torques (75, 74) representative of position and rate feedback information, respectively. A deadband (52) within each closed control loop can be widened during the spinup, to conserve fuel. Position feedback information (75) in each of the control loops is disabled upon saturation of the gyroscope associated with the roll axis (20).

Numerical simulations of loops heated to solar flare temperatures. III - Asymmetrical heating

NASA Technical Reports Server (NTRS)

Cheng, C.-C.; Doschek, G. A.; Karpen, J. T.

1984-01-01

A numerical model is defined for asymmetric full solar flare loop heating and comparisons are made with observational data. The Dynamic Flux Tube Model is used to describe the heating process in terms of one-dimensional, two fluid conservation equations of mass, energy and momentum. An adaptive grid allows for the downward movement of the transition region caused by an advancing conduction front. A loop 20,000 km long is considered, along with a flare heating system and the hydrodynamic evolution of the loop. The model was applied to generating line profiles and spatial X-ray and UV line distributions, which were compared with SMM, P78-1 and Hintori data for Fe, Ca and Mg spectra. Little agreement was obtained, and it is suggested that flares be treated as multi-loop phenomena. Finally, it is concluded that chromospheric evaporation is not an effective mechanism for generating the soft X-ray bursts associated with flares.

A deeper analysis of the epitope/paratope of PLY-5, a mouse monoclonal antibody which recognises the conserved undecapeptide tryptophan-rich loop (ECTGLAWEWWR) of bacterial cholesterol-dependent cytolysins.

PubMed

González-Menéndez, Pedro; García-Ocaña, Marcos; de los Toyos, Juan R

2013-01-04

A previous study showed that the minimal epitope recognised by the PLY-5 mAb in the conserved undecapeptide Trp-rich loop of bacterial CDCs should consist of WEWWRT (Jacobs et al., 1999) [5]. Now, through immunoscreening of amino acid substitution analogues, it is concluded that the second Trp and the Arg residues are essential in the PLY-5 epitope. The E residue is an auxiliary epitope contributor. Antibody modelling and docking simulations provided support for these findings. For recognition by the antibody, the Trp-rich loop flipped out, mimicking the mechanism of membrane insertion. The displaced second Trp was seen to establish aromatic stacking interactions with aromatic residues of the antibody paratope and the notably extruded guanidium tip of the arginine residue mediated electrostatic interactions with well-exposed carboxylic groups of glutamic residues on the surface of the paratope. Thus, the epitope/paratope interaction is mainly mediated by aromatic and by ionic interactions. Copyright © 2012 Elsevier Inc. All rights reserved.

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals

PubMed Central

Girard, Frederic C.; Ottink, Otmar M.; Ampt, Kirsten A.M.; Tessari, Marco; Wijmenga, Sybren S.

2007-01-01

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5′-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P–epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35°C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL. PMID:17430968

Slanted snaking of localized Faraday waves

NASA Astrophysics Data System (ADS)

Pradenas, Bastián; Araya, Isidora; Clerc, Marcel G.; Falcón, Claudio; Gandhi, Punit; Knobloch, Edgar

2017-06-01

We report on an experimental, theoretical, and numerical study of slanted snaking of spatially localized parametrically excited waves on the surface of a water-surfactant mixture in a Hele-Shaw cell. We demonstrate experimentally the presence of a hysteretic transition to spatially extended parametrically excited surface waves when the acceleration amplitude is varied, as well as the presence of spatially localized waves exhibiting slanted snaking. The latter extend outside the hysteresis loop. We attribute this behavior to the presence of a conserved quantity, the liquid volume trapped within the meniscus, and introduce a universal model based on symmetry arguments, which couples the wave amplitude with such a conserved quantity. The model captures both the observed slanted snaking and the presence of localized waves outside the hysteresis loop, as demonstrated by numerical integration of the model equations.

Insights into the fold organization of TIM barrel from interaction energy based structure networks.

PubMed

Vijayabaskar, M S; Vishveshwara, Saraswathi

2012-01-01

There are many well-known examples of proteins with low sequence similarity, adopting the same structural fold. This aspect of sequence-structure relationship has been extensively studied both experimentally and theoretically, however with limited success. Most of the studies consider remote homology or "sequence conservation" as the basis for their understanding. Recently "interaction energy" based network formalism (Protein Energy Networks (PENs)) was developed to understand the determinants of protein structures. In this paper we have used these PENs to investigate the common non-covalent interactions and their collective features which stabilize the TIM barrel fold. We have also developed a method of aligning PENs in order to understand the spatial conservation of interactions in the fold. We have identified key common interactions responsible for the conservation of the TIM fold, despite high sequence dissimilarity. For instance, the central beta barrel of the TIM fold is stabilized by long-range high energy electrostatic interactions and low-energy contiguous vdW interactions in certain families. The other interfaces like the helix-sheet or the helix-helix seem to be devoid of any high energy conserved interactions. Conserved interactions in the loop regions around the catalytic site of the TIM fold have also been identified, pointing out their significance in both structural and functional evolution. Based on these investigations, we have developed a novel network based phylogenetic analysis for remote homologues, which can perform better than sequence based phylogeny. Such an analysis is more meaningful from both structural and functional evolutionary perspective. We believe that the information obtained through the "interaction conservation" viewpoint and the subsequently developed method of structure network alignment, can shed new light in the fields of fold organization and de novo computational protein design.

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase.

PubMed

La Sala, Giuseppina; Riccardi, Laura; Gaspari, Roberto; Cavalli, Andrea; Hantschel, Oliver; De Vivo, Marco

2016-11-08

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity. Another key factor for full Abl activity is the formation of active conformations of the catalytic DFG motif in the Abl kinase domain. Furthermore, binding of the SH2 domain to the N-lobe of the Abl kinase was recently demonstrated to have a long-range allosteric effect on the stabilization of the A-loop open state. Intriguingly, these distinct structural factors imply a complex signal transmission network for controlling the A-loop's flexibility and conformational preference for optimal Abl function. However, the exact dynamical features of this signal transmission network structure remain unclear. Here, we report on microsecond-long molecular dynamics coupled with enhanced sampling simulations of multiple Abl model systems, in the presence or absence of the SH2 domain and with the DFG motif flipped in two ways (in or out conformation). Through comparative analysis, our simulations augment the interpretation of the existing Abl experimental data, revealing a dynamical network of interactions that interconnect SH2 domain binding with A-loop plasticity and Y412 autophosphorylation in Abl. This signaling network engages the DFG motif and, importantly, other conserved structural elements of the kinase domain, namely, the EPK-ELK H-bond network and the HRD motif. Our results show that the signal propagation for modulating the A-loop spatial localization is highly dependent on the HRD motif conformation, which thus acts as the central hub of this (allosteric) signaling network controlling Abl activation and function.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de

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

Loop conformation and dynamics of the Escherichia coli HPPK apo-enzyme and its binary complex with MgATP.

PubMed

Yang, Rong; Lee, Matthew C; Yan, Honggao; Duan, Yong

2005-07-01

Comparison of the crystallographic and NMR structures of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) suggests that the enzyme may undergo significant conformational change upon binding to its first substrate, ATP. Two of the three surface loops (loop 2 and loop 3) accounting for most of the conformational differences appear to be confined by crystal contacts, raising questions about the putative large-scale induced-fit conformational change of HPPK and the functional roles of the conserved side-chain residues on the loops. To investigate the loop dynamics in crystal-free environment, we carried out molecular dynamics and locally enhanced sampling simulations of the apo-enzyme and the HPPK.MgATP complex. Our simulations showed that the crystallographic B-factors underestimated the loop dynamics considerably. We found that the open-conformation of loop 3 in the binary complex is accessible to the apo-enzyme and is the favored conformation in solution phase. These results revise our previous view of HPPK-substrate interactions and the associated functional mechanism of conformational change. The lessons learned here offer valuable structural insights into the workings of HPPK and should be useful for structure-based drug design.

A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

PubMed

Seal, R P; Amara, S G

1998-12-01

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.

A stretch of 11 amino acids in the betaB-betaC loop of the coat protein of grapevine fanleaf virus is essential for transmission by the nematode Xiphinema index.

PubMed

Schellenberger, Pascale; Andret-Link, Peggy; Schmitt-Keichinger, Corinne; Bergdoll, Marc; Marmonier, Aurélie; Vigne, Emmanuelle; Lemaire, Olivier; Fuchs, Marc; Demangeat, Gérard; Ritzenthaler, Christophe

2010-08-01

Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the betaB-betaC loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the betaE-alphaB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.

A Triple Mutant in the Ω-loop of TEM-1 β-Lactamase Changes the Substrate Profile via a Large Conformational Change and an Altered General Base for Catalysis*

PubMed Central

Stojanoski, Vlatko; Chow, Dar-Chone; Hu, Liya; Sankaran, Banumathi; Gilbert, Hiram F.; Prasad, B. V. Venkataram; Palzkill, Timothy

2015-01-01

β-Lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. TEM-1 is a prevalent plasmid-encoded β-lactamase in Gram-negative bacteria that efficiently catalyzes the hydrolysis of penicillins and early cephalosporins but not oxyimino-cephalosporins. A previous random mutagenesis study identified a W165Y/E166Y/P167G triple mutant that displays greatly altered substrate specificity with increased activity for the oxyimino-cephalosporin, ceftazidime, and decreased activity toward all other β-lactams tested. Surprisingly, this mutant lacks the conserved Glu-166 residue critical for enzyme function. Ceftazidime contains a large, bulky side chain that does not fit optimally in the wild-type TEM-1 active site. Therefore, it was hypothesized that the substitutions in the mutant expand the binding site in the enzyme. To investigate structural changes and address whether there is an enlargement in the active site, the crystal structure of the triple mutant was solved to 1.44 Å. The structure reveals a large conformational change of the active site Ω-loop structure to create additional space for the ceftazidime side chain. The position of the hydroxyl group of Tyr-166 and an observed shift in the pH profile of the triple mutant suggests that Tyr-166 participates in the hydrolytic mechanism of the enzyme. These findings indicate that the highly conserved Glu-166 residue can be substituted in the mechanism of serine β-lactamases. The results reveal that the robustness of the overall β-lactamase fold coupled with the plasticity of an active site loop facilitates the evolution of enzyme specificity and mechanism. PMID:25713062

Investigating the effect of key mutations on the conformational dynamics of toll-like receptor dimers through molecular dynamics simulations and protein structure networks.

PubMed

Mahita, Jarjapu; Sowdhamini, Ramanathan

2018-04-01

The Toll-like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen-associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain-containing proteins. Mutations in the BB loop of the Toll-like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lps d mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain-containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein-protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild-type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways. © 2018 Wiley Periodicals, Inc.

Secondary structural analyses of ITS1 in Paramecium.

PubMed

Hoshina, Ryo

2010-01-01

The nuclear ribosomal RNA gene operon is interrupted by internal transcribed spacer (ITS) 1 and ITS2. Although the secondary structure of ITS2 has been widely investigated, less is known about ITS1 and its structure. In this study, the secondary structure of ITS1 sequences for Paramecium and other ciliates was predicted. Each Paramecium ITS1 forms an open loop with three helices, A through C. Helix B was highly conserved among Paramecium, and similar helices were found in other ciliates. A phylogenetic analysis using the ITS1 sequences showed high-resolution, implying that ITS1 is a good tool for species-level analyses.

Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly.

PubMed

Gamalinda, Michael; Woolford, John L

2014-11-01

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function. © 2014 Gamalinda and Woolford; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates.

PubMed Central

Bell, J; Grass, S; Jeanteur, D; Munson, R S

1994-01-01

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein. PMID:8188390

Lysophospholipid presentation by CD1d and recognition by a human Natural Killer T-cell receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

López-Sagaseta, Jacinto; Sibener, Leah V.; Kung, Jennifer E.

2014-10-02

Invariant Natural Killer T (iNKT) cells use highly restricted {alpha}{beta} T cell receptors (TCRs) to probe the repertoire of lipids presented by CD1d molecules. Here, we describe our studies of lysophosphatidylcholine (LPC) presentation by human CD1d and its recognition by a native, LPC-specific iNKT TCR. Human CD1d presenting LPC adopts an altered conformation from that of CD1d presenting glycolipid antigens, with a shifted {alpha}1 helix resulting in an open A pocket. Binding of the iNKT TCR requires a 7-{angstrom} displacement of the LPC headgroup but stabilizes the CD1d-LPC complex in a closed conformation. The iNKT TCR CDR loop footprint onmore » CD1d-LPC is anchored by the conserved positioning of the CDR3{alpha} loop, whereas the remaining CDR loops are shifted, due in part to amino-acid differences in the CDR3{beta} and J{beta} segment used by this iNKT TCR. These findings provide insight into how lysophospholipids are presented by human CD1d molecules and how this complex is recognized by some, but not all, human iNKT cells.« less

Indicators for surgery in adhesive bowel obstruction.

PubMed

Rajanikmanth, P V; Kate, V; Ananthakrishnan, N

2001-01-01

There is lack of data on risk factors, which, if present, would indicate the need for surgery in patients with adhesive bowel obstruction. A Cohort of 100 consecutive patients with adhesive obstruction was studied prospectively to compare clinical and investigative parameters between the operative and conservative group. It was found that female gender, previous obstetric or gynaecological procedures, pulse and BP on admission, nature of nasogastric aspirate, single distended loop on abdominal x-ray as also predominant ileal distension were independent factors indicating a high probability of surgical intervention. Patients with 2 or more risk factors had 12 times higher probability of surgery and in those with 3 or more the relative risk was 30 times. Patients with such risk factors should be monitored closely after admission and should be taken for surgery after an initial short trial of conservative measures.

High genetic diversity and demographic history of captive Siamese and Saltwater crocodiles suggest the first step toward the establishment of a breeding and reintroduction program in Thailand

PubMed Central

Lapbenjakul, Sorravis; Thapana, Watcharaporn; Twilprawat, Panupon; Muangmai, Narongrit; Kanchanaketu, Thiti; Temsiripong, Yosapong; Unajak, Sasimanas; Peyachoknagul, Surin

2017-01-01

The Siamese crocodile (Crocodylus siamensis) and Saltwater crocodile (C. porosus) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924–0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578–0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (M ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between C. siamensis and C. porosus. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported their genetic integrity and the viability of a breeding and reintroduction management plan. This work constitutes the first step in establishing an appropriate source population from a scientifically managed perspective for an in situ/ex situ conservation program and reintroduction of crocodile individuals to the wild in Thailand. PMID:28953895

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

PubMed

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

SA-Mot: a web server for the identification of motifs of interest extracted from protein loops

PubMed Central

Regad, Leslie; Saladin, Adrien; Maupetit, Julien; Geneix, Colette; Camproux, Anne-Claude

2011-01-01

The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr. PMID:21665924

SA-Mot: a web server for the identification of motifs of interest extracted from protein loops.

PubMed

Regad, Leslie; Saladin, Adrien; Maupetit, Julien; Geneix, Colette; Camproux, Anne-Claude

2011-07-01

The detection of functional motifs is an important step for the determination of protein functions. We present here a new web server SA-Mot (Structural Alphabet Motif) for the extraction and location of structural motifs of interest from protein loops. Contrary to other methods, SA-Mot does not focus only on functional motifs, but it extracts recurrent and conserved structural motifs involved in structural redundancy of loops. SA-Mot uses the structural word notion to extract all structural motifs from uni-dimensional sequences corresponding to loop structures. Then, SA-Mot provides a description of these structural motifs using statistics computed in the loop data set and in SCOP superfamily, sequence and structural parameters. SA-Mot results correspond to an interactive table listing all structural motifs extracted from a target structure and their associated descriptors. Using this information, the users can easily locate loop regions that are important for the protein folding and function. The SA-Mot web server is available at http://sa-mot.mti.univ-paris-diderot.fr.

Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

PubMed

Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

2014-01-01

Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes

PubMed Central

Tiwari, Sandhya P.; Reuter, Nathalie

2016-01-01

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 β-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the β-barrel core is highly rigid, while the α-helices that flank the β-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design. PMID:27015412

Genetic analysis of the Hungarian draft horse population using partial mitochondrial DNA D-loop sequencing

PubMed Central

2018-01-01

Background The Hungarian draft is a horse breed with a recent mixed ancestry created in the 1920s by crossing local mares with draught horses imported from France and Belgium. The interest in its conservation and characterization has increased over the last few years. The aim of this work is to contribute to the characterization of the endangered Hungarian heavy draft horse populations in order to obtain useful information to implement conservation strategies for these genetic stocks. Methods To genetically characterize the breed and to set up the basis for a conservation program, in the present study a hypervariable region of the mitochrondial DNA (D-loop) was used to assess genetic diversity in Hungarian draft horses. Two hundred and eighty five sequences obtained in our laboratory and 419 downloaded sequences available from Genbank were analyzed. Results One hundred and sixty-four haplotypes and thirty-six polymorphic sites were observed. High haplotype and nucleotide diversity values (Hd = 0.954 ± 0.004; π = 0.028 ± 0.0004) were identified in Hungarian population, although they were higher within than among the different populations (Hd = 0.972 ± 0.002; π = 0.03097 ± 0.002). Fourteen of the previously observed seventeen haplogroups were detected. Discussion Our samples showed a large intra- and interbreed variation. There was no clear clustering on the median joining network figure. The overall information collected in this work led us to consider that the genetic scenario observed for Hungarian draft breed is more likely the result of contributions from ‘ancestrally’ different genetic backgrounds. This study could contribute to the development of a breeding plan for Hungarian draft horses and help to formulate a genetic conservation plan, avoiding inbreeding while. PMID:29404201

A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla

2012-04-18

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change ofmore » a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.« less

Multiple maternal origins of Indonesian crowing chickens revealed by mitochondrial DNA analysis.

PubMed

Ulfah, Maria; Perwitasari, Dyah; Jakaria, Jakaria; Muladno, Muhammad; Farajallah, Achmad

2017-03-01

The utilization of Indonesian crowing chickens is increasing; as such, assessing their genetic structures is important to support the conservation of their genetic resources. This study analyzes the matrilineal evolution of Indonesian crowing chickens based on the mtDNA displacement loop D-loop region to clarify their phylogenetic relationships, possible maternal origin, and possible routes of chicken dispersal. The neighbor-joining tree reveals that the majority of Indonesian crowing chickens belong to haplogroups B, D, and E, but haplogroup D harbored most of them. The Bayesian analysis also reveals that Indonesian crowing chickens derive from Bekisar chicken, a hybrid of the green junglefowl, suggesting the possible contribution of green junglefowl to chicken domestication. There appear at least three maternal lineages of Indonesian chicken origins indicated by the median network profile of mtDNA D-loop haplotypes, namely (1) Chinese; (2) Chinese, Indian, and other Southeast Asian chickens; and (3) Indian, Chinese, Southeast Asian, Japanese, and European chickens. Chicken domestication might be centered in China, India, Indonesia, and other Southeast Asian countries, supporting multiple maternal origins of Indonesian crowing chickens. A systematic breeding program of indigenous chickens will be very important to retain the genetic diversity for future use and conservation.

ATM Substrate Chk2-interacting Zn2+ Finger (ASCIZ) Is a Bi-functional Transcriptional Activator and Feedback Sensor in the Regulation of Dynein Light Chain (DYNLL1) Expression*

PubMed Central

Jurado, Sabine; Conlan, Lindus A.; Baker, Emma K.; Ng, Jane-Lee; Tenis, Nora; Hoch, Nicolas C.; Gleeson, Kimberly; Smeets, Monique; Izon, David; Heierhorst, Jörg

2012-01-01

The highly conserved DYNLL1 (LC8) protein was originally discovered as a light chain of the dynein motor complex, but is increasingly emerging as a sequence-specific regulator of protein dimerization with hundreds of targets and wide-ranging cellular functions. Despite its important roles, DYNLL1's own regulation remains poorly understood. Here we identify ASCIZ (ATMIN/ZNF822), an essential Zn2+ finger protein with dual roles in the DNA base damage response and as a developmental transcription factor, as a conserved regulator of Dynll1 gene expression. DYNLL1 levels are reduced by ∼10-fold in the absence of ASCIZ in human, mouse and chicken cells. ASCIZ binds directly to the Dynll1 promoter and regulates its activity in a Zn2+ finger-dependent manner. DYNLL1 protein in turn interacts with ten binding sites in the ASCIZ transcription activation domain, and high DYNLL1 levels inhibit the transcriptional activity of ASCIZ. In addition, DYNLL1 was also required for DNA damage-induced ASCIZ focus formation. The dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. The ASCIZ-DYNLL1 feedback loop represents a novel mechanism for auto-regulation of gene expression, where the gene product directly inhibits the transcriptional activator while bound at its own promoter. PMID:22167198

New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases

PubMed Central

Tu, Tao; Meng, Kun; Luo, Huiying; Turunen, Ossi; Zhang, Lujia; Cheng, Yanli; Su, Xiaoyun; Ma, Rui; Shi, Pengjun; Wang, Yaru; Yang, Peilong; Yao, Bin

2015-01-01

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered K m values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered k cat (~3–20,000 fold) and k cat/K m (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues. PMID:26327390

Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

PubMed Central

Schibler, Manuel; Martinez, Yannick; Gerlach, Daniel; van Belle, Sandra; Turin, Lara; Zdobnov, Evgeny; Kaiser, Laurent; Tapparel, Caroline

2012-01-01

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient. PMID:22910880

An investigation of the impact of Melbourne Zoo's "Seal-the-Loop" donate call-to-action on visitor satisfaction and behavior.

PubMed

Mellish, Sarah; Sanders, Ben; Litchfield, Carla A; Pearson, Elissa L

2017-05-01

Modern zoos are uniquely positioned to educate the public about environmental issues and promote conservation action. This report investigates the introduction of a donation request during an interactive fur seal presentation (as part of Melbourne Zoo's "Seal-the-Loop" initiative) on visitor satisfaction, perceptions of donation as a way to help wild fur seals, and donation behaviors. Comparisons are made between three groups surveyed upon exit: (1) viewed the interactive fur seal presentation prior to the donation request implementation (pledge-presentation: N = 86; see Mellish, Pearson, Sanders, and Litchfield []; International Zoo Yearbook 129:129-154); (2) viewed the interactive fur seal presentation including the donation request (donate-presentation: N = 82); and (3) viewed the fur seal exhibit and donation point but not the presentation and were not directly asked to make a donation (donate-exhibit: N = 82). Findings demonstrate visitor satisfaction with the interactive fur seal presentation was not negatively impacted following the implementation of the donate request (with >92% of pledge-presentation and donate-presentation visitors providing a "satisfied" or "very satisfied" rating). Only the donate-presentation visitors reported donation as a conservation action to help wild fur seals (19.18%; 0% for pledge-presentation visitors). While both donate-exhibit (39.51%) and donate-presentation visitors (60.75%) self-reported making donations or intending to do so, donation behavior was significantly increased for visitors who had viewed the fur seal presentation. Findings provide preliminary support that zoos may utilize interactive educational presentations to effectively ask visitors for donations to support specific conservation projects, without negatively impacting on satisfaction and with a relatively high level of visitor engagement. © 2017 Wiley Periodicals, Inc.

Origin and Diversification of Basic-Helix-Loop-Helix Proteins in Plants

PubMed Central

Pires, Nuno; Dolan, Liam

2010-01-01

Basic helix-loop-helix (bHLH) proteins are a class of transcription factors found throughout eukaryotic organisms. Classification of the complete sets of bHLH proteins in the sequenced genomes of Arabidopsis thaliana and Oryza sativa (rice) has defined the diversity of these proteins among flowering plants. However, the evolutionary relationships of different plant bHLH groups and the diversity of bHLH proteins in more ancestral groups of plants are currently unknown. In this study, we use whole-genome sequences from nine species of land plants and algae to define the relationships between these proteins in plants. We show that few (less than 5) bHLH proteins are encoded in the genomes of chlorophytes and red algae. In contrast, many bHLH proteins (100–170) are encoded in the genomes of land plants (embryophytes). Phylogenetic analyses suggest that plant bHLH proteins are monophyletic and constitute 26 subfamilies. Twenty of these subfamilies existed in the common ancestors of extant mosses and vascular plants, whereas six further subfamilies evolved among the vascular plants. In addition to the conserved bHLH domains, most subfamilies are characterized by the presence of highly conserved short amino acid motifs. We conclude that much of the diversity of plant bHLH proteins was established in early land plants, over 440 million years ago. PMID:19942615

Highly conserved sequences mediate the dynamic interplay of basic helix-loop-helix proteins regulating retinogenesis.

PubMed

Hernandez, Julio; Matter-Sadzinski, Lidia; Skowronska-Krawczyk, Dorota; Chiodini, Florence; Alliod, Christine; Ballivet, Marc; Matter, Jean-Marc

2007-12-28

The atonal homolog 5 (ATH5) protein is central to the transcriptional network regulating the specification of retinal ganglion cells, and its expression comes under the spatiotemporal control of several basic helix-loop-helix (bHLH) proteins in the course of retina development. Monitoring the in vivo occupancy of the ATH5 promoter by the ATH5, Ngn2, and NeuroM proteins and analyzing the DNA motifs they bind, we show that three evolutionarily conserved E-boxes are required for the bHLH proteins to control the different phases of ATH5 expression. E-box 4 mediates the activity of Ngn2, ATH5, and NeuroM along the pathway leading to the conversion of progenitors into newborn neurons. E-box 1, by mediating the antagonistic effects of Ngn2 and HES1 in proliferating progenitors, controls the expansion of the ATH5 expression domain in early retina. E-box 2 is required for the positive feedback by ATH5 that underlies the up-regulation of ATH5 expression when progenitors are going through their last cell cycle. The combinatorial nature of the regulation of the ATH5 promoter suggests that the bHLH proteins involved have no assigned E-boxes but use a common set at which they either cooperate or compete to finely tune ATH5 expression as development proceeds.

Mutagenesis Studies of the H5 Influenza Hemagglutinin Stem Loop Region*

PubMed Central

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L.; Mishra, Rama K.; Rong, Lijun; Caffrey, Michael

2014-01-01

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. PMID:24947513

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

PubMed

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L; Mishra, Rama K; Rong, Lijun; Caffrey, Michael

2014-08-08

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cloning, expression and phylogenetic analysis of Hemolin, from the Chinese oak silkmoth, Antheraea pernyi.

PubMed

Li, Wenli; Terenius, Olle; Hirai, Makoto; Nilsson, Anders S; Faye, Ingrid

2005-01-01

The Chinese oak silk moth Antheraea pernyi is an important silk producer. To understand microbial resistance of this moth, we cloned Hemolin, encoding a multifunctional immune protein belonging to the immunoglobulin superfamily, and examined the expression in gonads and fat body. The ApHemolin amino acid sequence was compared to other Hemolin sequences in order to predict functional sites. Several sites were conserved; among them a phosphate binding site, which according to 3D structure modelling does not appear in neuroglian, the phylogenetically closest related protein. In addition, two conserved KDG sequences in the C-C' loop of immunoglobulin domains 1 and 3, give rise to gamma-turns, which is a common motif in the C'-C'' loop of the hypervariable region L2 in vertebrate immunoglobulins. The comparisons also show variable regions of specific interest for future studies of hemolin and its interaction with microbial entities.

Studying citizen science through adaptive management and learning feedbacks as mechanisms for improving conservation.

PubMed

Jordan, Rebecca; Gray, Steven; Sorensen, Amanda; Newman, Greg; Mellor, David; Newman, Greg; Hmelo-Silver, Cindy; LaDeau, Shannon; Biehler, Dawn; Crall, Alycia

2016-06-01

Citizen science has generated a growing interest among scientists and community groups, and citizen science programs have been created specifically for conservation. We examined collaborative science, a highly interactive form of citizen science, which we developed within a theoretically informed framework. In this essay, we focused on 2 aspects of our framework: social learning and adaptive management. Social learning, in contrast to individual-based learning, stresses collaborative and generative insight making and is well-suited for adaptive management. Adaptive-management integrates feedback loops that are informed by what is learned and is guided by iterative decision making. Participants engaged in citizen science are able to add to what they are learning through primary data collection, which can result in the real-time information that is often necessary for conservation. Our work is particularly timely because research publications consistently report a lack of established frameworks and evaluation plans to address the extent of conservation outcomes in citizen science. To illustrate how our framework supports conservation through citizen science, we examined how 2 programs enacted our collaborative science framework. Further, we inspected preliminary conservation outcomes of our case-study programs. These programs, despite their recent implementation, are demonstrating promise with regard to positive conservation outcomes. To date, they are independently earning funds to support research, earning buy-in from local partners to engage in experimentation, and, in the absence of leading scientists, are collecting data to test ideas. We argue that this success is due to citizen scientists being organized around local issues and engaging in iterative, collaborative, and adaptive learning. © 2016 Society for Conservation Biology.

Model-Driven Safety Analysis of Closed-Loop Medical Systems

PubMed Central

Pajic, Miroslav; Mangharam, Rahul; Sokolsky, Oleg; Arney, David; Goldman, Julian; Lee, Insup

2013-01-01

In modern hospitals, patients are treated using a wide array of medical devices that are increasingly interacting with each other over the network, thus offering a perfect example of a cyber-physical system. We study the safety of a medical device system for the physiologic closed-loop control of drug infusion. The main contribution of the paper is the verification approach for the safety properties of closed-loop medical device systems. We demonstrate, using a case study, that the approach can be applied to a system of clinical importance. Our method combines simulation-based analysis of a detailed model of the system that contains continuous patient dynamics with model checking of a more abstract timed automata model. We show that the relationship between the two models preserves the crucial aspect of the timing behavior that ensures the conservativeness of the safety analysis. We also describe system design that can provide open-loop safety under network failure. PMID:24177176

Model-Driven Safety Analysis of Closed-Loop Medical Systems.

PubMed

Pajic, Miroslav; Mangharam, Rahul; Sokolsky, Oleg; Arney, David; Goldman, Julian; Lee, Insup

2012-10-26

In modern hospitals, patients are treated using a wide array of medical devices that are increasingly interacting with each other over the network, thus offering a perfect example of a cyber-physical system. We study the safety of a medical device system for the physiologic closed-loop control of drug infusion. The main contribution of the paper is the verification approach for the safety properties of closed-loop medical device systems. We demonstrate, using a case study, that the approach can be applied to a system of clinical importance. Our method combines simulation-based analysis of a detailed model of the system that contains continuous patient dynamics with model checking of a more abstract timed automata model. We show that the relationship between the two models preserves the crucial aspect of the timing behavior that ensures the conservativeness of the safety analysis. We also describe system design that can provide open-loop safety under network failure.

Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Critton; L Tautz; R Page

2011-12-31

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loopmore » in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.« less

Molecular genetic analysis of circadian timekeeping in Drosophila

PubMed Central

Hardin, Paul E.

2014-01-01

A genetic screen for mutants that alter circadian rhythms in Drosophila identified the first clock gene - the period (per) gene. The per gene is a central player within a transcriptional feedback loop that represents the core mechanism for keeping circadian time in Drosophila and other animals. The per feedback loop, or core loop, is interlocked with the Clock (Clk) feedback loop, but whether the Clk feedback loop contributes to circadian timekeeping is not known. A series of distinct molecular events are thought to control transcriptional feedback in the core loop. The time it takes to complete these events should take much less than 24h, thus delays must be imposed at different steps within the core loop. As new clock genes are identified, the molecular mechanisms responsible for these delays have been revealed in ever-increasing detail, and provide an in depth accounting of how transcriptional feedback loops keep circadian time. The phase of these feedback loops shift to maintain synchrony with environmental cycles, the most reliable of which is light. Although a great deal is known about cell-autonomous mechanisms of light-induced phase shifting by CRYPTOCHROME (CRY), much less is known about non-cell autonomous mechanisms. CRY mediates phase shifts through an uncharacterized mechanism in certain brain oscillator neurons, and carries out a dual role as a photoreceptor and transcription factor in other tissues. Here I will review how transcriptional feedback loops function to keep time in Drosophila, how they impose delays to maintain a 24h cycle, and how they maintain synchrony with environmental light:dark cycles. The transcriptional feedback loops that keep time in Drosophila are well conserved in other animals, thus what we learn about these loops in Drosophila should continue to provide insight into the operation of analogous transcriptional feedback loops in other animals. PMID:21924977

Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

PubMed

Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

1998-02-01

RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

Scattering of glue by glue on the light-cone worldsheet. II. Helicity conserving amplitudes

NASA Astrophysics Data System (ADS)

Chakrabarti, D.; Qiu, J.; Thorn, C. B.

2006-08-01

This is the second of a pair of articles on scattering of glue by glue, in which we give the light-cone gauge calculation of the one-loop on-shell helicity conserving scattering amplitudes for gluon-gluon scattering (neglecting quark loops). The 1/p+ factors in the gluon propagator are regulated by replacing p+ integrals with discretized sums omitting the p+=0 terms in each sum. We also employ a novel ultraviolet regulator that is convenient for the light-cone worldsheet description of planar Feynman diagrams. The helicity conserving scattering amplitudes are divergent in the infrared. The infrared divergences in the elastic one-loop amplitude are shown to cancel, in their contribution to cross sections, against ones in the cross section for unseen bremsstrahlung gluons. We include here the explicit calculation of the latter, because it assumes an unfamiliar form due to the peculiar way discretization of p+ regulates infrared divergences. In resolving the infrared divergences we employ a covariant definition of jets, which allows a transparent demonstration of the Lorentz invariance of our final results. Because we use an explicit cutoff of the ultraviolet divergences in exactly four spacetime dimensions, we must introduce explicit counterterms to achieve this final covariant result. These counterterms are polynomials in the external momenta of the precise order dictated by power counting. We discuss the modifications they entail for the light-cone worldsheet action that reproduces the bare planar diagrams of the gluonic sector of QCD. The simplest way to do this is to interpret the QCD string as moving in six spacetime dimensions.

The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD

NASA Astrophysics Data System (ADS)

Calmettes, Charles; Ing, Christopher; Buckwalter, Carolyn M.; El Bakkouri, Majida; Chieh-Lin Lai, Christine; Pogoutse, Anastassia; Gray-Owen, Scott D.; Pomès, Régis; Moraes, Trevor F.

2015-08-01

Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Gohda, Keigo; Hakoshima, Toshio

2008-11-01

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

LOOP- SIMULATION OF THE AUTOMATIC FREQUENCY CONTROL SUBSYSTEM OF A DIFFERENTIAL MINIMUM SHIFT KEYING RECEIVER

NASA Technical Reports Server (NTRS)

Davarian, F.

1994-01-01

The LOOP computer program was written to simulate the Automatic Frequency Control (AFC) subsystem of a Differential Minimum Shift Keying (DMSK) receiver with a bit rate of 2400 baud. The AFC simulated by LOOP is a first order loop configuration with a first order R-C filter. NASA has been investigating the concept of mobile communications based on low-cost, low-power terminals linked via geostationary satellites. Studies have indicated that low bit rate transmission is suitable for this application, particularly from the frequency and power conservation point of view. A bit rate of 2400 BPS is attractive due to its applicability to the linear predictive coding of speech. Input to LOOP includes the following: 1) the initial frequency error; 2) the double-sided loop noise bandwidth; 3) the filter time constants; 4) the amount of intersymbol interference; and 5) the bit energy to noise spectral density. LOOP output includes: 1) the bit number and the frequency error of that bit; 2) the computed mean of the frequency error; and 3) the standard deviation of the frequency error. LOOP is written in MS SuperSoft FORTRAN 77 for interactive execution and has been implemented on an IBM PC operating under PC DOS with a memory requirement of approximately 40K of 8 bit bytes. This program was developed in 1986.

Cross-Cohort Analysis Identifies a TEAD4-MYCN Positive Feedback Loop as the Core Regulatory Element of High-Risk Neuroblastoma.

PubMed

Rajbhandari, Presha; Lopez, Gonzalo; Capdevila, Claudia; Salvatori, Beatrice; Yu, Jiyang; Rodriguez-Barrueco, Ruth; Martinez, Daniel; Yarmarkovich, Mark; Weichert-Leahey, Nina; Abraham, Brian J; Alvarez, Mariano J; Iyer, Archana; Harenza, Jo Lynne; Oldridge, Derek; De Preter, Katleen; Koster, Jan; Asgharzadeh, Shahab; Seeger, Robert C; Wei, Jun S; Khan, Javed; Vandesompele, Jo; Mestdagh, Pieter; Versteeg, Rogier; Look, A Thomas; Young, Richard A; Iavarone, Antonio; Lasorella, Anna; Silva, Jose M; Maris, John M; Califano, Andrea

2018-05-01

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN -amplified ( MYCN Amp ) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCN Amp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 517 . ©2018 American Association for Cancer Research.

Phylogenetic Analysis and Classification of the Fungal bHLH Domain

PubMed Central

Sailsbery, Joshua K.; Atchley, William R.; Dean, Ralph A.

2012-01-01

The basic Helix-Loop-Helix (bHLH) domain is an essential highly conserved DNA-binding domain found in many transcription factors in all eukaryotic organisms. The bHLH domain has been well studied in the Animal and Plant Kingdoms but has yet to be characterized within Fungi. Herein, we obtained and evaluated the phylogenetic relationship of 490 fungal-specific bHLH containing proteins from 55 whole genome projects composed of 49 Ascomycota and 6 Basidiomycota organisms. We identified 12 major groupings within Fungi (F1–F12); identifying conserved motifs and functions specific to each group. Several classification models were built to distinguish the 12 groups and elucidate the most discerning sites in the domain. Performance testing on these models, for correct group classification, resulted in a maximum sensitivity and specificity of 98.5% and 99.8%, respectively. We identified 12 highly discerning sites and incorporated those into a set of rules (simplified model) to classify sequences into the correct group. Conservation of amino acid sites and phylogenetic analyses established that like plant bHLH proteins, fungal bHLH–containing proteins are most closely related to animal Group B. The models used in these analyses were incorporated into a software package, the source code for which is available at www.fungalgenomics.ncsu.edu. PMID:22114358

Osmoregulation Requires Brain Expression of the Renal Na-K-2Cl Cotransporter NKCC2

PubMed Central

Konopacka, Agnieszka; Qiu, Jing; Yao, Song T.; Greenwood, Michael P.; Greenwood, Mingkwan; Lancaster, Thomas; Inoue, Wataru; de Souza Mecawi, Andre; Vechiato, Fernanda M.V.; de Lima, Juliana B.M.; Coletti, Ricardo; Hoe, See Ziau; Martin, Andrew; Lee, Justina; Joseph, Marina; Hindmarch, Charles; Paton, Julian; Antunes-Rodrigues, Jose; Bains, Jaideep

2015-01-01

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution—rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats. PMID:25834041

The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis[W

PubMed Central

Jégu, Teddy; Latrasse, David; Delarue, Marianne; Hirt, Heribert; Domenichini, Séverine; Ariel, Federico; Crespi, Martin; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2014-01-01

SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus. PMID:24510722

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

PubMed

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-05-02

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Low-energy effective field theory below the electroweak scale: operators and matching

NASA Astrophysics Data System (ADS)

Jenkins, Elizabeth E.; Manohar, Aneesh V.; Stoffer, Peter

2018-03-01

The gauge-invariant operators up to dimension six in the low-energy effective field theory below the electroweak scale are classified. There are 70 Hermitian dimension-five and 3631 Hermitian dimension-six operators that conserve baryon and lepton number, as well as Δ B = ±Δ L = ±1, Δ L = ±2, and Δ L = ±4 operators. The matching onto these operators from the Standard Model Effective Field Theory (SMEFT) up to order 1 /Λ2 is computed at tree level. SMEFT imposes constraints on the coefficients of the low-energy effective theory, which can be checked experimentally to determine whether the electroweak gauge symmetry is broken by a single fundamental scalar doublet as in SMEFT. Our results, when combined with the one-loop anomalous dimensions of the low-energy theory and the one-loop anomalous dimensions of SMEFT, allow one to compute the low-energy implications of new physics to leading-log accuracy, and combine them consistently with high-energy LHC constraints.

Development of a Novel Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Rickettsia spp.

PubMed

Hanaoka, Nozomu; Matsutani, Minenosuke; Satoh, Masaaki; Ogawa, Motohiko; Shirai, Mutsunori; Ando, Shuji

2017-01-24

We developed a novel loop-mediated isothermal amplification (LAMP) method to detect Rickettsia spp., including Rickettsia prowazekii and R. typhi. Species-specific LAMP primers were developed for orthologous genes conserved among Rickettsia spp. The selected modified primers could detect all the Rickettsia spp. tested. The LAMP method was successfully used to detect 100 DNA copies of Rickettsia spp. within approximately 60 min at 63℃. Therefore, this method may be an excellent tool for the early diagnosis of rickettsiosis in a laboratory or in the field.

Digital Baseband Architecture For Transponder

NASA Technical Reports Server (NTRS)

Nguyen, Tien M.; Yeh, Hen-Geul

1995-01-01

Proposed advanced transponder for long-distance radio communication system with turnaround ranging contains carrier-signal-tracking loop including baseband digital "front end." For reduced cost, transponder includes analog intermediate-frequency (IF) section and analog automatic gain control (AGC) loop at first of two IF mixers. However, second IF mixer redesigned to ease digitization of baseband functions. To conserve power and provide for simpler and smaller transponder hardware, baseband digital signal-processing circuits designed to implement undersampling scheme. Furthermore, sampling scheme and sampling frequency chosen so redesign involves minimum modification of command-detector unit (CDU).

Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meshcheryakov, Vladimir A.; Kitao, Akio; Core Research for Evolutionary Science and Technology, Tokyo 113-0032

2013-05-01

Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, themore » crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.« less

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed Central

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-01-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding. PMID:11350033

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

PubMed Central

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-01-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family.

PubMed

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C; Dénervaud-Tendon, Valérie; Vermeer, Joop E M; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-08-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. © 2014 American Society of Plant Biologists. All Rights Reserved.

Targeting p53-MDM2-MDMX Loop for Cancer Therapy

PubMed Central

Zhang, Qi; Zeng, Shelya X.

2015-01-01

The tumor suppressor p53 plays a central role in anti-tumorigenesis and cancer therapy. It has been described as “the guardian of the genome”, because it is essential for conserving genomic stability by preventing mutation, and its mutation and inactivation are highly related to all human cancers. Two important p53 regulators, MDM2 and MDMX, inactivate p53 by directly inhibiting its transcriptional activity and mediating its ubiquitination in a feedback fashion, as their genes are also the transcriptional targets of p53. On account of the importance of the p53-MDM2- MDMX loop in the initiation and development of wild type p53-containing tumors, intensive studies over the past decade have been aiming to identify small molecules or peptides that could specifically target individual protein molecules of this pathway for developing better anti-cancer therapeutics. In this chapter, we review the approaches for screening and discovering efficient and selective MDM2 inhibitors with emphasis on the most advanced synthetic small molecules that interfere with the p53-MDM2 interaction and are currently on Phase I clinical trials. Other therapeutically useful strategies targeting this loop, which potentially improve the prospects of cancer therapy and prevention, will also be discussed briefly. PMID:25201201

Decentralized Control of Sound Radiation from an Aircraft-Style Panel Using Iterative Loop Recovery

NASA Technical Reports Server (NTRS)

Schiller, Noah H.; Cabell, Randolph H.; Fuller, Chris R.

2008-01-01

A decentralized LQG-based control strategy is designed to reduce low-frequency sound transmission through periodically stiffened panels. While modern control strategies have been used to reduce sound radiation from relatively simple structural acoustic systems, significant implementation issues have to be addressed before these control strategies can be extended to large systems such as the fuselage of an aircraft. For instance, centralized approaches typically require a high level of connectivity and are computationally intensive, while decentralized strategies face stability problems caused by the unmodeled interaction between neighboring control units. Since accurate uncertainty bounds are not known a priori, it is difficult to ensure the decentralized control system will be robust without making the controller overly conservative. Therefore an iterative approach is suggested, which utilizes frequency-shaped loop recovery. The approach accounts for modeling error introduced by neighboring control loops, requires no communication between subsystems, and is relatively simple. The control strategy is validated using real-time control experiments performed on a built-up aluminum test structure representative of the fuselage of an aircraft. Experiments demonstrate that the iterative approach is capable of achieving 12 dB peak reductions and a 3.6 dB integrated reduction in radiated sound power from the stiffened panel.

Structural re-alignment in an immunologic surface region of ricin A chain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zemla, A T; Zhou, C E

2007-07-24

We compared structure alignments generated by several protein structure comparison programs to determine whether existing methods would satisfactorily align residues at a highly conserved position within an immunogenic loop in ribosome inactivating proteins (RIPs). Using default settings, structure alignments generated by several programs (CE, DaliLite, FATCAT, LGA, MAMMOTH, MATRAS, SHEBA, SSM) failed to align the respective conserved residues, although LGA reported correct residue-residue (R-R) correspondences when the beta-carbon (Cb) position was used as the point of reference in the alignment calculations. Further tests using variable points of reference indicated that points distal from the beta carbon along a vector connectingmore » the alpha and beta carbons yielded rigid structural alignments in which residues known to be highly conserved in RIPs were reported as corresponding residues in structural comparisons between ricin A chain, abrin-A, and other RIPs. Results suggest that approaches to structure alignment employing alternate point representations corresponding to side chain position may yield structure alignments that are more consistent with observed conservation of functional surface residues than do standard alignment programs, which apply uniform criteria for alignment (i.e., alpha carbon (Ca) as point of reference) along the entirety of the peptide chain. We present the results of tests that suggest the utility of allowing user-specified points of reference in generating alternate structural alignments, and we present a web server for automatically generating such alignments.« less

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase*

PubMed Central

Chen, Yaozong; Sun, Yueru; Song, Haigang; Guo, Zhihong

2015-01-01

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes, including acyl/aryl-CoA synthetases, adenylation domains of nonribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes. PMID:26276389

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.

PubMed

Shen, Xin-Ming; Brengman, Joan; Neubauer, David; Sine, Steven M; Engel, Andrew G

2016-02-12

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Conservative Treatment Seems the Best Choice in Adenocarcinoma In Situ of the Cervix Uteri.

PubMed

Baalbergen, Astrid; Molijn, Anco C; Quint, Wim G V; Smedts, Frank; Helmerhorst, Theo J M

2015-07-01

To study diagnostic and therapeutic strategies, outcomes, and follow-up in a large series of women with adenocarcinoma in situ (AIS) of the uterine cervix and investigate if human papillomavirus (HPV) typing among women with negative cytology reports would have helped with early AIS detection. Records of 132 AIS cases diagnosed between 1989 and 2012 were retrieved. Clinical and pathological data were reviewed and analyzed. Mean age at diagnosis was 37 years. Seventy-two percent (n = 95) of all patients were asymptomatic; diagnosis was established using cytology and biopsy. Primary treatment for 124 patents was cold knife cone or loop electrosurgical excision procedure (LEEP). Positive margins were found in 18% of those women treated with CKC versus 40% in those treated with LEEP. The mean follow-up time was 62 months (range, 2-217 months; median, 46 months). Three recurrences were found after conservative treatment in 86 patients. High-risk HPV (hrHPV) positivity was detected in 115 (96%) of 120 patients, with HPV-18 being the most commonly occurring subtype (51%). There is a small risk of relapse after conservative therapy with cold knife cone or LEEP when resection margins are negative in women with AIS. Patients should be given the options of hysterectomy or conservative therapy with strict follow-up.

UNSTEADY DISPERSION IN RANDOM INTERMITTENT FLOW

EPA Science Inventory

The longitudinal dispersion coefficient of a conservative tracer was calculated from flow tests in a dead-end pipe loop system. Flow conditions for these tests ranged from laminar to transitional flow, and from steady to intermittent and random. Two static mixers linked in series...

A Broadly Flavivirus Cross-Neutralizing Monoclonal Antibody that Recognizes a Novel Epitope within the Fusion Loop of E Protein

PubMed Central

Jiang, Tao; Wang, Hua-Jing; Yang, Hai-ou; Tan, Weng-Long; Liu, Ran; Yu, Man; Ge, Bao-Xue; Zhu, Qing-Yu; Qin, E-De; Guo, Ya-Jun; Qin, Cheng-Feng

2011-01-01

Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV), yellow fever (YFV), West Nile (WNV), and Japanese encephalitis (JEV) viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1–4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the 98DRXW101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1–4, YFV, and WNV and confers protection from lethal challenge with DENV 1–4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans. PMID:21264311

Genetic characterization of the chemokine receptor CXCR4 gene in lagomorphs: comparison between the families Ochotonidae and Leporidae.

PubMed

Abrantes, J; Esteves, P J; Carmo, C R; Müller, A; Thompson, G; van der Loo, W

2008-04-01

Chemokines receptors are transmembrane proteins that bind chemokines. Chemokines and their receptors are known to play a crucial role in the immune system and in pathogen entry. There is evidence that myxoma virus, the causative agent of myxomatosis, can use the chemokine receptor CXCR4 to infect cells. This virus causes a benign disease in its natural host, Sylvilagus, but in the European rabbit (Oryctolagus cuniculus) it causes a highly fatal and infectious disease known as myxomatosis. We have characterized the chemokine receptor CXCR4 gene in five genera of the order Lagomorpha, Ochotona (Ochotonidae), and Oryctolagus, Lepus, Bunolagus and Sylvilagus (Leporidae). In lagomorphs, the CXCR4 is highly conserved, with most of the protein diversity found at surface regions. Five amino acid replacements were observed, two in the intracellular loops, one in the transmembrane domain and two in the extracellular loops. Oryctolagus features unique amino acid changes at the intracellular domains, putting this genus apart of all other lagomorphs. Furthermore, in the 37 European rabbits analysed, which included healthy rabbits and rabbits with clinical symptoms of myxomatosis, 14 nucleotide substitutions were obtained but no amino acid differences were observed.

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE PAGES

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.; ...

2015-10-23

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

Coupled dual loop absorption heat pump

DOEpatents

Sarkisian, Paul H.; Reimann, Robert C.; Biermann, Wendell J.

1985-01-01

A coupled dual loop absorption system which utilizes two separate complete loops. Each individual loop operates at three temperatures and two pressures. This low temperature loop absorber and condenser are thermally coupled to the high temperature loop evaporator, and the high temperature loop condenser and absorber are thermally coupled to the low temperature generator.

Structural Determinants of Oligomerization of the Aquaporin-4 Channel.

PubMed

Kitchen, Philip; Conner, Matthew T; Bill, Roslyn M; Conner, Alex C

2016-03-25

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of a novel domain ‘GATE’ in the ABC protein DrrA and its role in drug efflux by the DrrAB complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Han; Rahman, Sadia; Li, Wen

2015-03-27

A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homologmore » MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis. - Highlights: • A novel domain ‘GATE’ is identified in the ABC protein DrrA. • GATE shows high sequence and structural conservation among diverse ABC proteins. • GATE is located outside of the previously studied ATP binding and hydrolysis motifs. • Conserved GATE residues are critical for stability of DrrAB and for ATP catalysis.« less

Chromatin looping defines expression of TAL1, its flanking genes, and regulation in T-ALL.

PubMed

Zhou, Yan; Kurukuti, Sreenivasulu; Saffrey, Peter; Vukovic, Milica; Michie, Alison M; Strogantsev, Ruslan; West, Adam G; Vetrie, David

2013-12-19

TAL1 is an important regulator of hematopoiesis and its expression is tightly controlled despite complexities in its genomic organization. It is frequently misregulated in T-cell acute lymphoblastic leukemia (T-ALL), often due to deletions between TAL1 and the neighboring STIL gene. To better understand the events that lead to TAL1 expression in hematopoiesis and in T-ALL, we studied looping interactions at the TAL1 locus. In TAL1-expressing erythroid cells, the locus adopts a looping "hub" which brings into close physical proximity all known TAL1 cis-regulatory elements including CTCF-bound insulators. Loss of GATA1 results in disassembly of the hub and loss of CTCF/RAD21 from one of its insulators. Genes flanking TAL1 are partly dependent on hub integrity for their transcriptional regulation. We identified looping patterns unique to TAL1-expressing T-ALL cells, and, intriguingly, loops occurring between the TAL1 and STIL genes at the common TAL1/STIL breakpoints found in T-ALL. These findings redefine how TAL1 and neighboring genes communicate within the nucleus, and indicate that looping facilitates both normal and aberrant TAL1 expression and may predispose to structural rearrangements in T-ALL. We also propose that GATA1-dependent looping mechanisms may facilitate the conservation of TAL1 regulation despite cis-regulatory remodeling during vertebrate evolution.

Affine q-deformed symmetry and the classical Yang-Baxter σ-model

NASA Astrophysics Data System (ADS)

Delduc, F.; Kameyama, T.; Magro, M.; Vicedo, B.

2017-03-01

The Yang-Baxter σ-model is an integrable deformation of the principal chiral model on a Lie group G. The deformation breaks the G × G symmetry to U(1)rank( G) × G. It is known that there exist non-local conserved charges which, together with the unbroken U(1)rank( G) local charges, form a Poisson algebra [InlineMediaObject not available: see fulltext.], which is the semiclassical limit of the quantum group {U}_q(g) , with g the Lie algebra of G. For a general Lie group G with rank( G) > 1, we extend the previous result by constructing local and non-local conserved charges satisfying all the defining relations of the infinite-dimensional Poisson algebra [InlineMediaObject not available: see fulltext.], the classical analogue of the quantum loop algebra {U}_q(Lg) , where Lg is the loop algebra of g. Quite unexpectedly, these defining relations are proved without encountering any ambiguity related to the non-ultralocality of this integrable σ-model.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalentmore » intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.« less

Gauge and integrable theories in loop spaces

NASA Astrophysics Data System (ADS)

Ferreira, L. A.; Luchini, G.

2012-05-01

We propose an integral formulation of the equations of motion of a large class of field theories which leads in a quite natural and direct way to the construction of conservation laws. The approach is based on generalized non-abelian Stokes theorems for p-form connections, and its appropriate mathematical language is that of loop spaces. The equations of motion are written as the equality of a hyper-volume ordered integral to a hyper-surface ordered integral on the border of that hyper-volume. The approach applies to integrable field theories in (1+1) dimensions, Chern-Simons theories in (2+1) dimensions, and non-abelian gauge theories in (2+1) and (3+1) dimensions. The results presented in this paper are relevant for the understanding of global properties of those theories. As a special byproduct we solve a long standing problem in (3+1)-dimensional Yang-Mills theory, namely the construction of conserved charges, valid for any solution, which are invariant under arbitrary gauge transformations.

Mapping the Ca(2+) induced structural change in calreticulin.

PubMed

Boelt, Sanne Grundvad; Norn, Christoffer; Rasmussen, Morten Ib; André, Ingemar; Čiplys, Evaldas; Slibinskas, Rimantas; Houen, Gunnar; Højrup, Peter

2016-06-16

Calreticulin is a highly conserved multifunctional protein implicated in many different biological systems and has therefore been the subject of intensive research. It is primarily present in the endoplasmatic reticulum where its main functions are to regulate Ca(2+) homeostasis, act as a chaperone and stabilize the MHC class I peptide-loading complex. Although several high-resolution structures of calreticulin exist, these only cover three-quarters of the entire protein leaving the extended structures unsolved. Additionally, the structure of calreticulin is influenced by the presence of Ca(2+). The conformational changes induced by Ca(2+) have not been determined yet as they are hard to study with traditional approaches. Here, we investigated the Ca(2+)-induced conformational changes with a combination of chemical cross-linking, mass spectrometry, bioinformatics analysis and modelling in Rosetta. Using a bifunctional linker, we found a large Ca(2+)-induced change to the cross-linking pattern in calreticulin. Our results are consistent with a high flexibility in the P-loop, a stabilization of the acidic C-terminal and a relatively close interaction of the P-loop and the acidic C-terminal. The function of calreticulin, an endoplasmatic reticulin chaperone, is affected by fluctuations in Ca(2+)concentration, but the structural mechanism is unknown. The present work suggests that Ca(2+)-dependent regulation is caused by different conformations of a long proline-rich loop that changes the accessibility to the peptide/lectin-binding site. Our results indicate that the binding of Ca(2+) to calreticulin may thus not only just be a question of Ca(2+) storage but is likely to have an impact on the chaperone activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Loop electrostatics modulates the intersubunit interactions in ferritin.

PubMed

Bernacchioni, Caterina; Ghini, Veronica; Pozzi, Cecilia; Di Pisa, Flavio; Theil, Elizabeth C; Turano, Paola

2014-11-21

Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.

Conserved water mediated recognition and the dynamics of active site Cys 331 and Tyr 411 in hydrated structure of human IMPDH-II.

PubMed

Bairagya, Hridoy R; Mukhopadhyay, Bishnu P; Bera, Asim K

2011-01-01

Inosine monophosphate dehydrogenase (IMPDH) of human is involved in GMP biosynthesis pathway, increased level of IMPDH-II (an isoform of enzyme) activity have found in leukemic and sarcoma cells. Modeling and extensive molecular dynamics simulation (15 ns) studies of IMPDH-II (1B3O PDB structure) have indicated the intricate involvement of four conserved water molecules (W 1, W 2, W 3, and W 4) in the conformational transition or the mobilities of "flap" (residues 400-450) and "loop" (residues 325-342) regions in enzyme. The stabilization of active site residues Asn 303, Gly 324, Ser 329, Cys 331, Asp 364, and Tyr 411 through variable H-bonding coordination from the conserved water molecular center seems interesting in the uninhibited hydrated form of human IMPDH-II structures. This conformational transition or the flexibility of mobile regions, water molecular recognition to active site residues Cys 331 and Tyr 411, and the presence of a hydrophilic cavity approximately 540 Å(3) (enclaved by the loop and flap region) near the C-terminal surface of this enzyme may explore a rational hope toward the water mimic inhibitor or anticancer agent design for human. 2010 John Wiley & Sons, Ltd.

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

PubMed Central

Zhou, Zheng; Feng, Hanqiao; Zhou, Bing-Rui; Ghirlando, Rodolfo; Hu, Kaifeng; Zwolak, Adam; Miller Jenkins, Lisa M.; Xiao, Hua; Tjandra, Nico; Wu, Carl; Bai, Yawen

2011-01-01

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore1. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A2. A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH33, 4. The structural basis of this specification is of outstanding interest. Yeast Scm3 and human HJURP are conserved nonhistone proteins that interact physically with the (CenH3-H4)2 heterotetramer and are required for the deposition of CenH3 at centromeres in vivo5, 6, 7, 8, 9, 10, 11, 12, 13. Here we have elucidated the structural basis for recognition of budding yeast CenH3 (Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 complexed with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved N-terminus and a shorter α-helix at the C-terminus of Scm3-CBD wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3-CBD induces major conformational changes and sterically occludes DNA binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome. PMID:21412236

Unified Framework for Deriving Simultaneous Equation Algorithms for Water Distribution Networks

EPA Science Inventory

The known formulations for steady state hydraulics within looped water distribution networks are re-derived in terms of linear and non-linear transformations of the original set of partly linear and partly non-linear equations that express conservation of mass and energy. All of ...

Tracheophytes Contain Conserved Orthologs of a Basic Helix-Loop-Helix Transcription Factor That Modulate ROOT HAIR SPECIFIC Genes[OPEN

PubMed Central

Cho, Hyun-Min

2017-01-01

ROOT HAIR SPECIFIC (RHS) genes, which contain the root hair-specific cis-element (RHE) in their regulatory regions, function in root hair morphogenesis. Here, we demonstrate that an Arabidopsis thaliana basic helix-loop-helix transcription factor, ROOT HAIR DEFECTVE SIX-LIKE4 (RSL4), directly binds to the RHE in vitro and in vivo, upregulates RHS genes, and stimulates root hair formation in Arabidopsis. Orthologs of RSL4 from a eudicot (poplar [Populus trichocarpa]), a monocot (rice [Oryza sativa]), and a lycophyte (Selaginella moellendorffii) each restored root hair growth in the Arabidopsis rsl4 mutant. In addition, the rice and S. moellendorffii RSL4 orthologs bound to the RHE in in vitro and in vivo assays. The RSL4 orthologous genes contain RHEs in their promoter regions, and RSL4 was able to bind to its own RHEs in vivo and amplify its own expression. This process likely provides a positive feedback loop for sustainable root hair growth. When RSL4 and its orthologs were expressed in cells in non-root-hair positions, they induced ectopic root hair growth, indicating that these genes are sufficient to specify root hair formation. Our results suggest that RSL4 mediates root hair formation by regulating RHS genes and that this mechanism is conserved throughout the tracheophyte (vascular plant) lineage. PMID:28087829

Mapping and characterization of vicriviroc resistance mutations from HIV-1 isolated from treatment-experienced subjects enrolled in a phase II study (VICTOR-E1).

PubMed

McNicholas, Paul M; Mann, Paul A; Wojcik, Lisa; Qiu, Ping; Lee, Erin; McCarthy, Michael; Shen, Junwu; Black, Todd A; Strizki, Julie M

2011-03-01

In the phase 2 VICTOR-E1 study, treatment-experienced subjects receiving 20 mg or 30 mg of the CCR5 antagonist vicriviroc (VCV), with a boosted protease containing optimized background regimen, experienced significantly greater reductions in HIV-1 viral load compared with control subjects. Among the 79 VCV-treated subjects, 15 experienced virologic failure, and of these 5 had VCV-resistant virus. This study investigated the molecular basis for the changes in susceptibility to VCV in these subjects. Sequence analysis and phenotypic susceptibility testing was performed on envelope clones from VCV-resistant virus. For select clones, an exchange of mutations in the V3 loop was performed between phenotypically resistant clones and the corresponding susceptible clones. Phenotypic resistance was manifest by reductions in the maximum percent inhibition. Clonal analysis of envelopes from the 5 subjects identified multiple amino acid changes in gp160 that were exclusive to the resistant clones, however, none of the changes were conserved between subjects. Introduction of V3 loop substitutions from the resistant clones into the matched susceptible clones was not sufficient to reproduce the resistant phenotype. Likewise, changing the substitutions in the V3 loops from resistant clones to match susceptible clones only restored susceptibility in 1 clone. There were no clearly conserved patterns of mutations in gp160 associated with phenotypic resistance to VCV and mutations both within and outside of the V3 loop contributed to the resistance phenotype. These data suggest that genotypic tests for VCV susceptibility may require larger training sets and additional information beyond V3 sequences.

Identification of a Novel EF-Loop in the N-terminus of TRPM2 Channel Involved in Calcium Sensitivity

PubMed Central

Luo, Yuhuan; Yu, Xiafei; Ma, Cheng; Luo, Jianhong; Yang, Wei

2018-01-01

As an oxidative stress sensor, transient receptor potential melastatin 2 (TRPM2) channel is involved in many physiological and pathological processes including warmth sensing, ischemia injury, inflammatory diseases and diabetes. Intracellular calcium is critical for TRPM2 channel activation and the IQ-like motif in the N-terminus has been shown to be important by mediating calmodulin binding. Sequence analysis predicted two potential EF-loops in the N-terminus of TRPM2. Site-directed mutagenesis combining with functional assay showed that substitution with alanine of several residues, most of which are conserved in the typical EF-loop, including D267, D278, D288, and E298 dramatically reduced TRPM2 channel currents. By further changing the charges or side chain length of these conserved residues, our results indicate that the negative charge of D267 and the side chain length of D278 are critical for calcium-induced TRPM2 channel activation. G272I mutation also dramatically reduced the channel currents, suggesting that this site is critical for calcium-induced TRPM2 channel activation. Furthermore, D267A mutant dramatically reduced the currents induced by calcium alone compared with that by ADPR, indicating that D267 residue in D267–D278 motif is the most important site for calcium sensitivity of TRPM2. In addition, inside-out recordings showed that mutations at D267, G272, D278, and E298 had no effect on single-channel conductance. Taken together, our data indicate that D267–D278 motif in the N-terminus as a novel EF-loop is critical for calcium-induced TRPM2 channel activation.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A conserved threonine in the S1-S2 loop of KV7.2 and K V7.3 channels regulates voltage-dependent activation.

PubMed

Füll, Yvonne; Seebohm, Guiscard; Lerche, Holger; Maljevic, Snezana

2013-06-01

The voltage-gated potassium channels KV7.2 and KV7.3 (KCNQ2/3 genes) play an important role in regulating neuronal excitability. More than 50 KCNQ2/3 mutations have been identified to cause an inherited form of epilepsy in newborns. For two of those (E119G and S122L) found in the S1-S2 region of KV7.2, we previously showed a decreased channel availability mainly at action potential subthreshold voltages caused by a slight depolarizing shift of the activation curve. Interestingly, recent studies revealed that a threonine residue within the S1-S2 loop, highly conserved among different classes of KV channels, is crucial for both their function and surface expression. To investigate the functional role of the homologous threonine residues in KV7.2 (T114) and KV7.3 (T144) channels, we replaced them with alanine and examined the electrophysiological properties using heterologous expression in CHO cells and whole cell patch clamping. Channels comprising mutant subunits yielded decreased potassium currents with slowed activation and accelerated deactivation kinetics. However, the most striking effect was a depolarizing shift in the voltage dependence of activation reaching +30 mV upon co-expression of both mutant subunits. Potential interactions of T114 within the channel were analyzed by creating a 3D homology model of KV7.2 in an open state suggesting that this residue plays a central role in the formation of a stable interface between the S1-S2 and the S5 segment helices. This could be the explanation why substitution of the conserved threonine in KV7.2 and KV7.3 channels destabilizes the open and favors the closed state of these channels.

Dynamic conformational switching in the chemokine ligand is essential for G-protein-coupled receptor activation

PubMed Central

Joseph, Prem Raj B.; Sawant, Kirti V.; Isley, Angela; Pedroza, Mesias; Garofalo, Roberto P.; Richardson, Ricardo M.; Rajarathnam, Krishna

2014-01-01

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. Mutations in the GP motif caused various differences from native-like function to complete loss of activity that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity. PMID:24032673

Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cairns, S.S.

1987-01-01

In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage ofmore » the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.« less

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

PubMed

Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

2017-06-29

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

PubMed Central

Jha, Sujata; Rollins, Madeline G.; Fuchs, Gabriele; Procter, Dean J.; Hall, Elizabeth A.; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N.; Walsh, Derek

2017-01-01

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements1. However, beyond differential subunit expression during development2,3, evidence for regulated ribosome specification within individual cells has remained elusive1. Here, we report that a poxvirus kinase phosphorylates serine/threonine residues in the small ribosomal subunit protein, Receptor for Activated C Kinase (RACK1) that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs whose 5’ untranslated regions (UTRs) contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analysis revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, where these leaders act as translational enhancers for poorly understood reasons. Phosphomimetics and inter-species chimeras demonstrated that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase4 confer a translational advantage. Our findings uncover ribosome customization through a novel trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie the enigmatic poxvirus polyA-leaders4. PMID:28636603

Immunomodulation: A definitive role of microRNA-142.

PubMed

Sharma, Salil

2017-12-01

Majority of microRNAs are evolutionarily conserved in vertebrates. This is suggestive of their similar roles in regulation of gene networks. In addition to their conserved mature sequences and regulatory roles, a few microRNAs show very cell or tissue specific expression. These microRNAs are highly enriched in some cell types or organs. One such microRNA is microRNA-142 (miR-142). The classical stem-loop structure of miR142 encodes for two species of mature microRNAs; miR142-5p and miR142-3p. MiR-142 is abundant in cells of hematopoietic origin, and therefore, aptly plays a role in lineage differentiation of hematopoietic cells. Interestingly, over the years, miR-142 has gained considerable attention for its quintessential role in regulating immune response. This mini-review discusses the important functional roles of miR-142 in inflammatory and immune response in different physiological and disease setting. Copyright © 2017 Elsevier Ltd. All rights reserved.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.

PubMed Central

Honda, M; Brown, E A; Lemon, S M

1996-01-01

The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence. PMID:8849773

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide.

PubMed

Pérez Sirkin, Daniela I; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M; Vissio, Paula G; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation.

Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide

PubMed Central

Pérez Sirkin, Daniela I.; Lafont, Anne-Gaëlle; Kamech, Nédia; Somoza, Gustavo M.; Vissio, Paula G.; Dufour, Sylvie

2017-01-01

GnRH-associated peptide (GAP) is the C-terminal portion of the gonadotropin-releasing hormone (GnRH) preprohormone. Although it was reported in mammals that GAP may act as a prolactin-inhibiting factor and can be co-secreted with GnRH into the hypophyseal portal blood, GAP has been practically out of the research circuit for about 20 years. Comparative studies highlighted the low conservation of GAP primary amino acid sequences among vertebrates, contributing to consider that this peptide only participates in the folding or carrying process of GnRH. Considering that the three-dimensional (3D) structure of a protein may define its function, the aim of this study was to evaluate if GAP sequences and 3D structures are conserved in the vertebrate lineage. GAP sequences from various vertebrates were retrieved from databases. Analysis of primary amino acid sequence identity and similarity, molecular phylogeny, and prediction of 3D structures were performed. Amino acid sequence comparison and phylogeny analyses confirmed the large variation of GAP sequences throughout vertebrate radiation. In contrast, prediction of the 3D structure revealed a striking conservation of the 3D structure of GAP1 (GAP associated with the hypophysiotropic type 1 GnRH), despite low amino acid sequence conservation. This GAP1 peptide presented a typical helix-loop-helix (HLH) structure in all the vertebrate species analyzed. This HLH structure could also be predicted for GAP2 in some but not all vertebrate species and in none of the GAP3 analyzed. These results allowed us to infer that selective pressures have maintained GAP1 HLH structure throughout the vertebrate lineage. The conservation of the HLH motif, known to confer biological activity to various proteins, suggests that GAP1 peptides may exert some hypophysiotropic biological functions across vertebrate radiation. PMID:28878737

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

PubMed Central

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-01-01

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

Two AFC Loops For Low CNR And High Dynamics

NASA Technical Reports Server (NTRS)

Hinedi, Sami M.; Aguirre, Sergio

1992-01-01

Two alternative digital automatic-frequency-control (AFC) loops proposed to acquire (or reacquire) and track frequency of received carrier radio signal. Intended for use where carrier-to-noise ratios (CNR's) low and carrier frequency characterized by high Doppler shift and Doppler rate because of high relative speed and acceleration, respectively, between transmitter and receiver. Either AFC loops used in place of phase-locked loop. New loop concepts integrate ideas from classical spectrum-estimation, digital-phase-locked-loop, and Kalman-Filter theories.

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

PubMed

Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

1996-12-24

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Nuclear axial currents in chiral effective field theory

DOE PAGES

Baroni, Alessandro; Girlanda, Luca; Pastore, Saori; ...

2016-01-11

Two-nucleon axial charge and current operators are derived in chiral effective field theory up to one loop. The derivation is based on time-ordered perturbation theory and accounts for cancellations between the contributions of irreducible diagrams and the contributions owing to nonstatic corrections from energy denominators of reducible diagrams. Ultraviolet divergencies associated with the loop corrections are isolated in dimensional regularization. The resulting axial current is finite and conserved in the chiral limit, while the axial charge requires renormalization. As a result, a complete set of contact terms for the axial charge up to the relevant order in the power countingmore » is constructed.« less

Telomere biology of trypanosomatids: beginning to answer some questions.

PubMed

Lira, Cristina B B; Giardini, Miriam A; Neto, Jair L Siqueira; Conte, Fábio F; Cano, Maria Isabel N

2007-08-01

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Constraining the axion-photon coupling with massive stars.

PubMed

Friedland, Alexander; Giannotti, Maurizio; Wise, Michael

2013-02-08

We point out that stars in the mass window ~8-12M([circumpunct]) can serve as sensitive probes of the axion-photon interaction, g(Aγγ). Specifically, for these stars axion energy losses from the helium-burning core would shorten and eventually eliminate the blue loop phase of the evolution. This would contradict observational data, since the blue loops are required, e.g., to account for the existence of Cepheid stars. Using the MESA stellar evolution code, modified to include the extra cooling, we conservatively find g(Aγγ)

Extended Impact of Pin1 Catalytic Loop Phosphorylation Revealed by S71E Phosphomimetic.

PubMed

Mahoney, Brendan J; Zhang, Meiling; Zintsmaster, John S; Peng, Jeffrey W

2018-03-02

Pin1 is a two-domain human protein that catalyzes the cis-trans isomerization of phospho-Ser/Thr-Pro (pS/T-P) motifs in numerous cell-cycle regulatory proteins. These pS/T-P motifs bind to Pin1's peptidyl-prolyl isomerase (PPIase) domain in a catalytic pocket, between an extended catalytic loop and the PPIase domain core. Previous studies showed that post-translational phosphorylation of S71 in the catalytic loop decreases substrate binding affinity and isomerase activity. To define the origins for these effects, we investigated a phosphomimetic Pin1 mutant, S71E-Pin1, using solution NMR. We find that S71E perturbs not only its host loop but also the nearby PPIase core. The perturbations identify a local network of hydrogen bonds and salt bridges that is more extended than previously thought, and includes interactions between the catalytic loop and the α2/α3 turn in the PPIase core. Explicit-solvent molecular dynamics simulations and phylogenetic analysis suggest that these interactions act as conserved "latches" between the loop and PPIase core that enhance binding of phosphorylated substrates, as they are absent in PPIases lacking pS/T-P specificity. Our results suggest that S71 is a hub residue within an electrostatic network primed for phosphorylation, and may illustrate a common mechanism of phosphorylation-mediated allostery. Copyright © 2018 Elsevier Ltd. All rights reserved.

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

PubMed

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

Identification and characterization of the sodium-binding site of activated protein C.

PubMed

He, X; Rezaie, A R

1999-02-19

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

Solution structure of CXCL5--a novel chemokine and adipokine implicated in inflammation and obesity.

PubMed

Sepuru, Krishna Mohan; Poluri, Krishna Mohan; Rajarathnam, Krishna

2014-01-01

The chemokine CXCL5 is selectively expressed in highly specialized cells such as epithelial type II cells in the lung and white adipose tissue macrophages in muscle, where it mediates diverse functions from combating microbial infections by regulating neutrophil trafficking to promoting obesity by inhibiting insulin signaling. Currently very little is known regarding the structural basis of how CXCL5 mediates its novel functions. Towards this missing knowledge, we have solved the solution structure of the CXCL5 dimer by NMR spectroscopy. CXCL5 is a member of a subset of seven CXCR2-activating chemokines (CAC) that are characterized by the highly conserved ELR motif in the N-terminal tail. The structure shows that CXCL5 adopts the typical chemokine fold, but also reveals several distinct differences in the 30 s loop and N-terminal residues; not surprisingly, crosstalk between N-terminal and 30 s loop residues have been implicated as a major determinant of receptor activity. CAC function also involves binding to highly sulfated glycosaminoglycans (GAG), and the CXCL5 structure reveals a distinct distribution of positively charged residues, suggesting that differences in GAG interactions also influence function. The availability of the structure should now facilitate the design of experiments to better understand the molecular basis of various CXCL5 functions, and also serve as a template for the design of inhibitors for use in a clinical setting.

Establishment and Application of a Loop-Mediated Isothermal Amplification Method for Simple, Specific, Sensitive and Rapid Detection of Toxoplasma gondii

PubMed Central

CAO, Lili; CHENG, Ronghua; YAO, Lin; YUAN, Shuxian; YAO, Xinhua

2013-01-01

ABSTRACT The Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high simply, specificity, sensitivity and rapidity. In this study, A LAMP assay with 6 primers targeting a highly conserved region of the GRA1 gene was developed to diagnose Toxoplasma gondii. The reaction time of the LAMP assay was shortened to 30 min after optimizing the reaction system. The LAMP assay was found to be highly specific and stable. The detection limit of the LAMP assay was 10 copies, the same as that of the conventional PCR. We used the LAMP assay to develop a real-time fluorogenic protocol to quantitate T. gondii DNA and generated a log-linear regression plot by plotting the time-to-threshold values against genomic equivalent copies. Furthermore, the LAMP assay was applied to detect T. gondii DNA in 423 blood samples and 380 lymph node samples from 10 pig farms, and positive results were obtained for 7.8% and 8.2% of samples, respectively. The results showed that the LAMP method is slightly more sensitive than conventional PCR (6.1% and 7.6%). Positive samples obtained from 6 pig farms. The LAMP assay established in this study resulted in simple, specific, sensitive and rapid detection of T. gondii DNA and is expected to play an important role in clinical detection of T. gondii. PMID:23965849

SigFlux: a novel network feature to evaluate the importance of proteins in signal transduction networks.

PubMed

Liu, Wei; Li, Dong; Zhang, Jiyang; Zhu, Yunping; He, Fuchu

2006-11-27

Measuring each protein's importance in signaling networks helps to identify the crucial proteins in a cellular process, find the fragile portion of the biology system and further assist for disease therapy. However, there are relatively few methods to evaluate the importance of proteins in signaling networks. We developed a novel network feature to evaluate the importance of proteins in signal transduction networks, that we call SigFlux, based on the concept of minimal path sets (MPSs). An MPS is a minimal set of nodes that can perform the signal propagation from ligands to target genes or feedback loops. We define SigFlux as the number of MPSs in which each protein is involved. We applied this network feature to the large signal transduction network in the hippocampal CA1 neuron of mice. Significant correlations were simultaneously observed between SigFlux and both the essentiality and evolutionary rate of genes. Compared with another commonly used network feature, connectivity, SigFlux has similar or better ability as connectivity to reflect a protein's essentiality. Further classification according to protein function demonstrates that high SigFlux, low connectivity proteins are abundant in receptors and transcriptional factors, indicating that SigFlux candescribe the importance of proteins within the context of the entire network. SigFlux is a useful network feature in signal transduction networks that allows the prediction of the essentiality and conservation of proteins. With this novel network feature, proteins that participate in more pathways or feedback loops within a signaling network are proved far more likely to be essential and conserved during evolution than their counterparts.

Genome-wide analysis of basic helix-loop-helix (bHLH) transcription factors in Brachypodium distachyon.

PubMed

Niu, Xin; Guan, Yuxiang; Chen, Shoukun; Li, Haifeng

2017-08-15

As a superfamily of transcription factors (TFs), the basic helix-loop-helix (bHLH) proteins have been characterized functionally in many plants with a vital role in the regulation of diverse biological processes including growth, development, response to various stresses, and so on. However, no systemic analysis of the bHLH TFs has been reported in Brachypodium distachyon, an emerging model plant in Poaceae. A total of 146 bHLH TFs were identified in the Brachypodium distachyon genome and classified into 24 subfamilies. BdbHLHs in the same subfamily share similar protein motifs and gene structures. Gene duplication events showed a close relationship to rice, maize and sorghum, and segment duplications might play a key role in the expansion of this gene family. The amino acid sequence of the bHLH domains were quite conservative, especially Leu-27 and Leu-54. Based on the predicted binding activities, the BdbHLHs were divided into DNA binding and non-DNA binding types. According to the gene ontology (GO) analysis, BdbHLHs were speculated to function in homodimer or heterodimer manner. By integrating the available high throughput data in public database and results of quantitative RT-PCR, we found the expression profiles of BdbHLHs were different, implying their differentiated functions. One hundred fourty-six BdbHLHs were identified and their conserved domains, sequence features, phylogenetic relationship, chromosomal distribution, GO annotations, gene structures, gene duplication and expression profiles were investigated. Our findings lay a foundation for further evolutionary and functional elucidation of BdbHLH genes.

New constraints on dark matter effective theories from standard model loops.

PubMed

Crivellin, Andreas; D'Eramo, Francesco; Procura, Massimiliano

2014-05-16

We consider an effective field theory for a gauge singlet Dirac dark matter particle interacting with the standard model fields via effective operators suppressed by the scale Λ ≳ 1 TeV. We perform a systematic analysis of the leading loop contributions to spin-independent Dirac dark matter-nucleon scattering using renormalization group evolution between Λ and the low-energy scale probed by direct detection experiments. We find that electroweak interactions induce operator mixings such that operators that are naively velocity suppressed and spin dependent can actually contribute to spin-independent scattering. This allows us to put novel constraints on Wilson coefficients that were so far poorly bounded by direct detection. Constraints from current searches are already significantly stronger than LHC bounds, and will improve in the near future. Interestingly, the loop contribution we find is isospin violating even if the underlying theory is isospin conserving.

Decentralized control of sound radiation using iterative loop recovery.

PubMed

Schiller, Noah H; Cabell, Randolph H; Fuller, Chris R

2010-10-01

A decentralized model-based control strategy is designed to reduce low-frequency sound radiation from periodically stiffened panels. While decentralized control systems tend to be scalable, performance can be limited due to modeling error introduced by the unmodeled interaction between neighboring control units. Since bounds on modeling error are not known in advance, it is difficult to ensure the decentralized control system will be robust without making the controller overly conservative. Therefore an iterative approach is suggested, which utilizes frequency-shaped loop recovery. The approach accounts for modeling error introduced by neighboring control loops, requires no communication between subsystems, and is relatively simple. The control strategy is evaluated numerically using a model of a stiffened aluminum panel that is representative of the sidewall of an aircraft. Simulations demonstrate that the iterative approach can achieve significant reductions in radiated sound power from the stiffened panel without destabilizing neighboring control units.

Decentralized Control of Sound Radiation Using Iterative Loop Recovery

NASA Technical Reports Server (NTRS)

Schiller, Noah H.; Cabell, Randolph H.; Fuller, Chris R.

2009-01-01

A decentralized model-based control strategy is designed to reduce low-frequency sound radiation from periodically stiffened panels. While decentralized control systems tend to be scalable, performance can be limited due to modeling error introduced by the unmodeled interaction between neighboring control units. Since bounds on modeling error are not known in advance, it is difficult to ensure the decentralized control system will be robust without making the controller overly conservative. Therefore an iterative approach is suggested, which utilizes frequency-shaped loop recovery. The approach accounts for modeling error introduced by neighboring control loops, requires no communication between subsystems, and is relatively simple. The control strategy is evaluated numerically using a model of a stiffened aluminum panel that is representative of the sidewall of an aircraft. Simulations demonstrate that the iterative approach can achieve significant reductions in radiated sound power from the stiffened panel without destabilizing neighboring control units.

A novel actin binding site of myosin required for effective muscle contraction.

PubMed

Várkuti, Boglárka H; Yang, Zhenhui; Kintses, Bálint; Erdélyi, Péter; Bárdos-Nagy, Irén; Kovács, Attila L; Hári, Péter; Kellermayer, Miklós; Vellai, Tibor; Málnási-Csizmadia, András

2012-02-12

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

Performance of the Extravehicular Mobility Unit (EMU) Airlock Coolant Loop Remediation (A/L CLR) Hardware - Final

NASA Technical Reports Server (NTRS)

Steele, John W.; Rector, Tony; Gazda, Daniel; Lewis, John

2011-01-01

An EMU water processing kit (Airlock Coolant Loop Recovery -- A/L CLR) was developed as a corrective action to Extravehicular Mobility Unit (EMU) coolant flow disruptions experienced on the International Space Station (ISS) in May of 2004 and thereafter. A conservative duty cycle and set of use parameters for A/L CLR use and component life were initially developed and implemented based on prior analysis results and analytical modeling. Several initiatives were undertaken to optimize the duty cycle and use parameters of the hardware. Examination of post-flight samples and EMU Coolant Loop hardware provided invaluable information on the performance of the A/L CLR and has allowed for an optimization of the process. The intent of this paper is to detail the evolution of the A/L CLR hardware, efforts to optimize the duty cycle and use parameters, and the final recommendations for implementation in the post-Shuttle retirement era.

Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

PubMed Central

Stephens, Robert F.; Guan, W.; Zhorov, Boris S.; Spafford, J. David

2015-01-01

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel. PMID:26042044

Structural and dynamic determinants of ligand binding and regulation of cyclin-dependent kinase 5 by pathological activator p25 and inhibitory peptide CIP.

PubMed

Cardone, A; Hassan, S A; Albers, R W; Sriram, R D; Pant, H C

2010-08-20

The crystal structure of the cdk5/p25 complex has provided information on possible molecular mechanisms of the ligand binding, specificity, and regulation of the kinase. Comparative molecular dynamics simulations are reported here for physiological conditions. This study provides new insight on the mechanisms that modulate such processes, which may be exploited to control pathological activation by p25. The structural changes observed in the kinase are stabilized by a network of interactions involving highly conserved residues within the cyclin-dependent kinase (cdk) family. Collective motions of the proteins (cdk5, p25, and CIP) and their complexes are identified by principal component analysis, revealing two conformational states of the activation loop upon p25 complexation, which are absent in the uncomplexed kinase and not apparent from the crystal. Simulations of the uncomplexed inhibitor CIP show structural rearrangements and increased flexibility of the interfacial loop containing the critical residue E240, which becomes fully hydrated and available for interactions with one of several positively charged residues in the kinase. These changes provide a rationale for the observed high affinity and enhanced inhibitory action of CIP when compared to either p25 or the physiological activators of cdk5. Published by Elsevier Ltd.

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

PubMed

Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

2014-07-29

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy.

PubMed

de Medeiros, Luciano Neves; Angeli, Renata; Sarzedas, Carolina G; Barreto-Bergter, Eliana; Valente, Ana Paula; Kurtenbach, Eleonora; Almeida, Fabio C L

2010-02-01

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized alpha/beta motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 (15)N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36-Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the mus-ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH. Copyright 2009 Elsevier B.V. All rights reserved.

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Dietary modulation and structure prediction of rat mucosal pentraxin (Mptx) protein and loss of function in humans

PubMed Central

van der Meer-van Kraaij, Cindy; Siezen, Roland; Kramer, Evelien; Reinders, Marjolein; Blokzijl, Hans; van der Meer, Roelof

2007-01-01

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), C-reactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrient-sensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive “top” face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents. PMID:18850182

Closing the Loop: Exploring Integrated Waste Management and Resource Conservation, Kindergarten through Grade Six. 2000 Edition.

ERIC Educational Resources Information Center

Clymire, Olga

This document is designed to teach concepts of source reduction, recycling, composting, and integrated waste management to kindergarten through grade six students. The lessons correlate to grade level and include sections on the lesson's concepts, purpose, overview, correlations to California's content standards and frameworks, scientific thinking…

Insights into substrate binding and catalysis in bacterial type I dehydroquinase.

PubMed

Maneiro, María; Peón, Antonio; Lence, Emilio; Otero, José M; Van Raaij, Mark J; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

2014-09-15

Structural, biochemical and computational studies to study substrate binding and the role of the conserved residues of the DHQ1 (type I dehydroquinase) enzyme active site are reported in the present paper. The crystal structure of DHQ1 from Salmonella typhi in complex with (2R)-2-methyl-3-dehydroquinic acid, a substrate analogue, was solved at 1.5 Å. The present study reveals a previously unknown key role for conserved Glu46, Phe145 and Met205 and Gln236, Pro234 and Ala233 residues, with the latter three being located in the flexible substrate-covering loop. Gln236 was shown to be responsible for the folding of this loop and for the dramatic reduction of its flexibility, which triggers active site closure. Glu46 was found to be key in bringing the substrate close to the lysine/histidine catalytic pocket to initiate catalysis. The present study could be useful in the rational design of inhibitors of this challenging and recognized target for the development of novel herbicides and antimicrobial agents.

Subspecies identification of captive Orang Utan in Melaka based on D-loop mitochondria DNA

NASA Astrophysics Data System (ADS)

Kamaluddin, Siti Norsyuhada; Yaakop, Salmah; Idris, Wan Mohd Razi; Rovie-Ryan, Jeffrine Japning; Md-Zain, Badrul Munir

2018-04-01

Mitochondrial DNA of Bornean Orang Utan populations suggests that there are three different subspecies (Pongo pygmaeus pygmaeus; Sarawak & Northwest Kalimantan, P. p. wurmbii; Southern West Kalimantan and Central Kalimantan, P. p. morio; East Kalimantan and Sabah). The subspecies of Orang Utans in captivity are difficult to determine through morphological observation. Thus, misidentification by ranger or zoo staffs leads to unwanted consequences especially towards conservation efforts of Orang Utan. The main objective of this study was to identify the subspecies and the geographic origin of 10 Orang Utans in Zoo Melaka and A' Famosa by using partial mitochondrial D-loop gene sequences. DNA of all individuals was extracted from FTA Card. Data analyses were performed using Maximum Parsimony, MP and Neighbor Joining, NJ. Molecular phylogeny analysis revealed that all the samples likely belong to one species of Sumatran Orang Utan (P. abelii) and three different subspecies of Bornean Orang Utans (P. p. pygmaeus, P. p. morio, and P. p. wurmbii). The results obtained in this study indirectly help the management of zoos in term of conservation and visitor's education.

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology?

PubMed Central

Janecek, S.

1995-01-01

Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed. PMID:7549888

Similarity of different beta-strands flanked in loops by glycines and prolines from distinct (alpha/beta)8-barrel enzymes: chance or a homology?

PubMed

Janecek, S

1995-06-01

Many (alpha/beta)8-barrel enzymes contain their conserved sequence regions at or around the beta-strand segments that are often preceded and succeeded by glycines and prolines, respectively. alpha-Amylase is one of these enzymes. Its sequences exhibit a very low degree of similarity, but strong conservation is seen around its beta-strands. These conserved regions were used in the search for similarities with beta-strands of other (alpha/beta)8-barrel enzymes. The analysis revealed an interesting similarity between the segment around the beta 2-strand of alpha-amylase and the one around the beta 4-strand of glycolate oxidase that are flanked in loops by glycines and prolines. The similarity can be further extended on other members of the alpha-amylase and glycolate oxidase subfamilies, i.e., cyclodextrin glycosyltransferase and oligo-1,6-glucosidase, and flavocytochrome b2, respectively. Moreover, the alpha-subunit of tryptophan synthase, the (alpha/beta)8-barrel enzyme belonging to the other subfamily of (alpha/beta)8-barrels, has both investigated strands, beta 2 and beta 4, similar to beta 2 of alpha-amylase and beta 4 of glycolate oxidase. The possibilities of whether this similarity exists only by chance or is a consequence of some processes during the evolution of (alpha/beta)8-barrel proteins are briefly discussed.

Rice Cellulose SynthaseA8 Plant-Conserved Region Is a Coiled-Coil at the Catalytic Core Entrance1[OPEN

PubMed Central

Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee; Badger, John

2017-01-01

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecular envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery. PMID:27879387

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling.

PubMed

Asha, Srinivasan; Sreekumar, Sweda; Soniya, E V

2016-01-01

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

PubMed Central

Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

2016-01-01

ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

Planning fuel-conservative descents in an airline environmental using a small programmable calculator: Algorithm development and flight test results

NASA Technical Reports Server (NTRS)

Knox, C. E.; Vicroy, D. D.; Simmon, D. A.

1985-01-01

A simple, airborne, flight-management descent algorithm was developed and programmed into a small programmable calculator. The algorithm may be operated in either a time mode or speed mode. The time mode was designed to aid the pilot in planning and executing a fuel-conservative descent to arrive at a metering fix at a time designated by the air traffic control system. The speed model was designed for planning fuel-conservative descents when time is not a consideration. The descent path for both modes was calculated for a constant with considerations given for the descent Mach/airspeed schedule, gross weight, wind, wind gradient, and nonstandard temperature effects. Flight tests, using the algorithm on the programmable calculator, showed that the open-loop guidance could be useful to airline flight crews for planning and executing fuel-conservative descents.

Planning fuel-conservative descents in an airline environmental using a small programmable calculator: algorithm development and flight test results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knox, C.E.; Vicroy, D.D.; Simmon, D.A.

A simple, airborne, flight-management descent algorithm was developed and programmed into a small programmable calculator. The algorithm may be operated in either a time mode or speed mode. The time mode was designed to aid the pilot in planning and executing a fuel-conservative descent to arrive at a metering fix at a time designated by the air traffic control system. The speed model was designed for planning fuel-conservative descents when time is not a consideration. The descent path for both modes was calculated for a constant with considerations given for the descent Mach/airspeed schedule, gross weight, wind, wind gradient, andmore » nonstandard temperature effects. Flight tests, using the algorithm on the programmable calculator, showed that the open-loop guidance could be useful to airline flight crews for planning and executing fuel-conservative descents.« less

Endo-β-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

PubMed

Gonçalves, Ana Maria D; Silva, Catarina S; Madeira, Tânia I; Coelho, Ricardo; de Sanctis, Daniele; San Romão, Maria Vitória; Bento, Isabel

2012-11-01

The crystal structure of wild-type endo-β-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (β/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations.

PubMed

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S; SantaLucia, John

2014-04-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson-Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.

Interactions driving the collapse of islet amyloid polypeptide: Implications for amyloid aggregation

NASA Astrophysics Data System (ADS)

Cope, Stephanie M.

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable beta-turn fibers. These non-amyloid fibers are present in the 10 muM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.

Fertility conserving management of early cervical cancer: our experience of LLETZ and pelvic lymph node dissection.

PubMed

Lindsay, Rhona; Burton, Kevin; Shanbhag, Smruta; Tolhurst, Jenny; Millan, David; Siddiqui, Nadeem

2014-01-01

Presently, for those diagnosed with early cervical cancer who wish to conserve their fertility, there is the option of radical trachelectomy. Although successful, this procedure is associated with significant obstetric morbidity. The recurrence risk of early cervical cancer is low and in tumors measuring less than 2 cm; if the lymphatics are negative, the likelihood of parametrial involvement is less than 1%. Therefore, pelvic lymph nodes are a surrogate marker of parametrial involvement and radical excision of the parametrium can be omitted if they are negative. The aim of this study was to report our experience of the fertility conserving management of early cervical cancer with repeat large loop excision of the transformation zone and laparoscopic pelvic lymph node dissection. Between 2004 and 2011, a retrospective review of cases of early cervical cancer who had fertility conserving management within Glasgow Royal Infirmary was done. Forty-three patients underwent fertility conserving management of early cervical cancer. Forty were screen-detected cancers; 2 were stage IA1, 4 were stage IA2, and 37 were stage IB1. There were 2 central recurrences during the follow-up period. There have been 15 live children to 12 women and there are 4 ongoing pregnancies. To our knowledge, this is the largest case series described and confirms the low morbidity and mortality of this procedure. However, even within our highly select group, there have been 2 cases of central recurrent disease. We, therefore, are urging caution in the global adoption of this technique and would welcome a multicenter multinational randomized controlled trial.

Identification and Characterization of MicroRNAs in Small Brown Planthopper (Laodephax striatellus) by Next-Generation Sequencing

PubMed Central

Lou, Yonggen; Cheng, Jia'an; Zhang, Hengmu; Xu, Jian-Hong

2014-01-01

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level and are thought to play critical roles in many metabolic activities in eukaryotes. The small brown planthopper (Laodephax striatellus Fallén), one of the most destructive agricultural pests, causes great damage to crops including rice, wheat, and maize. However, information about the genome of L. striatellus is limited. In this study, a small RNA library was constructed from a mixed L. striatellus population and sequenced by Solexa sequencing technology. A total of 501 mature miRNAs were identified, including 227 conserved and 274 novel miRNAs belonging to 125 and 250 families, respectively. Sixty-nine conserved miRNAs that are included in 38 families are predicted to have an RNA secondary structure typically found in miRNAs. Many miRNAs were validated by stem-loop RT-PCR. Comparison with the miRNAs in 84 animal species from miRBase showed that the conserved miRNA families we identified are highly conserved in the Arthropoda phylum. Furthermore, miRanda predicted 2701 target genes for 378 miRNAs, which could be categorized into 52 functional groups annotated by gene ontology. The function of miRNA target genes was found to be very similar between conserved and novel miRNAs. This study of miRNAs in L. striatellus will provide new information and enhance the understanding of the role of miRNAs in the regulation of L. striatellus metabolism and development. PMID:25057821

A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

DTIC Science & Technology

2015-05-01

structures that we reported earlier (Kryger et al [2000] Acta Crystallogr D Biol Crystallogr 56:1385-1394) were of complexes with the snake venom...interactions between conserved residues in the loop connecting α13 to α14 and residues from helices α18’-α19’, and, conversely, between residues in the...residues in the 4-helix bundle, including Glu376, Thr383, Asp384, Trp385, Gln508, Gln527, Phe535 and Lys538 (hAChE numbering), are strictly conserved in

Genetic distance of Malaysian mousedeer based on mitochondrial DNA cytochrome oxidase I (COI) and D-loop region sequences

NASA Astrophysics Data System (ADS)

Bakar, Mohamad-Azam Akmal Abu; Rovie-Ryan, Jeffrine Japning; Ampeng, Ahmad; Yaakop, Salmah; Nor, Shukor Md; Md-Zain, Badrul Munir

2018-04-01

Mousedeer is one of the primitive mammals that can be found mainly in Southeast-Asia region. There are two species of mousedeer in Malaysia which are Tragulus kanchil and Tragulus napu. Both species can be distinguish by size, coat coloration, and throat pattern but clear diagnosis still cannot be found. The objective of the study is to show the genetic distance relationship between T. kanchil and T. napu and their population based on mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and D-loop region. There are 42 sample of mousedeer were used in this study collected by PERHILITAN from different locality. Another 29 D-loop sequence were retrieved from Genbank for comparative analysis. All sample were amplified using universal primer and species-specific primer for COI and D-loop genes via PCR process. The amplified sequences were analyzed to determine genetic distance of T. kanchil and T. napu. From the analysis, the average genetic distance between T. kanchil and T. napu based on locus COI and D-loop were 0.145 and 0.128 respectively. The genetic distance between populations of T. kanchil based on locus COI was between 0.003-0.013. For locus D-loop, genetic distance analysis showed distance in relationship between west-coast populations to east-coast population of T. kanchil. COI and D-loop mtDNA region provided a clear picture on the relationship within the mousedeer species. Last but not least, conservation effort toward protecting this species can be done by study the molecular genetics and prevent the extinction of this species.

Low Speed and High Speed Correlation of SMART Active Flap Rotor Loads

NASA Technical Reports Server (NTRS)

Kottapalli, Sesi B. R.

2010-01-01

Measured, open loop and closed loop data from the SMART rotor test in the NASA Ames 40- by 80- Foot Wind Tunnel are compared with CAMRAD II calculations. One open loop high-speed case and four closed loop cases are considered. The closed loop cases include three high-speed cases and one low-speed case. Two of these high-speed cases include a 2 deg flap deflection at 5P case and a test maximum-airspeed case. This study follows a recent, open loop correlation effort that used a simple correction factor for the airfoil pitching moment Mach number. Compared to the earlier effort, the current open loop study considers more fundamental corrections based on advancing blade aerodynamic conditions. The airfoil tables themselves have been studied. Selected modifications to the HH-06 section flap airfoil pitching moment table are implemented. For the closed loop condition, the effect of the flap actuator is modeled by increased flap hinge stiffness. Overall, the open loop correlation is reasonable, thus confirming the basic correctness of the current semi-empirical modifications; the closed loop correlation is also reasonable considering that the current flap model is a first generation model. Detailed correlation results are given in the paper.

Positive selection in the SLC11A1 gene in the family Equidae.

PubMed

Bayerova, Zuzana; Janova, Eva; Matiasovic, Jan; Orlando, Ludovic; Horin, Petr

2016-05-01

Immunity-related genes are a suitable model for studying effects of selection at the genomic level. Some of them are highly conserved due to functional constraints and purifying selection, while others are variable and change quickly to cope with the variation of pathogens. The SLC11A1 gene encodes a transporter protein mediating antimicrobial activity of macrophages. Little is known about the patterns of selection shaping this gene during evolution. Although it is a typical evolutionarily conserved gene, functionally important polymorphisms associated with various diseases were identified in humans and other species. We analyzed the genomic organization, genetic variation, and evolution of the SLC11A1 gene in the family Equidae to identify patterns of selection within this important gene. Nucleotide SLC11A1 sequences were shown to be highly conserved in ten equid species, with more than 97 % sequence identity across the family. Single nucleotide polymorphisms (SNPs) were found in the coding and noncoding regions of the gene. Seven codon sites were identified to be under strong purifying selection. Codons located in three regions, including the glycosylated extracellular loop, were shown to be under diversifying selection. A 3-bp indel resulting in a deletion of the amino acid 321 in the predicted protein was observed in all horses, while it has been maintained in all other equid species. This codon comprised in an N-glycosylation site was found to be under positive selection. Interspecific variation in the presence of predicted N-glycosylation sites was observed.

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

Two different groups of signal sequence in M-superfamily conotoxins.

PubMed

Wang, Qi; Jiang, Hui; Han, Yu-Hong; Yuan, Duo-Duo; Chi, Cheng-Wu

2008-04-01

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

Computer-aided active-site-directed modeling of the Herpes Simplex Virus 1 and human thymidine kinase

NASA Astrophysics Data System (ADS)

Folkers, Gerd; Trumpp-Kallmeyer, Susanne; Gutbrod, Oliver; Krickl, Sabine; Fetzer, Jürgen; Keil, Günther M.

1991-10-01

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core β-sheet structure, connected by loops and α-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.

Beyond singular values and loop shapes

NASA Technical Reports Server (NTRS)

Stein, G.

1985-01-01

The status of singular value loop-shaping as a design paradigm for multivariable feedback systems is reviewed. It shows that this paradigm is an effective design tool whenever the problem specifications are spacially round. The tool can be arbitrarily conservative, however, when they are not. This happens because singular value conditions for robust performance are not tight (necessary and sufficient) and can severely overstate actual requirements. An alternate paradign is discussed which overcomes these limitations. The alternative includes a more general problem formulation, a new matrix function mu, and tight conditions for both robust stability and robust performance. The state of the art currently permits analysis of feedback systems within this new paradigm. Synthesis remains a subject of research.

RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription

PubMed Central

Huang, Xuhui; Wang, Dong; Weiss, Dahlia R.; Bushnell, David A.; Kornberg, Roger D.; Levitt, Michael

2010-01-01

A structurally conserved element, the trigger loop, has been suggested to play a key role in substrate selection and catalysis of RNA polymerase II (pol II) transcription elongation. Recently resolved X-ray structures showed that the trigger loop forms direct interactions with the β-phosphate and base of the matched nucleotide triphosphate (NTP) through residues His1085 and Leu1081, respectively. In order to understand the role of these two critical residues in stabilizing active site conformation in the dynamic complex, we performed all-atom molecular dynamics simulations of the wild-type pol II elongation complex and its mutants in explicit solvent. In the wild-type complex, we found that the trigger loop is stabilized in the “closed” conformation, and His1085 forms a stable interaction with the NTP. Simulations of point mutations of His1085 are shown to affect this interaction; simulations of alternative protonation states, which are inaccessible through experiment, indicate that only the protonated form is able to stabilize the His1085-NTP interaction. Another trigger loop residue, Leu1081, stabilizes the incoming nucleotide position through interaction with the nucleotide base. Our simulations of this Leu mutant suggest a three-component mechanism for correctly positioning the incoming NTP in which (i) hydrophobic contact through Leu1081, (ii) base stacking, and (iii) base pairing work together to minimize the motion of the incoming NTP base. These results complement experimental observations and provide insight into the role of the trigger loop on transcription fidelity. PMID:20798057

Oligomerization state of water channels and glycerol facilitators. Involvement of loop E.

PubMed

Lagrée, V; Froger, A; Deschamps, S; Pellerin, I; Delamarche, C; Bonnec, G; Gouranton, J; Thomas, D; Hubert, J F

1998-12-18

The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast or Xenopus oocytes behaves as an homotetramer when extracted by n-octyl beta-D-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins.

Distinct families of cis-acting RNA replication elements epsilon from hepatitis B viruses

PubMed Central

Chen, Augustine; Brown, Chris

2012-01-01

The hepadnavirus encapsidation signal, epsilon (ε), is an RNA structure located at the 5′ end of the viral pregenomic RNA. It is essential for viral replication and functions in polymerase protein binding and priming. This structure could also have potential regulatory roles in controlling the expression of viral replicative proteins. In addition to its structure, the primary sequence of this RNA element has crucial functional roles in the viral lifecycle. Although the ε elements in hepadnaviruses share common critical functions, there are some significant differences in mammalian and avian hepadnaviruses, which include both sequence and structural variations.   Here we present several covariance models for ε elements from the Hepadnaviridae. The model building included experimentally determined data from previous studies using chemical probing and NMR analysis. These models have sufficient similarity to comprise a clan. The clan has in common a highly conserved overall structure consisting of a lower-stem, bulge, upper-stem and apical-loop. The models differ in functionally critical regions—notably the two types of avian ε elements have a tetra-loop (UGUU) including a non-canonical UU base pair, while the hepatitis B virus (HBV) epsilon has a tri-loop (UGU). The avian epsilon elements have a less stable dynamic structure in the upper stem. Comparisons between these models and all other Rfam models, and searches of genomes, showed these structures are specific to the Hepadnaviridae. Two family models and the clan are available from the Rfam database. PMID:22418844

Architecture of interstitial nodal spaces in the rodent renal inner medulla.

PubMed

Gilbert, Rebecca L; Pannabecker, Thomas L

2013-09-01

Every collecting duct (CD) of the rat inner medulla is uniformly surrounded by about four abutting ascending vasa recta (AVR) running parallel to it. One or two ascending thin limbs (ATLs) lie between and parallel to each abutting AVR pair, opposite the CD. These structures form boundaries of axially running interstitial compartments. Viewed in transverse sections, these compartments appear as four interstitial nodal spaces (INSs) positioned symmetrically around each CD. The axially running compartments are segmented by interstitial cells spaced at regular intervals. The pairing of ATLs and CDs bounded by an abundant supply of AVR carrying reabsorbed water, NaCl, and urea make a strong argument that the mixing of NaCl and urea within the INSs and countercurrent flows play a critical role in generating the inner medullary osmotic gradient. The results of this study fully support that hypothesis. We quantified interactions of all structures comprising INSs along the corticopapillary axis for two rodent species, the Munich-Wistar rat and the kangaroo rat. The results showed remarkable similarities in the configurations of INSs, suggesting that the structural arrangement of INSs is a highly conserved architecture that plays a fundamental role in renal function. The number density of INSs along the corticopapillary axis directly correlated with a loop population that declines exponentially with distance below the outer medullary-inner medullary boundary. The axial configurations were consistent with discrete association between near-bend loop segments and INSs and with upper loop segments lying distant from INSs.

Architecture of interstitial nodal spaces in the rodent renal inner medulla

PubMed Central

Gilbert, Rebecca L.

2013-01-01

Every collecting duct (CD) of the rat inner medulla is uniformly surrounded by about four abutting ascending vasa recta (AVR) running parallel to it. One or two ascending thin limbs (ATLs) lie between and parallel to each abutting AVR pair, opposite the CD. These structures form boundaries of axially running interstitial compartments. Viewed in transverse sections, these compartments appear as four interstitial nodal spaces (INSs) positioned symmetrically around each CD. The axially running compartments are segmented by interstitial cells spaced at regular intervals. The pairing of ATLs and CDs bounded by an abundant supply of AVR carrying reabsorbed water, NaCl, and urea make a strong argument that the mixing of NaCl and urea within the INSs and countercurrent flows play a critical role in generating the inner medullary osmotic gradient. The results of this study fully support that hypothesis. We quantified interactions of all structures comprising INSs along the corticopapillary axis for two rodent species, the Munich-Wistar rat and the kangaroo rat. The results showed remarkable similarities in the configurations of INSs, suggesting that the structural arrangement of INSs is a highly conserved architecture that plays a fundamental role in renal function. The number density of INSs along the corticopapillary axis directly correlated with a loop population that declines exponentially with distance below the outer medullary-inner medullary boundary. The axial configurations were consistent with discrete association between near-bend loop segments and INSs and with upper loop segments lying distant from INSs. PMID:23825077

Analysis of crystal structure of Arabidopsis MPK6 and generation of its mutants with higher activity

PubMed Central

Wang, Bo; Qin, Xinghua; Wu, Juan; Deng, Hongying; Li, Yuan; Yang, Hailian; Chen, Zhongzhou; Liu, Guoqin; Ren, Dongtao

2016-01-01

Mitogen-activated protein kinase (MAPK) cascades, which are the highly conserved signalling modules in eukaryotic organisms, have been shown to play important roles in regulating growth, development, and stress responses. The structures of various MAPKs from yeast and animal have been solved, and structure-based mutants were generated for their function analyses, however, the structures of plant MAPKs remain unsolved. Here, we report the crystal structure of Arabidopsis MPK6 at a 3.0 Å resolution. Although MPK6 is topologically similar to ERK2 and p38, the structures of the glycine-rich loop, MAPK insert, substrate binding sites, and L16 loop in MPK6 show notable differences from those of ERK2 and p38. Based on the structural comparison, we constructed MPK6 mutants and analyzed their kinase activity both in vitro and in planta. MPK6F364L and MPK6F368L mutants, in which Phe364 and Phe368 in the L16 loop were changed to Leu, respectively, acquired higher intrinsic kinase activity and retained the normal MAPKK activation property. The expression of MPK6 mutants with basal activity is sufficient to induce camalexin biosynthesis; however, to induce ethylene and leaf senescence, the expression of MPK6 mutants with higher activity is required. The results suggest that these mutants can be used to analyze the specific biological functions of MPK6. PMID:27160427

Investigation of Inner Loop Flight Control Strategies for High-Speed Research

NASA Technical Reports Server (NTRS)

Newman, Brett; Kassem, Ayman

1999-01-01

This report describes the activities and findings conducted under contract NAS1-19858 with NASA Langley Research Center. Subject matter is the investigation of suitable flight control design methodologies and solutions for large, flexible high-speed vehicles. Specifically, methodologies are to address the inner control loops used for stabilization and augmentation of a highly coupled airframe system possibly involving rigid-body motion, structural vibrations, unsteady aerodynamics, and actuator dynamics. Techniques considered in this body of work are primarily conventional-based, and the vehicle of interest is the High-Speed Civil Transport (HSCT). Major findings include 1) current aeroelastic vehicle modeling procedures require further emphasis and refinement, 2) traditional and nontraditional inner loop flight control strategies employing a single feedback loop do not appear sufficient for highly flexible HSCT class vehicles, 3) inner loop flight control systems will, in all likelihood, require multiple interacting feedback loops, and 4) Ref. H HSCT configuration presents major challenges to designing acceptable closed-loop flight dynamics.

Loop Evolution Observed with AIA and Hi-C

NASA Technical Reports Server (NTRS)

Mulu-Moore, Fana; Winebarger, Amy R.; Cirtain, Jonathan W.; Kobayashi, Ken; Korreck, Kelly E.; Golub, Leon; Kuzin, Sergei; Walsh, Robert William; DeForest, Craig E.; De Pontieu, Bart;

A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain

USDA-ARS?s Scientific Manuscript database

Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62...

Human Y-Family DNA Polymerase κ Is More Tolerant to Changes in Its Active Site Loop than Its Ortholog Escherichia coli DinB.

PubMed

Antczak, Nicole M; Packer, Morgan R; Lu, Xueguang; Zhang, Ke; Beuning, Penny J

2017-11-20

DNA damage is a constant threat and can be bypassed in a process called translesion synthesis, which is typically carried out by Y-family DNA polymerases. Y-family DNA polymerases are conserved in all domains of life and tend to have specificity for certain types of DNA damage. Escherichia coli DinB and its human ortholog pol κ can bypass specific minor groove deoxyguanine adducts efficiently and are inhibited by major groove adducts, as Y-family DNA polymerases make contacts with the minor groove side of the DNA substrate and lack contacts with the major groove at the nascent base pair. DinB is inhibited by major groove adducts more than pol κ, and they each have active site loops of different lengths, with four additional amino acids in the DinB loop. We previously showed that the R35A active site loop mutation in DinB allows for bypass of the major groove adduct N 6 -furfuryl-dA. These observations led us to investigate the different active site loops by creating loop swap chimeras of DinB with a pol κ loop and vice versa by changing the loop residues in a stepwise fashion. We then determined their activity with undamaged DNA or DNA containing N 2 -furfuryl-dG or N 6 -furfuryl-dA. The DinB proteins with the pol kappa loop have low activity on all templates but have decreased misincorporation compared to either wild-type protein. The kappa proteins with the DinB loop retain activity on all templates and have decreased misincorporation compared to either wild-type protein. We assessed the thermal stability of the proteins and observed an increase in stability in the presence of all DNA templates and additional increases generally only in the presence of the undamaged and N 2 -furfuryl-dG adduct and dCTP, which correlates with activity. Overall we find that pol κ is more tolerant to changes in the active site loop than DinB.

The Universally Conserved Prokaryotic GTPases

PubMed Central

Verstraeten, Natalie; Fauvart, Maarten; Versées, Wim; Michiels, Jan

2011-01-01

Summary: Members of the large superclass of P-loop GTPases share a core domain with a conserved three-dimensional structure. In eukaryotes, these proteins are implicated in various crucial cellular processes, including translation, membrane trafficking, cell cycle progression, and membrane signaling. As targets of mutation and toxins, GTPases are involved in the pathogenesis of cancer and infectious diseases. In prokaryotes also, it is hard to overestimate the importance of GTPases in cell physiology. Numerous papers have shed new light on the role of bacterial GTPases in cell cycle regulation, ribosome assembly, the stress response, and other cellular processes. Moreover, bacterial GTPases have been identified as high-potential drug targets. A key paper published over 2 decades ago stated that, “It may never again be possible to capture [GTPases] in a family portrait” (H. R. Bourne, D. A. Sanders, and F. McCormick, Nature 348:125-132, 1990) and indeed, the last 20 years have seen a tremendous increase in publications on the subject. Sequence analysis identified 13 bacterial GTPases that are conserved in at least 75% of all bacterial species. We here provide an overview of these 13 protein subfamilies, covering their cellular functions as well as cellular localization and expression levels, three-dimensional structures, biochemical properties, and gene organization. Conserved roles in eukaryotic homologs will be discussed as well. A comprehensive overview summarizing current knowledge on prokaryotic GTPases will aid in further elucidating the function of these important proteins. PMID:21885683

Enzymatic properties of a GH19 chitinase isolated from rice lacking a major loop structure involved in chitin binding.

PubMed

Tanaka, Jun; Fukamizo, Tamo; Ohnuma, Takayuki

2017-05-01

The catalytic domains of family GH19 chitinases have been found to consist of a conserved, α-helical core-region and different numbers (1-6) of loop structures, located at both ends of the substrate-binding groove and which extend over the glycon- and aglycon-binding sites. We expressed, purified and enzymatically characterized a GH19 chitinase from rice, Oryza sativa L. cv. Nipponbare (OsChia2a), lacking a major loop structure (loop III) connected to the functionally important β-stranded region. The new enzyme thus contained the five remaining loop structures (loops I, II, IV, V and C-term). The OsChia2a recombinant protein catalyzed hydrolysis of chitin oligosaccharides, (GlcNAc)n (n = 3-6), with inversion of anomeric configuration, indicating that OsChia2a correctly folded without loop III. From thermal unfolding experiments and calorimetric titrations using the inactive OsChia2a mutant (OsChia2a-E68Q), in which the catalytic residue Glu68 was mutated to glutamine, we found that the binding affinities towards (GlcNAc)n (n = 2-6) were almost proportional to the degree of polymerization of (GlcNAc)n, but were much lower than those obtained for a moss GH19 chitinase having only loop III [Ohnuma T, Sørlie M, Fukuda T, Kawamoto N, Taira T, Fukamizo T. 2011. Chitin oligosaccharide binding to a family GH19 chitinase from the moss, Bryum coronatum. FEBS J. 278:3991-4001]. Nevertheless, OsChia2a exhibited significant antifungal activity. It appears that loop III connected to the β-stranded region is important for (GlcNAc)n binding, but is not essential for antifungal activity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

groEL is a suitable genetic marker for detecting Vibrio parahaemolyticus by loop-mediated isothermal amplification assay.

PubMed

Siddique, M P; Jang, W J; Lee, J M; Ahn, S H; Suraiya, S; Kim, C H; Kong, I S

2017-08-01

A groEL gene-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Vibrio parahaemolyticus in contaminated seafood and water. The assay was optimized and conducted at 63°C for 40 min using Bacillus stearothermophilus (Bst) DNA polymerase, large fragment. Amplification was analysed via multiple detection methods, including opacity, formation of white precipitate, DNA intercalating dyes (ethidium bromide and SYBR Green I), metal ion-binding indicator dye, calcein, and 2% agarose gel electrophoresis. A characteristic ladder-like band pattern on agarose gel and the desired colour changes when using different dyes were observed in positive cases, and these were species-specific for V. parahaemolyticus when compared with other closely related Vibrio spp. The limit of detection (LoD) of this assay was 100 fg per reaction, 100-fold higher than that for conventional polymerase chain reaction (PCR). When tested on artificially contaminated seafood and seawater, the LoDs of the LAMP assay were 120 and 150 fg per reaction respectively, and those of conventional PCR were 120 and 150 pg per reaction respectively. Based on our results, the groEL gene-based LAMP assay is rapid, specific, sensitive, and reliable for detecting V. parahaemolyticus, and it could be used in field diagnosis. The loop-mediated isothermal amplification (LAMP) assay using groEL gene (an abundant, highly conserved gene and member of the groESL chaperone gene family) provided rapid, species-specific and highly sensitive method for detecting Vibrio parahaemolyticus, the leading causal agent of seafood-borne diseases worldwide. Moreover, groEL LAMP revealed high efficiency than conventional PCR assay for V. parahaemolyticus using template both from pure culture and artificially contaminated seafood and water, which indicated the applicability in the field and environmental screening purpose for the organism. © 2017 The Society for Applied Microbiology.

Connecting Active-Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme.

PubMed

Pareek, Vidhi; Samanta, Moumita; Joshi, Niranjan V; Balaram, Hemalatha; Murthy, Mathur R N; Balaram, Padmanabhan

2016-04-01

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop-6 (residues 166-176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98% of the 6277 unique TIM sequences), spatially proximal to E165 and the loop-6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue--phenylalanine--at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop-6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

Amino acid sequence analysis of the annexin super-gene family of proteins.

PubMed

Barton, G J; Newman, R H; Freemont, P S; Crumpton, M J

1991-06-15

The annexins are a widespread family of calcium-dependent membrane-binding proteins. No common function has been identified for the family and, until recently, no crystallographic data existed for an annexin. In this paper we draw together 22 available annexin sequences consisting of 88 similar repeat units, and apply the techniques of multiple sequence alignment, pattern matching, secondary structure prediction and conservation analysis to the characterisation of the molecules. The analysis clearly shows that the repeats cluster into four distinct families and that greatest variation occurs within the repeat 3 units. Multiple alignment of the 88 repeats shows amino acids with conserved physicochemical properties at 22 positions, with only Gly at position 23 being absolutely conserved in all repeats. Secondary structure prediction techniques identify five conserved helices in each repeat unit and patterns of conserved hydrophobic amino acids are consistent with one face of a helix packing against the protein core in predicted helices a, c, d, e. Helix b is generally hydrophobic in all repeats, but contains a striking pattern of repeat-specific residue conservation at position 31, with Arg in repeats 4 and Glu in repeats 2, but unconserved amino acids in repeats 1 and 3. This suggests repeats 2 and 4 may interact via a buried saltbridge. The loop between predicted helices a and b of repeat 3 shows features distinct from the equivalent loop in repeats 1, 2 and 4, suggesting an important structural and/or functional role for this region. No compelling evidence emerges from this study for uteroglobin and the annexins sharing similar tertiary structures, or for uteroglobin representing a derivative of a primordial one-repeat structure that underwent duplication to give the present day annexins. The analyses performed in this paper are re-evaluated in the Appendix, in the light of the recently published X-ray structure for human annexin V. The structure confirms most of the predictions and shows the power of techniques for the determination of tertiary structural information from the amino acid sequences of an aligned protein family.

Point mutations in the extracytosolic loop between transmembrane segments M5 and M6 of the yeast Pma1 H+-ATPase: alanine-scanning mutagenesis.

PubMed

Petrov, Valery V

2015-01-01

Membrane-spanning segments M4, M5, M6, and M8 of the H(+)-, Ca(2+)-, and K(+), Na(+)-ATPases, which belong to the P2-type pumps are the core through which cations are transported. M5 and M6 loop is a short extracytoplasmic stretch of the seven amino acid residues (714-DNSLDID) connecting two of these segments, M5 and M6, where residues involved in the formation of the proton-binding site(s) are located. In the present study, we have used alanine-scanning mutagenesis to explore the structural and functional relationships within this loop of the yeast plasma membrane Pma1 H(+)-ATPase. Of the 7 Ala mutants made, substitution for the most conserved residue (Leu-717) has led to a severe misfolding and complete block in biogenesis of the mutant enzyme. The replacement of Asp-714 has also caused misfolding leading to significant decrease in the expression of the mutant and loss of activity. The remaining mutants were expressed in secretory vesicles at 21-119% of the wild-type level and were active enough to be analyzed in detail. One of these mutants (I719A) showed five- to threefold decrease in both expression and ATP hydrolyzing and H(+) pumping activities and also threefold reduction in the coupling ratio between ATP hydrolysis and H(+) transport. Thus, Ala substitutions at three positions of the seven seriously affected biogenesis, folding, stability and/or functioning of the enzyme. Taken together, these results lead to suggestion that M5 and M6 loop play an important role in the protein stability and function and is responsible for proper arrangement of transmembrane segments M5 and M6 and probably other domains of the enzyme. Results for additional conserved substitutions (Asn and Glu) at Asp-714 and Asp-720 confirmed this suggestion.

Design and simulation of permanent magnet synchronous motor control system

NASA Astrophysics Data System (ADS)

Li, Li; Liu, Yongqiu

2018-06-01

In recent years, with the development of power electronics, microelectronics, new motor control theory and rare earth permanent magnet materials, permanent magnet synchronous motors have been rapidly applied. Permanent magnet synchronous motors have the advantages of small size, low loss and high efficiency. Today, energy conservation and environmental protection are increasingly valued. It is very necessary to study them. Permanent magnet synchronous motor control system has a wide range of application prospects in the fields of electric vehicles, ships and other transportation. Using the simulation function of MATLAB/SIMULINK, a modular design structure was used to simulate the whole system model of speed loop adjustment, current PI modulation, SVPWM (Space Vector Pulse Width Module) wave generation and double closed loop. The results show that this control method has good robustness, and this method can improve the design efficiency and shorten the system design time. In this article, the analysis of the control principle of modern permanent magnet synchronous motor and the various processes of MATLAB simulation application will be analyzed in detail. The basic theory, basic method and application technology of the permanent magnet synchronous motor control system are systematically introduced.

Rapid detection of contagious ecthyma by loop-mediated isothermal amplification and epidemiology in Jilin Province China.

PubMed

Wang, Kai; Shao, Hongze; Pei, Zhihua; Hu, Guixue

2016-01-01

The aim of this experiment was to develop a loop-mediated isothermal amplification (LAMP) assay and to research the recent epidemiology of contagious ecthyma in Jilin Province, China, using the assay. A LAMP assay targeting a highly conserved region of the F1L gene was developed to detect contagious ecthyma virus (CEV). Three hundred and sixty-five cases from 64 flocks in 9 different areas of Jilin Province, China, from 2011 to 2014 were tested using the LAMP assay. The results showed that the sensitivity of the LAMP assay was 100 copies of the standard plasmid, which is 100-fold higher than the sensitivity of PCR. No cross-reactivity was observed with capripoxvirus, fowlpox virus, foot-and-mouth disease virus serotype O, foot-and-mouth disease virus serotype Asia I and bluetongue virus. The average positive rate was 19.73% (72/365), and the positive rate was highest in lambs aged 1-6 months. Our results demonstrated that CEV infection was very widespread in the flocks of Jilin Province and that the LAMP assay allows for easy, rapid, accurate and sensitive detection of CEV infection.

Dynamics of protein-protein interactions at the MscL periplasmic-lipid interface.

PubMed

Zhong, Dalian; Yang, Li-Min; Blount, Paul

2014-01-21

MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Robust Translation of the Nucleoid Protein Fis Requires a Remote Upstream AU Element and Is Enhanced by RNA Secondary Structure

PubMed Central

Nafissi, Maryam; Chau, Jeannette; Xu, Jimin

2012-01-01

Synthesis of the Fis nucleoid protein rapidly increases in response to nutrient upshifts, and Fis is one of the most abundant DNA binding proteins in Escherichia coli under nutrient-rich growth conditions. Previous work has shown that control of Fis synthesis occurs at transcription initiation of the dusB-fis operon. We show here that while translation of the dihydrouridine synthase gene dusB is low, unusual mechanisms operate to enable robust translation of fis. At least two RNA sequence elements located within the dusB coding region are responsible for high fis translation. The most important is an AU element centered 35 nucleotides (nt) upstream of the fis AUG, which may function as a binding site for ribosomal protein S1. In addition, a 44-nt segment located upstream of the AU element and predicted to form a stem-loop secondary structure plays a prominent role in enhancing fis translation. On the other hand, mutations close to the AUG, including over a potential Shine-Dalgarno sequence, have little effect on Fis protein levels. The AU element and stem-loop regions are phylogenetically conserved within dusB-fis operons of representative enteric bacteria. PMID:22389479

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Yao; Tan, Kemin; Chhor, Gekleng

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

Activation-dependent intrachromosomal interactions formed by the TNF gene promoter and two distal enhancers

PubMed Central

Tsytsykova, Alla V.; Rajsbaum, Ricardo; Falvo, James V.; Ligeiro, Filipa; Neely, Simon R.; Goldfeld, Anne E.

2007-01-01

Here we provide a mechanism for specific, efficient transcription of the TNF gene and, potentially, other genes residing within multigene loci. We identify and characterize highly conserved noncoding elements flanking the TNF gene, which undergo activation-dependent intrachromosomal interactions. These elements, hypersensitive site (HSS)−9 and HSS+3 (9 kb upstream and 3 kb downstream of the TNF gene, respectively), contain DNase I hypersensitive sites in naive, T helper 1, and T helper 2 primary T cells. Both HSS-9 and HSS+3 inducibly associate with acetylated histones, indicative of chromatin remodeling, bind the transcription factor nuclear factor of activated T cells (NFAT)p in vitro and in vivo, and function as enhancers of NFAT-dependent transactivation mediated by the TNF promoter. Using the chromosome conformation capture assay, we demonstrate that upon T cell activation intrachromosomal looping occurs in the TNF locus. HSS-9 and HSS+3 each associate with the TNF promoter and with each other, circularizing the TNF gene and bringing NFAT-containing nucleoprotein complexes into close proximity. TNF gene regulation thus reveals a mode of intrachromosomal interaction that combines a looped gene topology with interactions between enhancers and a gene promoter. PMID:17940009

PsRobot: a web-based plant small RNA meta-analysis toolbox.

PubMed

Wu, Hua-Jun; Ma, Ying-Ke; Chen, Tong; Wang, Meng; Wang, Xiu-Jie

2012-07-01

Small RNAs (smRNAs) in plants, mainly microRNAs and small interfering RNAs, play important roles in both transcriptional and post-transcriptional gene regulation. The broad application of high-throughput sequencing technology has made routinely generation of bulk smRNA sequences in laboratories possible, thus has significantly increased the need for batch analysis tools. PsRobot is a web-based easy-to-use tool dedicated to the identification of smRNAs with stem-loop shaped precursors (such as microRNAs and short hairpin RNAs) and their target genes/transcripts. It performs fast analysis to identify smRNAs with stem-loop shaped precursors among batch input data and predicts their targets using a modified Smith-Waterman algorithm. PsRobot integrates the expression data of smRNAs in major plant smRNA biogenesis gene mutants and smRNA-associated protein complexes to give clues to the smRNA generation and functional processes. Besides improved specificity, the reliability of smRNA target prediction results can also be evaluated by mRNA cleavage (degradome) data. The cross species conservation statuses and the multiplicity of smRNA target sites are also provided. PsRobot is freely accessible at http://omicslab.genetics.ac.cn/psRobot/.

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

PubMed Central

Surana, Parag

2016-01-01

Abstract The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process. PMID:27111887

CO2 Transport by PIP2 Aquaporins of Barley

PubMed Central

Mori, Izumi C.; Rhee, Jiye; Shibasaka, Mineo; Sasano, Shizuka; Kaneko, Toshiyuki; Horie, Tomoaki; Katsuhara, Maki

2014-01-01

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity. PMID:24406630

Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

PubMed Central

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized. PMID:24688696

Conformational sampling in template-free protein loop structure modeling: an overview.

PubMed

Li, Yaohang

2013-01-01

Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

Study of mitochondria D-loop gene to detect the heterogeneity of gemak in Turnicidae family

NASA Astrophysics Data System (ADS)

Setiati, N.; Partaya

2018-03-01

As a part of life biodiversity, birds in Turnicidae family should be preserved from the extinction and its type heterogeneity decline. One effort for giving the strategic base of plasma nutfah conservation is through genetic heterogeneity study. The aim of the research is to analyze D-loop gen from DNA mitochondria of gemak bird in Turnicidae family molecularly. From the result of the analysis, it may be known the genetic heterogeneity of gemak bird based on the sequence of D-loop gen. The collection of both types of gemak of Turnicidae family is still easy since we can find them in ricefield area after harvest particularly for Gemakloreng (Turnix sylvatica), it means while gemak tegalan (Turnixsusciator) is getting difficult to find. Based on the above DNA quantification standard, the blood sample of Gemak in this research is mostly grouped into pure blood (ranges from 1,63 – 1,90), and it deserves to be used for PCR analysis. The sequencing analysis has not detected the sequence of nucleotide completely. However, it indicates sequence polymorphism of base as the arranger of D-loop gen. D-loop gen may identify genetic heterogeneity of gemak bird of Turnicidae family, but it is necessary to perform further sequencing analysis with PCR-RFLP technique. This complete nucleotide sequence is obtained and easy to detect after being cut restriction enzyme.

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

PubMed Central

Jiang, Jun; Aduri, Raviprasad; Chow, Christine S.; SantaLucia, John

2014-01-01

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson–Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity. PMID:24371282

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain.

PubMed

Shi, Hang; Rojas, Raul; Bonifacino, Juan S; Hurley, James H

2006-06-01

The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.

Mechanism of foreign DNA selection in a bacterial adaptive immune system

PubMed Central

Sashital, Dipali G.; Wiedenheft, Blake; Doudna, Jennifer A.

2012-01-01

Summary In bacterial and archaeal CRISPR immune pathways, DNA sequences from invading bacteriophage or plasmids are integrated into CRISPR loci within the host genome, conferring immunity against subsequent infections. The ribonucleoprotein complex Cascade utilizes RNAs generated from these loci to target complementary “non-self” DNA sequences for destruction, while avoiding binding to “self” sequences within the CRISPR locus. Here we show that CasA, the largest protein subunit of Cascade, is required for non-self target recognition and binding. Combining a 2.3 Å crystal structure of CasA with cryo-EM structures of Cascade, we have identified a loop that is required for viral defense. This loop contacts a conserved 3-base pair motif that is required for non-self target selection. Our data suggest a model in which the CasA loop scans DNA for this short motif prior to target destabilization and binding, maximizing the efficiency of DNA surveillance by Cascade. PMID:22521690

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions

PubMed Central

Rashu, Rasheduzzaman; Begum, Yasmin Ara; Ciorba, Matthew A.; Hultgren, Scott J.; Qadri, Firdausi

2017-01-01

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens. PMID:28531220

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R., E-mail: grw7@cornell.ed

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined withmore » cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.« less

A Conserved Structural Motif Mediates Retrograde Trafficking of Shiga Toxin Types 1 and 2.

PubMed

Selyunin, Andrey S; Mukhopadhyay, Somshuvra

2015-12-01

Shiga toxin-producing Escherichia coli (STEC) produce two types of Shiga toxin (STx): STx1 and STx2. The toxin A-subunits block protein synthesis, while the B-subunits mediate retrograde trafficking. STEC infections do not have definitive treatments, and there is growing interest in generating toxin transport inhibitors for therapy. However, a comprehensive understanding of the mechanisms of toxin trafficking is essential for drug development. While STx2 is more toxic in vivo, prior studies focused on STx1 B-subunit (STx1B) trafficking. Here, we show that, compared with STx1B, trafficking of the B-subunit of STx2 (STx2B) to the Golgi occurs with slower kinetics. Despite this difference, similar to STx1B, endosome-to-Golgi transport of STx2B does not involve transit through degradative late endosomes and is dependent on dynamin II, epsinR, retromer and syntaxin5. Importantly, additional experiments show that a surface-exposed loop in STx2B (β4-β5 loop) is required for its endosome-to-Golgi trafficking. We previously demonstrated that residues in the corresponding β4-β5 loop of STx1B are required for interaction with GPP130, the STx1B-specific endosomal receptor, and for endosome-to-Golgi transport. Overall, STx1B and STx2B share a common pathway and use a similar structural motif to traffic to the Golgi, suggesting that the underlying mechanisms of endosomal sorting may be evolutionarily conserved. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

Identification and characterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing

PubMed Central

2012-01-01

Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression. MiRNAs play essential roles in almost all plant biological processes. Currently, few miRNAs have been identified in the model food legume Phaseolus vulgaris (common bean). Recent advances in next generation sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used Illumina's sequencing by synthesis (SBS) technology to identify and characterize the miRNA population of Phaseolus vulgaris. Results Small RNA libraries were generated from roots, flowers, leaves, and seedlings of P. vulgaris. Based on similarity to previously reported plant miRNAs,114 miRNAs belonging to 33 conserved miRNA families were identified. Stem-loop precursors and target gene sequences for several conserved common bean miRNAs were determined from publicly available databases. Less conserved miRNA families and species-specific common bean miRNA isoforms were also characterized. Moreover, novel miRNAs based on the small RNAs were found and their potential precursors were predicted. In addition, new target candidates for novel and conserved miRNAs were proposed. Finally, we studied organ-specific miRNA family expression levels through miRNA read frequencies. Conclusions This work represents the first massive-scale RNA sequencing study performed in Phaseolus vulgaris to identify and characterize its miRNA population. It significantly increases the number of miRNAs, precursors, and targets identified in this agronomically important species. The miRNA expression analysis provides a foundation for understanding common bean miRNA organ-specific expression patterns. The present study offers an expanded picture of P. vulgaris miRNAs in relation to those of other legumes. PMID:22394504

Superheat recovery system shakes savings out of A/C systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

1985-09-01

One of the most productive and least expensive methods of recovering waste heat is a system that reclaims the excess energy or superheat generated by closed-loop-air conditioning or refrigeration cycles. Installed recently in 72 Steak N' Shakes restaurants as part of a total energy conservation package, it has helped cut gas bills by more than 70%.

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

PubMed

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Accumulation of dislocation loops in the α phase of Zr Excel alloy under heavy ion irradiation

NASA Astrophysics Data System (ADS)

Yu, Hongbing; Yao, Zhongwen; Idrees, Yasir; Zhang, He K.; Kirk, Mark A.; Daymond, Mark R.

2017-08-01

In-situ heavy ion irradiations were performed on the high Sn content Zr alloy 'Excel', measuring type dislocation loop accumulation up to irradiation damage doses of 10 dpa at a range of temperatures. The high content of Sn, which diffuses slowly, and the thin foil geometry of the sample provide a unique opportunity to study an extreme case where displacement cascades dominate the loop formation and evolution. The dynamic observation of dislocation loop evolution under irradiation at 200 °C reveals that type dislocation loops can form at very low dose (0.0025 dpa). The size of the dislocation loops increases slightly with irradiation damage dose. The mechanism controlling loop growth in this study is different from that in neutron irradiation; in this study, larger dislocation loops can condense directly from the interaction of displacement cascades and the high concentration of point defects in the matrix. The size of the dislocation loop is dependent on the point defect concentration in the matrix. A negative correlation between the irradiation temperature and the dislocation loop size was observed. A comparison between cascade dominated loop evolution (this study), diffusion dominated loop evolution (electron irradiation) and neutron irradiation suggests that heavy ion irradiation alone may not be enough to accurately reproduce neutron irradiation induced loop structures. An alternative method is proposed in this paper. The effects of Sn on the displacement cascades, defect yield, and the diffusion behavior of point defects are established.

Dual-circuit, multiple-effect refrigeration system and method

DOEpatents

DeVault, Robert C.

1995-01-01

A dual circuit absorption refrigeration system comprising a high temperature single-effect refrigeration loop and a lower temperature double-effect refrigeration loop separate from one another and provided with a double-condenser coupling therebetween. The high temperature condenser of the single-effect refrigeration loop is double coupled to both of the generators in the double-effect refrigeration loop to improve internal heat recovery and a heat and mass transfer additive such as 2-ethyl-1-hexanol is used in the lower temperature double-effect refrigeration loop to improve the performance of the absorber in the double-effect refrigeration loop.

High Torque-to-Inertia Servo System for Stabilizing Sensor Systems. Candidate Systems Include Missile Guidance, Surveillance, and Tracking

DTIC Science & Technology

1980-04-01

specifications ... 3-10 25. Typical isolation curve ... 3-12 26. Servo amp/motor/load frequency response (inner gimbal) ... 4-3 27. Slave loop ( open loop...slave loop ( open loop) frequency response (inner gimbal) . . . 4-4 30. Slave loop (closed loop) frequency response (inner gimbal) ... 4-5 3 . Slave...loop inner gimbal time response ... 4-5 32. Servo amp/motor/load frequency response (outer gimbal) ... 4-6 33. Slave loop ( open loop) uncompensated

A decision-directed network for dual-polarization crosstalk cancellation

NASA Technical Reports Server (NTRS)

Weber, W. J., III

1979-01-01

Frequency reuse in the specific form of dual-polarized microwave communication systems has grown in importance in recent years as a practical means of radio spectrum conservation. Ideally the capacity of a given frequency allocation can be doubled through dual-polarization. However, hardware imperfections and propagation effects, particularly rain depolarization, prevent the achievement of this doubling without severe system performance degradation. A decision-directed cross-polarization correction network is presented whose operation depends on only simple base-band signal processing. No pilot tones or frequency offsets are required. The loop can work with any two-dimensional signal set for digital data transmission. The loop has been experimentally verified and provides a means of doubling the data capacity with little performance degradation.

47 CFR 51.319 - Specific unbundling requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... copper loop and a second competitive LEC provides digital subscriber line service over the high frequency portion of that same loop. The high frequency portion of the loop consists of the frequency range on the... complete transmission path on the high frequency range between the incumbent LEC's distribution frame (or...

47 CFR 51.319 - Specific unbundling requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... copper loop and a second competitive LEC provides digital subscriber line service over the high frequency portion of that same loop. The high frequency portion of the loop consists of the frequency range on the... complete transmission path on the high frequency range between the incumbent LEC's distribution frame (or...

Paranodal reorganization results in the depletion of transverse bands in the aged central nervous system

PubMed Central

Shepherd, Mark N.; Pomicter, Anthony D.; Velazco, Cristine S.; Henderson, Scott C.; Dupree, Jeffrey L.

2012-01-01

Paranodal axo-glial junctional complexes anchor the myelin sheath to the axon and breakdown of these complexes presumably facilitates demyelination. Myelin deterioration is also prominent in the aging central nervous system (CNS); however, the stability of the paranodal complexes in the aged CNS has not been examined. Here, we show that transverse bands, prominent components of paranodal junctions, are significantly reduced in the aged CNS; however, the number of paired clusters of both myelin and axonal paranodal proteins is not altered. Ultrastructural analyses also reveal that thicker myelin sheaths display a “piling” of paranodal loops, the cytoplasm-containing sacs that demarcate the paranode. Loops involved in piling are observed throughout the paranode and are not limited to loops positioned in either the nodal- or juxtanodal-most regions. Here, we propose that as myelination continues, previously anchored loops lose their transverse bands and recede away from the axolemma. Newly juxtaposed loops then lose their transverse bands, move laterally to fill in the gap left by the receded loops and finally reform their transverse bands. This paranodal reorganization results in conservation of paranodal length, which may be important in maintaining ion channel spacing and axonal function. Furthermore, we propose that transverse band reformation is less efficient in the aged CNS, resulting in the significant reduction of these junctional components. Although demyelination was not observed, we propose that loss of transverse bands facilitates myelin degeneration and may predispose the aged CNS to a poorer prognosis following a secondary insult. PMID:20888080

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ling, Jiqiang; Peterson, Kaitlyn M.; Simonovic, Ivana

2014-03-12

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular ?? with a threonine (Thr) anticodon, MST1 also recognizes an unusual ??, which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employsmore » distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for ?? recognition, whereas the anticodon sequence is essential for aminoacylation of ??. The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate ??, reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix {alpha}11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.« less

Open-Loop HIRF Experiments Performed on a Fault Tolerant Flight Control Computer

NASA Technical Reports Server (NTRS)

Koppen, Daniel M.

1997-01-01

During the third quarter of 1996, the Closed-Loop Systems Laboratory was established at the NASA Langley Research Center (LaRC) to study the effects of High Intensity Radiated Fields on complex avionic systems and control system components. This new facility provided a link and expanded upon the existing capabilities of the High Intensity Radiated Fields Laboratory at LaRC that were constructed and certified during 1995-96. The scope of the Closed-Loop Systems Laboratory is to place highly integrated avionics instrumentation into a high intensity radiated field environment, interface the avionics to a real-time flight simulation that incorporates aircraft dynamics, engines, sensors, actuators and atmospheric turbulence, and collect, analyze, and model aircraft performance. This paper describes the layout and functionality of the Closed-Loop Systems Laboratory, and the open-loop calibration experiments that led up to the commencement of closed-loop real-time flight experiments.

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC. PMID:24754906

Sequence Comparisons of Odorant Receptors among Tortricid Moths Reveal Different Rates of Molecular Evolution among Family Members

PubMed Central

Carraher, Colm; Authier, Astrid; Steinwender, Bernd; Newcomb, Richard D.

2012-01-01

In insects, odorant receptors detect volatile cues involved in behaviours such as mate recognition, food location and oviposition. We have investigated the evolution of three odorant receptors from five species within the moth genera Ctenopseustis and Planotrotrix, family Tortricidae, which fall into distinct clades within the odorant receptor multigene family. One receptor is the orthologue of the co-receptor Or83b, now known as Orco (OR2), and encodes the obligate ion channel subunit of the receptor complex. In comparison, the other two receptors, OR1 and OR3, are ligand-binding receptor subunits, activated by volatile compounds produced by plants - methyl salicylate and citral, respectively. Rates of sequence evolution at non-synonymous sites were significantly higher in OR1 compared with OR2 and OR3. Within the dataset OR1 contains 109 variable amino acid positions that are distributed evenly across the entire protein including transmembrane helices, loop regions and termini, while OR2 and OR3 contain 18 and 16 variable sites, respectively. OR2 shows a high level of amino acid conservation as expected due to its essential role in odour detection; however we found unexpected differences in the rate of evolution between two ligand-binding odorant receptors, OR1 and OR3. OR3 shows high sequence conservation suggestive of a conserved role in odour reception, whereas the higher rate of evolution observed in OR1, particularly at non-synonymous sites, may be suggestive of relaxed constraint, perhaps associated with the loss of an ancestral role in sex pheromone reception. PMID:22701634

Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41

PubMed Central

Seaman, Mike S.; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

2010-01-01

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool. PMID:21124990

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

PubMed

Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

2010-11-18

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

Structural conservation, variability, and immunogenicity of the T6 backbone pilin of serotype M6 Streptococcus pyogenes.

PubMed

Young, Paul G; Moreland, Nicole J; Loh, Jacelyn M; Bell, Anita; Atatoa Carr, Polly; Proft, Thomas; Baker, Edward N

2014-07-01

Group A streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive human pathogen that causes a broad range of diseases ranging from acute pharyngitis to the poststreptococcal sequelae of acute rheumatic fever. GAS pili are highly diverse, long protein polymers that extend from the cell surface. They have multiple roles in infection and are promising candidates for vaccine development. This study describes the structure of the T6 backbone pilin (BP; Lancefield T-antigen) from the important M6 serotype. The structure reveals a modular arrangement of three tandem immunoglobulin-like domains, two with internal isopeptide bonds. The T6 pilin lysine, essential for polymerization, is located in a novel VAKS motif that is structurally homologous to the canonical YPKN pilin lysine in other three- and four-domain Gram-positive pilins. The T6 structure also highlights a conserved pilin core whose surface is decorated with highly variable loops and extensions. Comparison to other Gram-positive BPs shows that many of the largest variable extensions are found in conserved locations. Studies with sera from patients diagnosed with GAS-associated acute rheumatic fever showed that each of the three T6 domains, and the largest of the variable extensions (V8), are targeted by IgG during infection in vivo. Although the GAS BP show large variations in size and sequence, the modular nature of the pilus proteins revealed by the T6 structure may aid the future design of a pilus-based vaccine. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

PubMed Central

Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

2017-01-01

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

A Close Loop Low-Power and High Speed 130 nm CMOS Sample and Hold Circuit Based on Switched Capacitor for ADC Module

NASA Astrophysics Data System (ADS)

Nasir, Z.; Ruslan, S. H.

2017-08-01

A sample and hold (S/H) block is typically used as an analogue to digital interface in the analogue to digital converter (ADC) system. Since ADC is widely used in processing signals, the power consumption of the ADC must be lowered to conserve energy. Therefore the S/H circuit must be of a low powered too. Sampling phase and hold phase are the two phases of the operation cycle of the S/H circuit. Switched capacitor (SC) techniques have been developed in order to allow the integration on a single silicon chip of both digital and analogue functions. By controlling switches around the SC, the SC circuit works by passing charge into and out of a capacitor. SC circuits are suitable for on chip implementations because they replace a resistor with switches and capacitors. In this research, a closed-loop sample and hold circuit based on SC is designed and simulated with Cadence EDA tools. The schematic, layout, and simulation of the circuit is done using generic Silterra 130 nm technology file. All the analysis is done using Virtuoso Analog Design Environment. Layout and schematic are drawn using Virtuoso Schematic Editor and Virtuoso Layout Editor, Calibre is used for post layout simulation. The closed loop S/H circuit based on SC is successfully designed and able to sample and hold the analogue input waveform. The power consumption of the circuit is 0.919 mW and the propagation delay is 64.96 ps.

High-Throughput Sequencing Reveals Diverse Sets of Conserved, Nonconserved, and Species-Specific miRNAs in Jute

PubMed Central

Islam, Md. Tariqul; Ferdous, Ahlan Sabah; Najnin, Rifat Ara; Sarker, Suprovath Kumar; Khan, Haseena

2015-01-01

MicroRNAs play a pivotal role in regulating a broad range of biological processes, acting by cleaving mRNAs or by translational repression. A group of plant microRNAs are evolutionarily conserved; however, others are expressed in a species-specific manner. Jute is an agroeconomically important fibre crop; nonetheless, no practical information is available for microRNAs in jute to date. In this study, Illumina sequencing revealed a total of 227 known microRNAs and 17 potential novel microRNA candidates in jute, of which 164 belong to 23 conserved families and the remaining 63 belong to 58 nonconserved families. Among a total of 81 identified microRNA families, 116 potential target genes were predicted for 39 families and 11 targets were predicted for 4 among the 17 identified novel microRNAs. For understanding better the functions of microRNAs, target genes were analyzed by Gene Ontology and their pathways illustrated by KEGG pathway analyses. The presence of microRNAs identified in jute was validated by stem-loop RT-PCR followed by end point PCR and qPCR for randomly selected 20 known and novel microRNAs. This study exhaustively identifies microRNAs and their target genes in jute which will ultimately pave the way for understanding their role in this crop and other crops. PMID:25861616

Bandwidth controller for phase-locked-loop

NASA Technical Reports Server (NTRS)

Brockman, Milton H. (Inventor)

1992-01-01

A phase locked loop utilizing digital techniques to control the closed loop bandwidth of the RF carrier phase locked loop in a receiver provides high sensitivity and a wide dynamic range for signal reception. After analog to digital conversion, a digital phase locked loop bandwidth controller provides phase error detection with automatic RF carrier closed loop tracking bandwidth control to accommodate several modes of transmission.

Filter for third order phase locked loops

NASA Technical Reports Server (NTRS)

Crow, R. B.; Tausworthe, R. C. (Inventor)

1973-01-01

Filters for third-order phase-locked loops are used in receivers to acquire and track carrier signals, particularly signals subject to high doppler-rate changes in frequency. A loop filter with an open-loop transfer function and set of loop constants, setting the damping factor equal to unity are provided.

Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

PubMed Central

Lin, Jia-Hwei; Levin, Henry L.

1998-01-01

An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5′ end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function. PMID:9774699

Water Stream "Loop-the-Loop"

ERIC Educational Resources Information Center

Jefimenko, Oleg

1974-01-01

Discusses the design of a modified loop-the-loop apparatus in which a water stream is used to illustrate centripetal forces and phenomena of high-velocity hydrodynamics. Included are some procedures of carrying out lecture demonstrations. (CC)

A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pejchal, Robert; Doores, Katie J.; Walker, Laura M.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment ofmore » the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.« less

A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

PubMed

Ulbricht, David; Pippel, Jan; Schultz, Stephan; Meier, René; Sträter, Norbert; Heiker, John T

2015-09-15

SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin. © 2015 Authors; published by Portland Press Limited.

SL1 revisited: functional analysis of the structure and conformation of HIV-1 genome RNA.

PubMed

Sakuragi, Sayuri; Yokoyama, Masaru; Shioda, Tatsuo; Sato, Hironori; Sakuragi, Jun-Ichi

2016-11-11

The dimer initiation site/dimer linkage sequence (DIS/DLS) region of HIV is located on the 5' end of the viral genome and suggested to form complex secondary/tertiary structures. Within this structure, stem-loop 1 (SL1) is believed to be most important and an essential key to dimerization, since the sequence and predicted secondary structure of SL1 are highly stable and conserved among various virus subtypes. In particular, a six-base palindromic sequence is always present at the hairpin loop of SL1 and the formation of kissing-loop structure at this position between the two strands of genomic RNA is suggested to trigger dimerization. Although the higher-order structure model of SL1 is well accepted and perhaps even undoubted lately, there could be stillroom for consideration to depict the functional SL1 structure while in vivo (in virion or cell). In this study, we performed several analyses to identify the nucleotides and/or basepairing within SL1 which are necessary for HIV-1 genome dimerization, encapsidation, recombination and infectivity. We unexpectedly found that some nucleotides that are believed to contribute the formation of the stem do not impact dimerization or infectivity. On the other hand, we found that one G-C basepair involved in stem formation may serve as an alternative dimer interactive site. We also report on our further investigation of the roles of the palindromic sequences on viral replication. Collectively, we aim to assemble a more-comprehensive functional map of SL1 on the HIV-1 viral life cycle. We discovered several possibilities for a novel structure of SL1 in HIV-1 DLS. The newly proposed structure model suggested that the hairpin loop of SL1 appeared larger, and genome dimerization process might consist of more complicated mechanism than previously understood. Further investigations would be still required to fully understand the genome packaging and dimerization of HIV.

Role of loop L5-6 connecting transmembrane segments M5 and M6 in biogenesis and functioning of yeast Pma1 H+-ATPase.

PubMed

Petrov, V V

2015-01-01

The L5-6 loop is a short extracytoplasmic stretch (714-DNSLDID) connecting transmembrane segments M5 and M6 and forming along with segments M4 and M8 the core through which cations are transported by H+-, Ca2+-, K+,Na+-, H+,K+-, and other P2-ATPases. To study structure-function relationships within this loop of the yeast plasma membrane Pma1 H+-ATPase, alanine- and cysteine-scanning mutagenesis has been employed. Ala and Cys substitutions for the most conserved residue (Leu717) led to complete block in biogenesis preventing the enzyme from reaching secretory vesicles. The Ala replacement at Asp714 led to five-fold decrease in the mutant expression and loss of its activity, while the Cys substitution blocked biogenesis completely. Replacements of other residues did not lead to loss of enzymatic activity. Additional replacements were made for Asp714 and Asp720 (Asp®Asn/Glu). Of the substitutions made at Asp714, only D714N partially restored the mutant enzyme biogenesis and functioning. However, all mutant enzymes with substituted Asp720 were active. The expressed mutants (34-95% of the wild-type level) showed activity high enough (35-108%) to be analyzed in detail. One of the mutants (I719A) had three-fold reduced coupling ratio between ATP hydrolysis and H+ transport; however, the I719C mutation was rather indistinguishable from the wild-type enzyme. Thus, substitutions at two of the seven positions seriously affected biogenesis and/or functioning of the enzyme. Taken together, these results suggest that the M5-M6 loop residues play an important role in protein stability and function, and they are probably responsible for proper arrangement of transmembrane segments M5 and M6 and other domains of the enzyme. This might also be important for the regulation of the enzyme.

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits

PubMed Central

2016-01-01

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the “stiffness site” affects filament mechanical properties. Incorporating a magnesium ion in the “polymerization site” does not seem to require any large-scale change to an actin subunit’s conformation. Binding of a magnesium ion in the “stiffness site” adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids. PMID:27146246

Isolated and coupled superquadric loop antennas for mobile communications applications

NASA Technical Reports Server (NTRS)

Jensen, Michael A.; Rahmat-Samii, Yahya

1993-01-01

This work provides an investigation of the performance of loop antennas for use in mobile communications applications. The analysis tools developed allow for high flexibility by representing the loop antenna as a superquadric curve, which includes the case of circular, elliptical, and rectangular loops. The antenna may be in an isolated environment, located above an infinite ground plane, or placed near a finite conducting plate or box. In cases where coupled loops are used, the two loops may have arbitrary relative positions and orientations. Several design examples are included to illustrate the versatility of the analysis capabilities. The performance of coupled loops arranged in a diversity scheme is also evaluated, and it is found that high diversity gain can be achieved even when the antennas are closely spaced.

Structural variations of single and tandem mismatches in RNA duplexes: a joint MD simulation and crystal structure database analysis.

PubMed

Halder, Sukanya; Bhattacharyya, Dhananjay

2012-10-04

Internal loops within RNA duplex regions are formed by single or tandem basepairing mismatches with flanking canonical Watson-Crick basepairs on both sides. They are the most common motif observed in RNA secondary structures and play integral functional and structural roles. In this report, we have studied the structural features of 1 × 1, 2 × 2, and 3 × 3 internal loops using all-atom molecular dynamics (MD) simulation technique with explicit solvent model. As MD simulation is intricately dependent on the choice of force-field and these are often rather approximate, we have used both the most popular force-fields for nucleic acids-CHARMM27 and AMBER94-for a comparative analysis. We find that tandem noncanonical basepairs forming 2 × 2 and 3 × 3 internal loops are considerably more stable than the single mismatches forming 1 × 1 internal loops, irrespective of the force field. We have also analyzed crystal structure database to study the conservation of these helical fragments in the corresponding sets of RNA structures. We observe that the nature of stability in MD simulations mimic their fluctuating natures in crystal data sets also, probably indicating reliable natures of both the force fields to reproduce experimental results. We also notice significant structural changes in the wobble G:U basepairs present in these double helical stretches, leading to a biphasic stability for these wobble pairs to release the deformational strains introduced by internal loops within duplex regions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abraitiene, Asta; US Department of Agriculture, Agricultural Research Service, Molecular Plant Pathology Laboratory, Room 214 Building 004 BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705; Zhao Yan

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequentmore » GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.« less

Harwell high pressure heat transfer loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, A.W.; Keeys, R.K.F.

1967-12-15

A detailed description is presented of the Harwell (Chemical Engineering and Process Technology Division) high pressure, steam-water heat transfer loop; this description is aimed at supplementing the information given in reports on individual experiments. The operating instructions for the loop are given in an appendix. (auth)

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peisach, D.; Gee, P.; Kent, K.

2010-03-08

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline bindingmore » site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.« less

Closed coronal structures. V - Gasdynamic models of flaring loops and comparison with SMM observations

NASA Technical Reports Server (NTRS)

Peres, G.; Serio, S.; Vaiana, G.; Acton, L.; Leibacher, J.; Rosner, R.; Pallavicini, R.

1983-01-01

A time-dependent one-dimensional code incorporating energy, momentum and mass conservation equations, and taking the entire solar atmospheric structure into account, is used to investigate the hydrodynamic response of confined magnetic structures to strong heating perturbations. Model calculation results are compared with flare observations which include the light curves of spectral lines formed over a wide range of coronal flare temperatures, as well as determinations of Doppler shifts for the high temperature plasma. It is shown that the numerical simulation predictions are in good overall agreement with the observed flare coronal plasma evolution, correctly reproducing the temporal profile of X-ray spectral lines and their relative intensities. The predicted upflow velocities support the interpretation of the blueshifts as due to evaporation of chromospheric material.

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

PubMed Central

Conaty, J; Hendry, P; Lockett, T

1999-01-01

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system. PMID:10325431

Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

PubMed Central

Wheatley, Robert W.; Lo, Summie; Jancewicz, Larisa J.; Dugdale, Megan L.; Huber, Reuben E.

2013-01-01

β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution. PMID:23486479

Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate Euplotes octocarinatus centrin.

PubMed

Liu, Wen; Duan, Lian; Sun, Tijian; Yang, Binsheng

2016-12-01

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb 3+ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca 2+ and Tb 3+ ) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb 3+ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.

Crystal structures of the apo and ATP bound Mycobacterium tuberculosis nitrogen regulatory PII protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shetty, Nishant D.; Reddy, Manchi C.M.; Palaninathan, Satheesh K.

2010-10-11

PII constitutes a family of signal transduction proteins that act as nitrogen sensors in microorganisms and plants. Mycobacterium tuberculosis (Mtb) has a single homologue of PII whose precise role has as yet not been explored. We have solved the crystal structures of the Mtb PII protein in its apo and ATP bound forms to 1.4 and 2.4 {angstrom} resolutions, respectively. The protein forms a trimeric assembly in the crystal lattice and folds similarly to the other PII family proteins. The Mtb PII:ATP binary complex structure reveals three ATP molecules per trimer, each bound between the base of the T-loop ofmore » one subunit and the C-loop of the neighboring subunit. In contrast to the apo structure, at least one subunit of the binary complex structure contains a completely ordered T-loop indicating that ATP binding plays a role in orienting this loop region towards target proteins like the ammonium transporter, AmtB. Arg38 of the T-loop makes direct contact with the {gamma}-phosphate of the ATP molecule replacing the Mg{sup 2+} position seen in the Methanococcus jannaschii GlnK1 structure. The C-loop of a neighboring subunit encloses the other side of the ATP molecule, placing the GlnK specific C-terminal 3{sub 10} helix in the vicinity. Homology modeling studies with the E. coli GlnK:AmtB complex reveal that Mtb PII could form a complex similar to the complex in E. coli. The structural conservation and operon organization suggests that the Mtb PII gene encodes for a GlnK protein and might play a key role in the nitrogen regulatory pathway.« less

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

PubMed

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

Pub1p C-Terminal RRM Domain Interacts with Tif4631p through a Conserved Region Neighbouring the Pab1p Binding Site

PubMed Central

Rico-Lastres, Palma; Pérez-Cañadillas, José Manuel

2011-01-01

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1–402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role. PMID:21931728

Concerted action of the MutLβ heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion

PubMed Central

Duroc, Yann; Kumar, Rajeev; Ranjha, Lepakshi; Adam, Céline; Guérois, Raphaël; Md Muntaz, Khan; Marsolier-Kergoat, Marie-Claude; Dingli, Florent; Laureau, Raphaëlle; Loew, Damarys; Llorente, Bertrand; Charbonnier, Jean-Baptiste; Cejka, Petr; Borde, Valérie

2017-01-01

Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutLβ complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutLβ preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutLβ is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations. DOI: http://dx.doi.org/10.7554/eLife.21900.001 PMID:28051769

Homology modeling study toward identifying structural properties in the HA2 B-loop that would influence the HA1 receptor-binding site.

PubMed

Cueno, Marni E; Imai, Kenichi; Shimizu, Kazufumi; Ochiai, Kuniyasu

2013-07-01

Influenza hemagglutinin (HA) consists of a fibrous globular stem (HA2) inserted into the viral membrane supporting a globular head (HA1). HA1 receptor-binding has been hypothesized to be structurally correlated to the HA2 B-loop, however, this was never fully understood. Here, we elucidated the structural relationship between the HA2 B-loop and the HA1 receptor-binding site (RBS). Throughout this study, we analyzed 2486 H1N1 HA homology models obtained from human, swine and avian strains during 1976-2012. Quality of all homology models were verified before further analyses. We established that amino acid residue 882 is putatively strain-conserved and differs in the human (K882), swine (H882) and avian (N882) strains. Moreover, we observed that the amino acid at residue 882 and, similarly, its orientation has the potential to influence the HA1 RBS diameter measurements which we hypothesize may consequentially affect influenza H1N1 viral infectivity, immune escape, transmissibility, and evolution. Copyright © 2013 Elsevier Inc. All rights reserved.

Scattering of glue by glue on the light-cone worldsheet: Helicity nonconserving amplitudes

NASA Astrophysics Data System (ADS)

Chakrabarti, D.; Qiu, J.; Thorn, C. B.

2005-09-01

We give the light-cone gauge calculation of the one-loop on-shell scattering amplitudes for gluon-gluon scattering which violate helicity conservation. We regulate infrared divergences by discretizing the p+ integrations, omitting the terms with p+=0. Collinear divergences are absent diagram by diagram for the helicity nonconserving amplitudes. We also employ a novel ultraviolet regulator that is natural for the light-cone worldsheet description of planar Feynman diagrams. We show that these regulators give the known answers for the helicity nonconserving one-loop amplitudes, which do not suffer from the usual infrared vagaries of massless particle scattering. For the maximal helicity violating process we elucidate the physics of the remarkable fact that the loop momentum integrand for the on-shell Green function associated with this process, with a suitable momentum routing of the different contributing topologies, is identically zero. We enumerate the counterterms that must be included to give Lorentz covariant results to this order, and we show that they can be described locally in the light-cone worldsheet formulation of the sum of planar diagrams.

Regulated Formation of lncRNA-DNA Hybrids Enables Faster Transcriptional Induction and Environmental Adaptation.

PubMed

Cloutier, Sara C; Wang, Siwen; Ma, Wai Kit; Al Husini, Nadra; Dhoondia, Zuzer; Ansari, Athar; Pascuzzi, Pete E; Tran, Elizabeth J

2016-02-04

Long non-coding (lnc)RNAs, once thought to merely represent noise from imprecise transcription initiation, have now emerged as major regulatory entities in all eukaryotes. In contrast to the rapidly expanding identification of individual lncRNAs, mechanistic characterization has lagged behind. Here we provide evidence that the GAL lncRNAs in the budding yeast S. cerevisiae promote transcriptional induction in trans by formation of lncRNA-DNA hybrids or R-loops. The evolutionarily conserved RNA helicase Dbp2 regulates formation of these R-loops as genomic deletion or nuclear depletion results in accumulation of these structures across the GAL cluster gene promoters and coding regions. Enhanced transcriptional induction is manifested by lncRNA-dependent displacement of the Cyc8 co-repressor and subsequent gene looping, suggesting that these lncRNAs promote induction by altering chromatin architecture. Moreover, the GAL lncRNAs confer a competitive fitness advantage to yeast cells because expression of these non-coding molecules correlates with faster adaptation in response to an environmental switch. Copyright © 2016 Elsevier Inc. All rights reserved.

Helical unwinding and side-chain unlocking unravel the outward open conformation of the melibiose transporter

NASA Astrophysics Data System (ADS)

Wang, Li-Ying; Ravi, Vidhya M.; Leblanc, Gérard; Padrós, Esteve; Cladera, Josep; Perálvarez-Marín, Alex

2016-09-01

Molecular dynamics simulations have been used to study the alternate access mechanism of the melibiose transporter from Escherichia coli. Starting from the outward-facing partially occluded form, 2 out of 12 simulations produced an outward full open form and one partially open, whereas the rest yielded fully or partially occluded forms. The shape of the outward-open form resembles other outward-open conformations of secondary transporters. During the transporter opening, conformational changes in some loops are followed by changes in the periplasm region of transmembrane helix 7. Helical curvature relaxation and unlocking of hydrophobic and ionic locks promote the outward opening of the transporter making accessible the substrate binding site. In particular, FRET studies on mutants of conserved aromatic residues of extracellular loop 4 showed lack of substrate binding, emphasizing the importance of this loop for making crucial interactions that control the opening of the periplasmic side. This study indicates that the alternate access mechanism for the melibiose transporter fits better into a flexible gating mechanism rather than the archetypical helical rigid-body rocker-switch mechanism.

D-loop haplotype diversity in Brazilian horse breeds

PubMed Central

Ianella, Patrícia; Albuquerque, Maria do Socorro Maués; Paiva, Samuel Rezende; do Egito, Andréa Alves; Almeida, Leonardo Daniel; Sereno, Fabiana T. P. S.; Carvalho, Luiz Felipe Ramos; Mariante, Arthur da Silva; McManus, Concepta Margaret

2017-01-01

Abstract The first horses were brought to Brazil by the colonizers after 1534. Over the centuries, these animals evolved and adapted to local environmental conditions usually unsuitable for exotic breeds, thereby originating locally adapted Brazilian breeds. The present work represents the first description of maternal genetic diversity in these horse breeds based on D-loop sequences. A D-Loop HSV-I fragment of 252 bp, from 141 horses belonging to ten Brazilian breeds / genetic groups (locally adapted and specialized breeds) were analysed. Thirty-five different haplotypes belonging to 18 haplogroups were identified with 33 polymorphic sites. Haplotype diversity (varying from 0.20 to 0.96) and nucleotide diversity (varying from 0.0039 to 0.0239) was lower for locally adapted than for specialized breeds, with the same pattern observed for FST values. Haplogroups identified in Brazilian breeds are in agreement with previous findings in South American samples. The low variability observed mainly in locally adapted breeds, indicates that, to ensure conservation of these breeds, careful reproductive management is needed. Additional genetic characterization studies are required to support accurate decision-making. PMID:28863209

Energetics of codon-anticodon recognition on the small ribosomal subunit.

PubMed

Almlöf, Martin; Andér, Martin; Aqvist, Johan

2007-01-09

Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

Type 0 open string amplitudes and the tensionless limit

NASA Astrophysics Data System (ADS)

Rojas, Francisco

2014-12-01

The sum over planar multiloop diagrams in the NS + sector of type 0 open strings in flat spacetime has been proposed by Thorn as a candidate to resolve nonperturbative issues of gauge theories in the large N limit. With S U (N ) Chan-Paton factors, the sum over planar open string multiloop diagrams describes the 't Hooft limit N →∞ with N gs2 held fixed. By including only planar diagrams in the sum the usual mechanism for the cancellation of loop divergences (which occurs, for example, among the planar and Möbius strip diagrams by choosing a specific gauge group) is not available and a renormalization procedure is needed. In this article the renormalization is achieved by suspending total momentum conservation by an amount p ≡∑ i n ki≠0 at the level of the integrands in the integrals over the moduli and analytically continuing them to p =0 at the very end. This procedure has been successfully tested for the 2 and 3 gluon planar loop amplitudes by Thorn. Gauge invariance is respected and the correct running of the coupling in the limiting gauge field theory was also correctly obtained. In this article we extend those results in two directions. First, we generalize the renormalization method to an arbitrary n -gluon planar loop amplitude giving full details for the 4-point case. One of our main results is to provide a fully renormalized amplitude which is free of both UV and the usual spurious divergences leaving only the physical singularities in it. Second, using the complete renormalized amplitude, we extract the high-energy scattering regime at fixed angle (tensionless limit). Apart from obtaining the usual exponential falloff at high energies, we compute the full dependence on the scattering angle which shows the existence of a smooth connection between the Regge and hard scattering regimes.

Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

PubMed Central

Fong, Rachel H.; Banik, Soma S. R.; Mattia, Kimberly; Barnes, Trevor; Tucker, David; Liss, Nathan; Lu, Kai; Selvarajah, Suganya; Srinivasan, Surabhi; Mabila, Manu; Miller, Adam; Muench, Marcus O.; Michault, Alain; Rucker, Joseph B.; Paes, Cheryl; Simmons, Graham; Kahle, Kristen M.

2014-01-01

ABSTRACT Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response. PMID:25275138

Global Profiling of Rice and Poplar Transcriptomes Highlights Key Conserved Circadian-Controlled Pathways and cis-Regulatory Modules

PubMed Central

Filichkin, Sergei A.; Breton, Ghislain; Priest, Henry D.; Dharmawardhana, Palitha; Jaiswal, Pankaj; Fox, Samuel E.; Michael, Todd P.; Chory, Joanne; Kay, Steve A.; Mockler, Todd C.

2011-01-01

Background Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants. Methodology/Principal Findings Using a combination of oligonucleotide microarrays and data mining pipelines, we examined daily rhythms in gene expression in one monocotyledonous and one dicotyledonous plant, rice and poplar, respectively. Cycling transcriptomes were interrogated under different diurnal (driven) and circadian (free running) light and temperature conditions. Collectively, photocycles and thermocycles regulated about 60% of the expressed nuclear genes in rice and poplar. Depending on the condition tested, up to one third of oscillating Arabidopsis-poplar-rice orthologs were phased within three hours of each other suggesting a high degree of conservation in terms of rhythmic gene expression. We identified clusters of rhythmically co-expressed genes and searched their promoter sequences to identify phase-specific cis-elements, including elements that were conserved in the promoters of Arabidopsis, poplar, and rice. Conclusions/Significance Our results show that the cycling patterns of many circadian clock genes are highly conserved across poplar, rice, and Arabidopsis. The expression of many orthologous genes in key metabolic and regulatory pathways is diurnal and/or circadian regulated and phased to similar times of day. Our results confirm previous findings in Arabidopsis of three major classes of cis-regulatory modules within the plant circadian network: the morning (ME, GBOX), evening (EE, GATA), and midnight (PBX/TBX/SBX) modules. Identification of identical overrepresented motifs in the promoters of cycling genes from different species suggests that the core diurnal/circadian cis-regulatory network is deeply conserved between mono- and dicotyledonous species. PMID:21694767

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

DOE PAGES

Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

2015-07-03

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

PubMed Central

St John, Franz J.; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M.; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C.

2014-01-01

Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyro­solvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rushton, Phillip S.; Olek, Anna T.; Makowski, Lee

The crystallographic structure of a rice (Oryza sativa) cellulose synthase, OsCesA8, plant-conserved region (P-CR), one of two unique domains in the catalytic domain of plant CesAs, was solved to 2.4 Å resolution. Two antiparallel α-helices form a coiled-coil domain linked by a large extended connector loop containing a conserved trio of aromatic residues. The P-CR structure was fit into a molecular envelope for the P-CR domain derived from small-angle X-ray scattering data. The P-CR structure and molecular envelope, combined with a homology-based chain trace of the CesA8 catalytic core, were modeled into a previously determined CesA8 small-angle X-ray scattering molecularmore » envelope to produce a detailed topological model of the CesA8 catalytic domain. The predicted position for the P-CR domain from the molecular docking models places the P-CR connector loop into a hydrophobic pocket of the catalytic core, with the coiled-coil aligned near the entrance of the substrate UDP-glucose into the active site. In this configuration, the P-CR coiled-coil alone is unlikely to regulate substrate access to the active site, but it could interact with other domains of CesA, accessory proteins, or other CesA catalytic domains to control substrate delivery.« less

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-12-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit.

Detailed analysis of RNA-protein interactions within the bacterial ribosomal protein L5/5S rRNA complex.

PubMed Central

Perederina, Anna; Nevskaya, Natalia; Nikonov, Oleg; Nikulin, Alexei; Dumas, Philippe; Yao, Min; Tanaka, Isao; Garber, Maria; Gongadze, George; Nikonov, Stanislav

2002-01-01

The crystal structure of ribosomal protein L5 from Thermus thermophilus complexed with a 34-nt fragment comprising helix III and loop C of Escherichia coli 5S rRNA has been determined at 2.5 A resolution. The protein specifically interacts with the bulged nucleotides at the top of loop C of 5S rRNA. The rRNA and protein contact surfaces are strongly stabilized by intramolecular interactions. Charged and polar atoms forming the network of conserved intermolecular hydrogen bonds are located in two narrow planar parallel layers belonging to the protein and rRNA, respectively. The regions, including these atoms conserved in Bacteria and Archaea, can be considered an RNA-protein recognition module. Comparison of the T. thermophilus L5 structure in the RNA-bound form with the isolated Bacillus stearothermophilus L5 structure shows that the RNA-recognition module on the protein surface does not undergo significant changes upon RNA binding. In the crystal of the complex, the protein interacts with another RNA molecule in the asymmetric unit through the beta-sheet concave surface. This protein/RNA interface simulates the interaction of L5 with 23S rRNA observed in the Haloarcula marismortui 50S ribosomal subunit. PMID:12515387

Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements

NASA Astrophysics Data System (ADS)

Fernandez-Chamorro, Javier; Lozano, Gloria; Garcia-Martin, Juan Antonio; Ramajo, Jorge; Dotu, Ivan; Clote, Peter; Martinez-Salas, Encarnacion

2016-04-01

The function of Internal Ribosome Entry Site (IRES) elements is intimately linked to their RNA structure. Viral IRES elements are organized in modular domains consisting of one or more stem-loops that harbor conserved RNA motifs critical for internal initiation of translation. A conserved motif is the pyrimidine-tract located upstream of the functional initiation codon in type I and II picornavirus IRES. By computationally designing synthetic RNAs to fold into a structure that sequesters the polypyrimidine tract in a hairpin, we establish a correlation between predicted inaccessibility of the pyrimidine tract and IRES activity, as determined in both in vitro and in vivo systems. Our data supports the hypothesis that structural sequestration of the pyrimidine-tract within a stable hairpin inactivates IRES activity, since the stronger the stability of the hairpin the higher the inhibition of protein synthesis. Destabilization of the stem-loop immediately upstream of the pyrimidine-tract also decreases IRES activity. Our work introduces a hybrid computational/experimental method to determine the importance of structural motifs for biological function. Specifically, we show the feasibility of using the software RNAiFold to design synthetic RNAs with particular sequence and structural motifs that permit subsequent experimental determination of the importance of such motifs for biological function.

RELAP5-3D Modeling of Heat Transfer Components (Intermediate Heat Exchanger and Helical-Coil Steam Generator) for NGNP Application

DOE Office of Scientific and Technical Information (OSTI.GOV)

N. A. Anderson; P. Sabharwall

2014-01-01

The Next Generation Nuclear Plant project is aimed at the research and development of a helium-cooled high-temperature gas reactor that could generate both electricity and process heat for the production of hydrogen. The heat from the high-temperature primary loop must be transferred via an intermediate heat exchanger to a secondary loop. Using RELAP5-3D, a model was developed for two of the heat exchanger options a printed-circuit heat exchanger and a helical-coil steam generator. The RELAP5-3D models were used to simulate an exponential decrease in pressure over a 20 second period. The results of this loss of coolant analysis indicate thatmore » heat is initially transferred from the primary loop to the secondary loop, but after the decrease in pressure in the primary loop the heat is transferred from the secondary loop to the primary loop. A high-temperature gas reactor model should be developed and connected to the heat transfer component to simulate other transients.« less

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

PubMed

Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

2018-05-25

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Mitochondrial genome of Pteronotus personatus (Chiroptera: Mormoopidae): comparison with selected bats and phylogenetic considerations.

PubMed

López-Wilchis, Ricardo; Del Río-Portilla, Miguel Ángel; Guevara-Chumacero, Luis Manuel

2017-02-01

We described the complete mitochondrial genome (mitogenome) of the Wagner's mustached bat, Pteronotus personatus, a species belonging to the family Mormoopidae, and compared it with other published mitogenomes of bats (Chiroptera). The mitogenome of P. personatus was 16,570 bp long and contained a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). Most of the genes were encoded on the H-strand, except for eight tRNA and the ND6 genes. The order of protein-coding and rRNA genes was highly conserved in all mitogenomes. All protein-coding genes started with an ATG codon, except for ND2, ND3, and ND5, which initiated with ATA, and terminated with the typical stop codon TAA/TAG or the codon AGA. Phylogenetic trees constructed using Maximum Parsimony, Maximum Likelihood, and Bayesian inference methods showed an identical topology and indicated the monophyly of different families of bats (Mormoopidae, Phyllostomidae, Vespertilionidae, Rhinolophidae, and Pteropopidae) and the existence of two major clades corresponding to the suborders Yangochiroptera and Yinpterochiroptera. The mitogenome sequence provided here will be useful for further phylogenetic analyses and population genetic studies in mormoopid bats.

Structural Characterization of the 1918 Influenza H1N1 Neuraminidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, X.; Zhu, X.; Dwek, R.A.

2009-05-28

Influenza virus neuraminidase (NA) plays a crucial role in facilitating the spread of newly synthesized virus in the host and is an important target for controlling disease progression. The NA crystal structure from the 1918 'Spanish flu' (A/Brevig Mission/1/18 H1N1) and that of its complex with zanamivir (Relenza) at 1.65-{angstrom} and 1.45-{angstrom} resolutions, respectively, corroborated the successful expression of correctly folded NA tetramers in a baculovirus expression system. An additional cavity adjacent to the substrate-binding site is observed in N1, compared to N2 and N9 NAs, including H5N1. This cavity arises from an open conformation of the 150 loop (Gly147more » to Asp151) and appears to be conserved among group 1 NAs (N1, N4, N5, and N8). It closes upon zanamivir binding. Three calcium sites were identified, including a novel site that may be conserved in N1 and N4. Thus, these high-resolution structures, combined with our recombinant expression system, provide new opportunities to augment the limited arsenal of therapeutics against influenza.« less

EXAMINATION OF Zr AND Ti RECOMBINER LOOP SPECIMENS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rittenhouse, P.L.

1958-12-19

Cold-worked specimens of iodide zirconium, Zircaloy-2, iodide titanium, and A-55 titanium were tested in a high-pressure recombiner loop in an attempt to duplicate anomalous results obtained in a prior recombiner loop. Hydrogen analyses and metallographic examinations were made on all specimens. The titanium materials and Zircaloy-2 picked up major amounts of hydrogen in the cell section. None of the materials tested showed appreciable hydrogen absorption in the recombiner section. Complete recrystallization occurred in all cell specimens while only Zircaloy-2, of the recombiner specimens, showed any degree of recrystallization. No explanation for this behavior can be given. A survnnary of themore » data obtained in previous recombiner loops is compared with the results of this loop. Conclusions were based on the results of three recombiner loops. Primarlly because of the hydrogen absorption data obtained in all three recombiner loops it is recommended that the zirconium and titunium materials tested not be used in environments similar to those encountered in high pressure recombiner loops. (auth)« less

High-Performance Computing Data Center Waste Heat Reuse | Computational

Science.gov Websites

control room With heat exchangers, heat energy in the energy recovery water (ERW) loop becomes available to heat the facility's process hot water (PHW) loop. Once heated, the PHW loop supplies: Active loop in the courtyard of the ESIF's main entrance District heating loop: If additional heat is needed

Transcription of telomeric DNA leads to high levels of homologous recombination and t-loops.

PubMed

Kar, Anirban; Willcox, Smaranda; Griffith, Jack D

2016-11-02

The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex. The loops are very stable and some RNase H resistant TERRA remains at the t-loop, likely adding to their stability. Transcription of DNAs containing TTAGTG or TGAGTG repeats showed greatly reduced loop formation. While in the cell multiple pathways may lead to t-loop formation, the pathway revealed here does not depend on the shelterins but rather on the unique character of telomeric DNA when it is opened for transcription. Hence, telomeric sequences may have evolved to facilitate their ability to loop back on themselves. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, Rodrigo; Lan, Benson; Latif, Yama

2014-04-01

The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully understand the function of the acyl-group binding pocket in substrate specificity.« less

Differential α4(+)/(−)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms*

PubMed Central

Lucero, Linda M.; Weltzin, Maegan M.; Eaton, J. Brek; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.; Whiteaker, Paul

2016-01-01

Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(−)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(−)α4 site with lower agonist affinity than the α4(+)/(−)β2 sites. However, the relative roles of the conserved α4(+)/(−)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (−)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(−)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(−)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect. PMID:26644472

High frequency, high temperature specific core loss and dynamic B-H hysteresis loop characteristics of soft magnetic alloys

NASA Technical Reports Server (NTRS)

Wieserman, W. R.; Schwarze, G. E.; Niedra, J. M.

1990-01-01

Limited experimental data exists for the specific core loss and dynamic B-H loops for soft magnetic materials for the combined conditions of high frequency and high temperature. This experimental study investigates the specific core loss and dynamic B-H loop characteristics of Supermalloy and Metglas 2605SC over the frequency range of 1 to 50 kHz and temperature range of 23 to 300 C under sinusoidal voltage excitation. The experimental setup used to conduct the investigation is described. The effects of the maximum magnetic flux density, frequency, and temperature on the specific core loss and on the size and shape of the B-H loops are examined.

Loop gain stabilizing with an all-digital automatic-gain-control method for high-precision fiber-optic gyroscope.

PubMed

Zheng, Yue; Zhang, Chunxi; Li, Lijing; Song, Lailiang; Chen, Wen

2016-06-10

For a fiber-optic gyroscope (FOG) using electronic dithers to suppress the dead zone, without a fixed loop gain, the deterministic compensation for the dither signals in the control loop of the FOG cannot remain accurate, resulting in the dither residuals in the FOG rotation rate output and the navigation errors in the inertial navigation system. An all-digital automatic-gain-control method for stabilizing the loop gain of the FOG is proposed. By using a perturbation square wave to measure the loop gain of the FOG and adding an automatic gain control loop in the conventional control loop of the FOG, we successfully obtain the actual loop gain and make the loop gain converge to the reference value. The experimental results show that in the case of 20% variation in the loop gain, the dither residuals are successfully eliminated and the standard deviation of the FOG sampling outputs is decreased from 2.00  deg/h to 0.62  deg/h (sampling period 2.5 ms, 10 points smoothing). With this method, the loop gain of the FOG can be stabilized over the operation temperature range and in the long-time application, which provides a solid foundation for the engineering applications of the high-precision FOG.

Dislocation loops in ultra-high purity Fe(Cr) alloys after 7.2 MeV proton irradiation

NASA Astrophysics Data System (ADS)

Chen, J.; Duval, F.; Jung, P.; Schäublin, R.; Gao, N.; Barthe, M. F.

2018-05-01

Ultra-high purity Fe(Cr) alloys (from 0 wt% Cr to 14 wt% Cr) were 3D homogeneously irradiated by 0-7.2 MeV protons to 0.3 dpa at nominal temperatures from 270 °C to 500 °C. Microstructural changes were observed by transmission electron microscopy (TEM). The results showed that evolution of dislocation loops depends on the Cr content. Below 300 °C, large ½ a0 <111> loops are dominating. Above 300 °C, a0 <100> loops with a habit plane {100} appear. Loop sizes of both types are more or less the same. At temperatures from 310 °C to 400 °C, a0 <100> loops form clusters with the same {100} habit plane as the one of the loops forming them. This indicates that <100> loops of the same variant start gliding under mutual elastic interaction. At 500 °C, dislocation loops form disc shaped clusters about 1000 nm in diameter and sitting on {111} and/or {100} planes in the pure Fe samples. Based on these observations a quantitative analysis of the dislocation loops configurations and their temperature dependence is made, leading to an understanding of the basic mechanisms of formation of these loops.

Evolution of dislocation loops in austenitic stainless steels implanted with high concentration of hydrogen

NASA Astrophysics Data System (ADS)

Zheng, Zhongcheng; Gao, Ning; Tang, Rui; Yu, Yanxia; Zhang, Weiping; Shen, Zhenyu; Long, Yunxiang; Wei, Yaxia; Guo, Liping

2017-10-01

It has been found that under certain conditions, hydrogen retention would be strongly enhanced in irradiated austenitic stainless steels. To investigate the effect of the retained hydrogen on the defect microstructure, AL-6XN stainless steel specimens were irradiated with low energy (100 keV) H2+ so that high concentration of hydrogen was injected into the specimens while considerable displacement damage dose (up to 7 dpa) was also achieved. Irradiation induced dislocation loops and voids were characterised by transmission electron microscopy. For specimens irradiated to 7 dpa at 290 °C, dislocation loops with high number density were found and the void swelling was observed. At 380 °C, most of dislocation loops were unfaulted and tangled at 7 dpa, and the void swellings were observed at 5 dpa and above. Combining the data from low dose in previous work to high dose, four stages of dislocation loops evolution with hydrogen retention were suggested. Finally, molecular dynamics simulation was made to elucidate the division of large dislocation loops under irradiation.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.

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

Mapping social-ecological vulnerability to inform local decision making.

PubMed

Thiault, Lauric; Marshall, Paul; Gelcich, Stefan; Collin, Antoine; Chlous, Frédérique; Claudet, Joachim

2018-04-01

An overarching challenge of natural resource management and biodiversity conservation is that relationships between people and nature are difficult to integrate into tools that can effectively guide decision making. Social-ecological vulnerability offers a valuable framework for identifying and understanding important social-ecological linkages, and the implications of dependencies and other feedback loops in the system. Unfortunately, its implementation at local scales has hitherto been limited due at least in part to the lack of operational tools for spatial representation of social-ecological vulnerability. We developed a method to map social-ecological vulnerability based on information on human-nature dependencies and ecosystem services at local scales. We applied our method to the small-scale fishery of Moorea, French Polynesia, by combining spatially explicit indicators of exposure, sensitivity, and adaptive capacity of both the resource (i.e., vulnerability of reef fish assemblages to fishing) and resource users (i.e., vulnerability of fishing households to the loss of fishing opportunity). Our results revealed that both social and ecological vulnerabilities varied considerably through space and highlighted areas where sources of vulnerability were high for both social and ecological subsystems (i.e., social-ecological vulnerability hotspots) and thus of high priority for management intervention. Our approach can be used to inform decisions about where biodiversity conservation strategies are likely to be more effective and how social impacts from policy decisions can be minimized. It provides a new perspective on human-nature linkages that can help guide sustainability management at local scales; delivers insights distinct from those provided by emphasis on a single vulnerability component (e.g., exposure); and demonstrates the feasibility and value of operationalizing the social-ecological vulnerability framework for policy, planning, and participatory management decisions. © 2017 Society for Conservation Biology.

High precision locating control system based on VCM for Talbot lithography

NASA Astrophysics Data System (ADS)

Yao, Jingwei; Zhao, Lixin; Deng, Qian; Hu, Song

2016-10-01

Aiming at the high precision and efficiency requirements of Z-direction locating in Talbot lithography, a control system based on Voice Coil Motor (VCM) was designed. In this paper, we built a math model of VCM and its moving characteristic was analyzed. A double-closed loop control strategy including position loop and current loop were accomplished. The current loop was implemented by driver, in order to achieve the rapid follow of the system current. The position loop was completed by the digital signal processor (DSP) and the position feedback was achieved by high precision linear scales. Feed forward control and position feedback Proportion Integration Differentiation (PID) control were applied in order to compensate for dynamic lag and improve the response speed of the system. And the high precision and efficiency of the system were verified by simulation and experiments. The results demonstrated that the performance of Z-direction gantry was obviously improved, having high precision, quick responses, strong real-time and easily to expend for higher precision.

Interaction of Extracellular Domain 2 of the Human Retina-specific ATP-binding Cassette Transporter (ABCA4) with All-trans-retinal*

PubMed Central

Biswas-Fiss, Esther E.; Kurpad, Deepa S.; Joshi, Kinjalben; Biswas, Subhasis B.

2010-01-01

The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 ± 3% α-helix, 20 ± 3% β-pleated sheet, and 53 ± 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal. PMID:20404325

A Genetic Analysis of Taoyuan Pig and Its Phylogenetic Relationship to Eurasian Pig Breeds

PubMed Central

Li, Kuan-Yi; Li, Kuang-Ti; Cheng, Chun-Chun; Chen, Chia-Hsuan; Hung, Chien-Yi; Ju, Yu-Ten

2015-01-01

Taoyuan pig is a native Taiwan breed. According to the historical record, the breed was first introduced to Taiwan from Guangdong province, Southern China, around 1877. The breed played an important role in Taiwan’s early swine industry. It was classified as an indigenous breed in 1986. After 1987, a conserved population of Taoyuan pig was collected and reared in isolation. In this study, mitochondrial DNA sequences and 18 microsatellite markers were used to investigate maternal lineage and genetic diversity within the Taoyuan pig population. Population differentiation among Taoyuan, Asian type, and European type pig breeds was also evaluated using differentiation indices. Only one D-loop haplotype of the Taoyuan pig was found. It clustered with Lower Changjiang River Basin and Central China Type pig breeds. Based on the polymorphism of microsatellite markers, a positive fixation index value (FIS) indicates that the conserved Taoyuan population suffers from inbreeding. In addition, high FST values (>0.2105) were obtained, revealing high differentiation among these breeds. Non-metric multi-dimensional scaling showed a clear geometric structure among 7 breeds. Together these results indicate that maternally Taoyuan pig originated in the Lower Changjiang River Basin and Central China; however, since being introduced to Taiwan differentiation has occurred. In addition, Taoyuan pig has lost genetic diversity in both its mitochondrial and nuclear genomes. PMID:25656199

Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

NASA Astrophysics Data System (ADS)

Sielewiesiuk, Jan; Łopaciuk, Agata

2015-03-01

Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

Conservation success as a function of good alignment of social and ecological structures and processes.

PubMed

Bodin, Orjan; Crona, Beatrice; Thyresson, Matilda; Golz, Anna-Lea; Tengö, Maria

2014-10-01

How to create and adjust governing institutions so that they align (fit) with complex ecosystem processes and structures across scales is an issue of increasing concern in conservation. It is argued that lack of such social-ecological fit makes governance and conservation difficult, yet progress in explicitly defining and rigorously testing what constitutes a good fit has been limited. We used a novel modeling approach and data from case studies of fishery and forest conservation to empirically test presumed relationships between conservation outcomes and certain patterns of alignment of social-ecological interdependences. Our approach made it possible to analyze conservation outcome on a systems level while also providing information on how individual actors are positioned in the complex web of social-ecological interdependencies. We found that when actors who shared resources were also socially linked, conservation at the level of the whole social-ecological system was positively affected. When the scales at which individual actors used resources and the scale at which ecological resources were interconnected to other ecological resources were aligned through tightened feedback loops, conservation outcome was better than when they were not aligned. The analysis of individual actors' positions in the web of social-ecological interdependencies was helpful in understanding why a system has a certain level of social-ecological fit. Results of analysis of positions showed that different actors contributed in very different ways to achieve a certain fit and revealed some underlying difference between the actors, for example in terms of actors' varying rights to access and use different ecological resources. © 2014 Society for Conservation Biology.

Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci.

PubMed

Amaral, Paulo P; Leonardi, Tommaso; Han, Namshik; Viré, Emmanuelle; Gascoigne, Dennis K; Arias-Carrasco, Raúl; Büscher, Magdalena; Pandolfini, Luca; Zhang, Anda; Pluchino, Stefano; Maracaja-Coutinho, Vinicius; Nakaya, Helder I; Hemberg, Martin; Shiekhattar, Ramin; Enright, Anton J; Kouzarides, Tony

2018-03-15

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider promoter conservation and positional conservation as indicators of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human that are preserved in genomic position relative to orthologous coding genes. These positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are coexpressed in a tissue-specific manner. Over half of positionally conserved RNAs in this set are linked to chromatin organization structures, overlapping binding sites for the CTCF chromatin organiser and located at chromatin loop anchor points and borders of topologically associating domains (TADs). We define these RNAs as topological anchor point RNAs (tapRNAs). Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other's expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Furthermore, we find that tapRNAs contain conserved sequence domains that are enriched in motifs for zinc finger domain-containing RNA-binding proteins and transcription factors, whose binding sites are found mutated in cancers. This work leverages positional conservation to identify lncRNAs with potential importance in genome organization, development and disease. The evidence that many developmental transcription factors are physically and functionally connected to lncRNAs represents an exciting stepping-stone to further our understanding of genome regulation.

Diagrammatic representation of scalar QCD and sign problem at nonzero chemical potential

NASA Astrophysics Data System (ADS)

Bruckmann, Falk; Wellnhofer, Jacob

2018-01-01

We consider QCD at strong coupling with scalar quarks coupled to a chemical potential. Performing the link integrals we present a diagrammatic representation of the path integral weight. It is based on mesonic and baryonic building blocks, in close analogy to fermionic QCD. Likewise, the baryon loops are subject to a manifest conservation of the baryon number. The sign problem is expected to disappear in this representation and we do confirm this for three flavors, where a scalar baryon can be built and, thus, a dependence on the chemical potential occurs. For higher flavor number, we analyze examples for a potential sign problem in the baryon sector and conjecture that all weights are positive upon exploring the current conservation of each flavor.

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

PubMed

Zhang, Zheng Z; Pannunzio, Nicholas R; Lu, Zhengfei; Hsu, Ellen; Yu, Kefei; Lieber, Michael R

2015-10-01

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution. Copyright © 2015 Elsevier Ltd. All rights reserved.

The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.

PubMed

Pizzitutti, Francesco; Giansanti, Andrea; Ballario, Paola; Ornaghi, Prisca; Torreri, Paola; Ciccotti, Giovanni; Filetici, Patrizia

2006-01-01

Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain. Copyright 2005 John Wiley & Sons, Ltd.

Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.

PubMed

Crary, J I; Dean, D M; Maroof, F; Zimmerman, A L

2000-12-01

We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.

OncomiR Addiction Is Generated by a miR-155 Feedback Loop in Theileria-Transformed Leukocytes

PubMed Central

Medjkane, Souhila; Perichon, Martine; Yin, Qinyan; Flemington, Erik; Weitzman, Matthew D.; Weitzman, Jonathan B.

2013-01-01

The intracellular parasite Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet ‘oncogene addiction’ implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation. PMID:23637592

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

PubMed Central

Keller, Jenny; Leulliot, Nicolas; Cambillau, Christian; Campanacci, Valérie; Porciero, Stéphanie; Prangishvili, David; Forterre, Patrick; Cortez, Diego; Quevillon-Cheruel, Sophie; van Tilbeurgh, Herman

2007-01-01

The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues) encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes. PMID:17241456

Drive-amplitude-modulation atomic force microscopy: From vacuum to liquids

PubMed Central

Jaafar, Miriam; Cuenca, Mariano; Melcher, John; Raman, Arvind

2012-01-01

Summary We introduce drive-amplitude-modulation atomic force microscopy as a dynamic mode with outstanding performance in all environments from vacuum to liquids. As with frequency modulation, the new mode follows a feedback scheme with two nested loops: The first keeps the cantilever oscillation amplitude constant by regulating the driving force, and the second uses the driving force as the feedback variable for topography. Additionally, a phase-locked loop can be used as a parallel feedback allowing separation of the conservative and nonconservative interactions. We describe the basis of this mode and present some examples of its performance in three different environments. Drive-amplutide modulation is a very stable, intuitive and easy to use mode that is free of the feedback instability associated with the noncontact-to-contact transition that occurs in the frequency-modulation mode. PMID:22563531

Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

PubMed Central

Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

2014-01-01

The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

Evolutionary relationship and structural characterization of the EPF/EPFL gene family.

PubMed

Takata, Naoki; Yokota, Kiyonobu; Ohki, Shinya; Mori, Masashi; Taniguchi, Toru; Kurita, Manabu

2013-01-01

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes.

Evolutionary Relationship and Structural Characterization of the EPF/EPFL Gene Family

PubMed Central

Takata, Naoki; Yokota, Kiyonobu; Ohki, Shinya; Mori, Masashi; Taniguchi, Toru; Kurita, Manabu

2013-01-01

EPF1-EPF2 and EPFL9/Stomagen act antagonistically in regulating leaf stomatal density. The aim of this study was to elucidate the evolutionary functional divergence of EPF/EPFL family genes. Phylogenetic analyses showed that AtEPFL9/Stomagen-like genes are conserved only in vascular plants and are closely related to AtEPF1/EPF2-like genes. Modeling showed that EPF/EPFL peptides share a common 3D structure that is constituted of a scaffold and loop. Molecular dynamics simulation suggested that AtEPF1/EPF2-like peptides form an additional disulfide bond in their loop regions and show greater flexibility in these regions than AtEPFL9/Stomagen-like peptides. This study uncovered the evolutionary relationship and the conformational divergence of proteins encoded by the EPF/EPFL family genes. PMID:23755192

Severe coagulation factor VII deficiency caused by a novel homozygous mutation (p. Trp284Gly) in loop 140s.

PubMed

Hao, Xiuping; Cheng, XiaoLi; Ye, Jiajia; Wang, Yingyu; Yang, LiHong; Wang, Mingshan; Jin, Yanhui

2016-06-01

Congenital coagulation factor VII (FVII) deficiency is a rare disorder caused by mutation in F7 gene. Herein, we reported a patient who had unexplained hematuria and vertigo with consanguineous parents. He has been diagnosed as having FVII deficiency based on the results of reduced FVII activity (2.0%) and antigen (12.8%). The thrombin generation tests verified that the proband has obstacles in producing thrombin. Direct sequencing analysis revealed a novel homozygous missense mutation p.Trp284Gly. Also noteworthy is the fact that the mutational residue belongs to structurally conserved loop 140s, which majorly undergo rearrangement after FVII activation. Model analysis indicated that the substitution disrupts these native hydrophobic interactions, which are of great importance to the conformation in the activation domain of FVIIa.

What's New is What's Old: Use of Bode's Integral Theorem (circa 1945) to Provide Insight for 21st Century Spacecraft Attitude Control System Design Tuning

NASA Technical Reports Server (NTRS)

Ruth, Mike; Lebsock, Ken; Dennehy, Neil

2010-01-01

This paper revisits the Bode integral theorem, first described in 1945 for feedback amplifier design, in the context of modern satellite Attitude Control System (ACS) design tasks. Use of Bode's Integral clarifies in an elegant way the connection between open-loop stability margins and closed-loop bandwidth. More importantly it shows that there is a very strong tradeoff between disturbance rejection below the satellite controller design bandwidth, and disturbance amplification in the 'penalty region' just above the design bandwidth. This information has been successfully used to re-tune the control designs for several NASA science-mission satellites. The Appendix of this paper contains a complete summary of the relevant integral conservation theorems for stable, unstable, and non-minimum- phase plants.

The nuclear higher-order structure defined by the set of topological relationships between DNA and the nuclear matrix is species-specific in hepatocytes.

PubMed

Silva-Santiago, Evangelina; Pardo, Juan Pablo; Hernández-Muñoz, Rolando; Aranda-Anzaldo, Armando

2017-01-15

During the interphase the nuclear DNA of metazoan cells is organized in supercoiled loops anchored to constituents of a nuclear substructure or compartment known as the nuclear matrix. The stable interactions between DNA and the nuclear matrix (NM) correspond to a set of topological relationships that define a nuclear higher-order structure (NHOS). Current evidence suggests that the NHOS is cell-type-specific. Biophysical evidence and theoretical models suggest that thermodynamic and structural constraints drive the actualization of DNA-NM interactions. However, if the topological relationships between DNA and the NM were the subject of any biological constraint with functional significance then they must be adaptive and thus be positively selected by natural selection and they should be reasonably conserved, at least within closely related species. We carried out a coarse-grained, comparative evaluation of the DNA-NM topological relationships in primary hepatocytes from two closely related mammals: rat and mouse, by determining the relative position to the NM of a limited set of target sequences corresponding to highly-conserved genomic regions that also represent a sample of distinct chromosome territories within the interphase nucleus. Our results indicate that the pattern of topological relationships between DNA and the NM is not conserved between the hepatocytes of the two closely related species, suggesting that the NHOS, like the karyotype, is species-specific. Copyright © 2016 Elsevier B.V. All rights reserved.

Pathophysiology and Toxicokinetic Studies of Blue-Green Algae Intoxication in the Swine Model

DTIC Science & Technology

1987-08-31

bovine aer, albamin caused similar reductions !u toxicity, suggesting nonspecific protein binding. Saturation at the N-methyldehydroalanine of MCYST-A...need to conserve toxin and the goal of creating a model for naturally occurring toxicosis caused us to evaluate the oral administration of microcystin...the isolated ileal loop preparation. Pancreatic enzymes and protein-binding were examined as 2 possible causes for the low oral toxicity of MCYST-A

Closed-Loop Concepts for the Army: Water Conservation, Recycle, and Reuse.

DTIC Science & Technology

1984-11-01

generally small parcels of land used for out- door instruction and field maneuvers. Normally, no permanent improvements are made to the property . Contract...avoid the use of flush toilets. In many situations, this procedure may be necessary because of the physical difficulties with sewage disposal. Several...monitoring of the chemical-biological- physical treatment process, however, it may be prudent to limit their use to locations in the United States where

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin.

PubMed

Elgeti, Matthias; Kazmin, Roman; Rose, Alexander S; Szczepek, Michal; Hildebrand, Peter W; Bartl, Franz J; Scheerer, Patrick; Hofmann, Klaus Peter

2018-03-23

Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, G t α-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Threading structural model of the manganese-stabilizing protein PsbO reveals presence of two possible beta-sandwich domains.

PubMed

Pazos, F; Heredia, P; Valencia, A; de las Rivas, J

2001-12-01

The manganese-stabilizing protein (PsbO) is an essential component of photosystem II (PSII) and is present in all oxyphotosynthetic organisms. PsbO allows correct water splitting and oxygen evolution by stabilizing the reactions driven by the manganese cluster. Despite its important role, its structure and detailed functional mechanism are still unknown. In this article we propose a structural model based on fold recognition and molecular modeling. This model has additional support from a study of the distribution of characteristics of the PsbO sequence family, such as the distribution of conserved, apolar, tree-determinants, and correlated positions. Our threading results consistently showed PsbO as an all-beta (beta) protein, with two homologous beta domains of approximately 120 amino acids linked by a flexible Proline-Glycine-Glycine (PGG) motif. These features are compatible with a general elongated and flexible architecture, in which the two domains form a sandwich-type structure with Greek key topology. The first domain is predicted to include 8 to 9 beta-strands, the second domain 6 to 7 beta-strands. An Ig-like beta-sandwich structure was selected as a template to build the 3-D model. The second domain has, between the strands, long-loops rich in Pro and Gly that are difficult to model. One of these long loops includes a highly conserved region (between P148 and P174) and a short alpha-helix (between E181 and N188)). These regions are characteristic parts of PsbO and show that the second domain is not so similar to the template. Overall, the model was able to account for much of the experimental data reported by several authors, and it would allow the detection of key residues and regions that are proposed in this article as essential for the structure and function of PsbO. Copyright 2001 Wiley-Liss, Inc.

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

PubMed Central

Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

2016-01-01

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

Na(+)-D-glucose cotransporter in the kidney of Squalus acanthias: molecular identification and intrarenal distribution.

PubMed

Althoff, Thorsten; Hentschel, Hartmut; Luig, Jutta; Schütz, Hendrike; Kasch, Myriam; Kinne, Rolf K-H

2006-04-01

Using primers against conserved regions of mammalian Na(+)-d-glucose cotransporters (SGLT), a cDNA was cloned from the kidney of spiny dogfish shark (Squalus acanthias). On the basis of comparison of amino acid sequence, membrane topology, and putative glycosylation and phosphorylation sites, the cDNA could be shown to belong to the family of sglt genes. Indeed, Na(+)-dependent d-glucose uptake could be demonstrated after expression of the gene in Xenopus laevis oocytes. In a dendrogram, the SGLT from shark kidney has a high homology to the mammalian SGLT2. Computer analysis revealed that the elasmobranch protein is most similar to the mammalian proteins in the transmembrane regions and contains already all the amino acids identified to be functionally important, suggesting early conservation during evolution. Extramembraneous loops show larger variations. This holds especially for loop 13, which has been implied as a phlorizin-binding domain. Antibodies were generated and the intrarenal distribution of the SGLT was studied in cryosections. In parallel, the nephron segments were identified by lectins. Positive immunoreactions were found in the proximal tubule in the early parts PIa and PIb and the late segment PIIb. The large PIIa segment of the proximal tubule showed no reaction. In contrast to the mammalian kidney also the late distal tubule, the collecting tubule, and the collecting duct showed immunoreactivity. The molecular information confirms previous vesicle studies in which a low affinity SGLT with a low stoichiometry has been observed and supports the notion of a similarity of the shark kidney SGLT to the mammalian SGLT2. Despite its presence in the late parts of the nephron, the absence of SGLT in the major part of the proximal tubule, the relatively low affinity, and in particular the low stoichiometry might explain the lack of a T(m) for d-glucose in the shark kidney.

Innovative design of closing loops producing an optimal force system applicable in the 0.022-in bracket slot system.

PubMed

Sumi, Mayumi; Koga, Yoshiyuki; Tominaga, Jun-Ya; Hamanaka, Ryo; Ozaki, Hiroya; Chiang, Pao-Chang; Yoshida, Noriaki

2016-12-01

Most closing loops designed for producing higher moment-to-force (M/F) ratios require complex wire bending and are likely to cause hygiene problems and discomfort because of their complicated configurations. We aimed to develop a simple loop design that can produce optimal force and M/F ratio. A loop design that can generate a high M/F ratio and the ideal force level was investigated by varying the portion and length of the cross-sectional reduction of a teardrop loop and the loop position. The forces and moments acting on closing loops were calculated using structural analysis based on the tangent stiffness method. An M/F ratio of 9.3 (high enough to achieve controlled movement of the anterior teeth) and an optimal force level of approximately 250 g of force can be generated by activation of a 10-mm-high teardrop loop whose cross-section of 0.019 × 0.025 or 0.021 × 0.025 in was reduced in thickness by 50% for a distance of 3 mm from the apex, located between a quarter and a third of the interbracket distance from the canine bracket. The simple loop design that we developed delivers an optimal force and an M/F ratio for the retraction of anterior teeth, and is applicable in a 0.022-in slot system. Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

Unique Structural Features of Influenza Virus H15 Hemagglutinin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tzarum, Netanel; McBride, Ryan; Nycholat, Corwin M.

Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avianmore » receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted. IMPORTANCEIn the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.« less

The structure of cytomegalovirus immune modulator UL141 highlights structural Ig-fold versatility for receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemčovičová, Ivana; Slovak Academy of Sciences, Dúbravská cesta 9, SK 84505 Bratislava; Zajonc, Dirk M., E-mail: dzajonc@liai.org

2014-03-01

The crystal structure of Human cytomegalovirus immune modulator UL141 was solved at 3.25 Å resolution. Here, a detailed analysis of its intimate dimerization interface and the biophysical properties of its receptor (TRAIL-R2 and CD155) binding interactions are presented. Natural killer (NK) cells are critical components of the innate immune system as they rapidly detect and destroy infected cells. To avoid immune recognition and to allow long-term persistence in the host, Human cytomegalovirus (HCMV) has evolved a number of genes to evade or inhibit immune effector pathways. In particular, UL141 can inhibit cell-surface expression of both the NK cell-activating ligand CD155more » as well as the TRAIL death receptors (TRAIL-R1 and TRAIL-R2). The crystal structure of unliganded HCMV UL141 refined to 3.25 Å resolution allowed analysis of its head-to-tail dimerization interface. A ‘dimerization-deficient’ mutant of UL141 (ddUL141) was further designed, which retained the ability to bind to TRAIL-R2 or CD155 while losing the ability to cross-link two receptor monomers. Structural comparison of unliganded UL141 with UL141 bound to TRAIL-R2 further identified a mobile loop that makes intimate contacts with TRAIL-R2 upon receptor engagement. Superposition of the Ig-like domain of UL141 on the CD155 ligand T-cell immunoreceptor with Ig and ITIM domains (TIGIT) revealed that UL141 can potentially engage CD155 similar to TIGIT by using the C′C′′ and GF loops. Further mutations in the TIGIT binding site of CD155 (Q63R and F128R) abrogated UL141 binding, suggesting that the Ig-like domain of UL141 is a viral mimic of TIGIT, as it targets the same binding site on CD155 using similar ‘lock-and-key’ interactions. Sequence alignment of the UL141 gene and its orthologues also showed conservation in this highly hydrophobic (L/A)X{sub 6}G ‘lock’ motif for CD155 binding as well as conservation of the TRAIL-R2 binding patches, suggesting that these host–receptor interactions are evolutionary conserved.« less

Stabilization of the second oxyanion intermediate by 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase of the menaquinone pathway: spectroscopic evidence of the involvement of a conserved aspartic acid.

PubMed

Chen, Minjiao; Jiang, Ming; Sun, Yueru; Guo, Zu-Feng; Guo, Zhihong

2011-07-05

1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.

Loop models of low coronal structures observed by the Normal Incidence X-Ray Telescope (NIXT)

NASA Technical Reports Server (NTRS)

Peres, G.; Reale, F.; Golub, L.

1994-01-01

The X-ray pictures obtained with the Normal Incidence X-Ray Telescope (NIXT), apart from the ubiquitous coronal loops well known from previous X-ray observations, show a new and peculiar morphology: in many active regions there are wide and apparently low-lying areas of intense emission which resemble H alpha plages. By means of hydrostatic models of coronal arches, we analyze the distribution of temperature, density, emission measure, and plasma emissivity in the spectral band to which NIXT is sensitive, and we show that the above morphology can be explained by the characteristics of high pressure loops having a thin region of high surface brightness at the base. We therefore propose that this finding might help to identify high-pressure X-ray emitting coronal regions in NIXT images, and it is in principle applicable to any imaging instrument which has high sensitivity to 10(exp 4) - 10(exp 6) K plasma within a narrow coronal-temperature passband. As a more general result of this study, we propose that the comparison of NIXT observations with models of stationary loops might provide a new diagnostic: the determination of the loop plasma pressure from measurements of brightness distribution along the loop.

How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA.

PubMed

Hur, Sun; Stroud, Robert M

2007-04-27

Translational accuracy and efficiency depend upon modification of uridines in the tRNA anticodon stem loop (ASL) by a highly conserved pseudouridine synthase TruA. TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures through a poorly characterized mechanism that differs from previously studied RNA-modifying enzymes. The molecular basis for the site and substrate "promiscuity" was studied by determining the crystal structures of E. coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA*tRNA reaction, revealing the mechanism by which TruA selects the target site. We propose that TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function.

Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein

NASA Astrophysics Data System (ADS)

Bokori-Brown, Monika; Martin, Thomas G.; Naylor, Claire E.; Basak, Ajit K.; Titball, Richard W.; Savva, Christos G.

2016-04-01

Lysenin from the coelomic fluid of the earthworm Eisenia fetida belongs to the aerolysin family of small β-pore-forming toxins (β-PFTs), some members of which are pathogenic to humans and animals. Despite efforts, a high-resolution structure of a channel for this family of proteins has been elusive and therefore the mechanism of activation and membrane insertion remains unclear. Here we determine the pore structure of lysenin by single particle cryo-EM, to 3.1 Å resolution. The nonameric assembly reveals a long β-barrel channel spanning the length of the complex that, unexpectedly, includes the two pre-insertion strands flanking the hypothetical membrane-insertion loop. Examination of other members of the aerolysin family reveals high structural preservation in this region, indicating that the membrane-insertion pathway in this family is conserved. For some toxins, proteolytic activation and pro-peptide removal will facilitate unfolding of the pre-insertion strands, allowing them to form the β-barrel of the channel.

A role of SCN9A in human epilepsies, as a cause of febrile seizures and as a potential modifier of Dravet syndrome.

PubMed

Singh, Nanda A; Pappas, Chris; Dahle, E Jill; Claes, Lieve R F; Pruess, Timothy H; De Jonghe, Peter; Thompson, Joel; Dixon, Missy; Gurnett, Christina; Peiffer, Andy; White, H Steve; Filloux, Francis; Leppert, Mark F

2009-09-01

A follow-up study of a large Utah family with significant linkage to chromosome 2q24 led us to identify a new febrile seizure (FS) gene, SCN9A encoding Na(v)1.7. In 21 affected members, we uncovered a potential mutation in a highly conserved amino acid, p.N641Y, in the large cytoplasmic loop between transmembrane domains I and II that was absent from 586 ethnically matched population control chromosomes. To establish a functional role for this mutation in seizure susceptibility, we introduced the orthologous mutation into the murine Scn9a ortholog using targeted homologous recombination. Compared to wild-type mice, homozygous Scn9a(N641Y/N641Y) knockin mice exhibit significantly reduced thresholds to electrically induced clonic and tonic-clonic seizures, and increased corneal kindling acquisition rates. Together, these data strongly support the SCN9A p.N641Y mutation as disease-causing in this family. To confirm the role of SCN9A in FS, we analyzed a collection of 92 unrelated FS patients and identified additional highly conserved Na(v)1.7 missense variants in 5% of the patients. After one of these children with FS later developed Dravet syndrome (severe myoclonic epilepsy of infancy), we sequenced the SCN1A gene, a gene known to be associated with Dravet syndrome, and identified a heterozygous frameshift mutation. Subsequent analysis of 109 Dravet syndrome patients yielded nine Na(v)1.7 missense variants (8% of the patients), all in highly conserved amino acids. Six of these Dravet syndrome patients with SCN9A missense variants also harbored either missense or splice site SCN1A mutations and three had no SCN1A mutations. This study provides evidence for a role of SCN9A in human epilepsies, both as a cause of FS and as a partner with SCN1A mutations.

High-Performance Computing Data Center Efficiency Dashboard | Computational

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recovery water (ERW) loop Heat exchanger for energy recovery Thermosyphon Heat exchanger between ERW loop and cooling tower loop Evaporative cooling towers Learn more about our energy-efficient facility

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

PubMed Central

Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

2015-01-01

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

Structural Insights into the Allosteric Operation of the Lon AAA+ Protease.

PubMed

Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I

2016-05-03

The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. Copyright © 2016 Elsevier Ltd. All rights reserved.

The implications of free 3D scanning in the conservation state assessment of old wood painted icon

NASA Astrophysics Data System (ADS)

Munteanu, Marius; Sandu, Ion

2016-06-01

The present paper presents the conservation state and the making of a 3D model of a XVIII-th century orthodox icon on wood support, using free available software and cloud computing. In order to create the 3D model of the painting layer of the icon a number of 70 pictures were taken using a Nikon DSLR D3300, 24.2 MP in setup with a Hama Star 75 photo tripod, in loops 360° around the painting, at three different angles. The pictures were processed with Autodesk I23D Catch, which automatically finds and matches common features among all of the uploaded photographs in order to create the 3D scene, using the power and speed of cloud computing. The obtained 3D model was afterwards analyzed and processed in order to obtain a final version, which can now be use to better identify, to map and to prioritize the future conservation processes and finally can be shared online as an animation.

Closed-Loop Control of Vortex Formation in Separated Flows

NASA Technical Reports Server (NTRS)

Colonius, Tim; Joe, Won Tae; MacMynowski, Doug; Rowley, Clancy; Taira, Sam; Ahuja, Sunil

2010-01-01

In order to phase lock the flow at the desired shedding cycle, particularly at Phi,best, We designed a feedback compensator. (Even though the open-loop forcing at Wf below Wn can lead to phase-locked limit cycles with a high average lift,) This feedback controller resulted in the phase-locked limit cycles that the open-loop control could not achieve for alpha=30 and 40 Particularly for alpha=40, the feedback was able to stabilize the limit cycle that was not stable with any of the open-loop periodic forcing. This results in stable phase-locked limit cycles for a larger range of forcing frequencies than the open-loop control. Also, it was shown that the feedback achieved the high-lift unsteady flow states that open-loop control could not sustain even after the states have been achieved for a long period of time.

Incorporating parametric uncertainty into population viability analysis models

USGS Publications Warehouse

McGowan, Conor P.; Runge, Michael C.; Larson, Michael A.

2011-01-01

Uncertainty in parameter estimates from sampling variation or expert judgment can introduce substantial uncertainty into ecological predictions based on those estimates. However, in standard population viability analyses, one of the most widely used tools for managing plant, fish and wildlife populations, parametric uncertainty is often ignored in or discarded from model projections. We present a method for explicitly incorporating this source of uncertainty into population models to fully account for risk in management and decision contexts. Our method involves a two-step simulation process where parametric uncertainty is incorporated into the replication loop of the model and temporal variance is incorporated into the loop for time steps in the model. Using the piping plover, a federally threatened shorebird in the USA and Canada, as an example, we compare abundance projections and extinction probabilities from simulations that exclude and include parametric uncertainty. Although final abundance was very low for all sets of simulations, estimated extinction risk was much greater for the simulation that incorporated parametric uncertainty in the replication loop. Decisions about species conservation (e.g., listing, delisting, and jeopardy) might differ greatly depending on the treatment of parametric uncertainty in population models.

Toward a blueprint for UDP-glucose pyrophosphorylase structure/function properties: homology-modeling analyses.

PubMed

Geisler, Matt; Wilczynska, Malgorzata; Karpinski, Stanislaw; Kleczkowski, Leszek A

2004-11-01

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of synthesis of sucrose, cellulose, and several other polysaccharides in all plants. The protein is evolutionarily conserved among eukaryotes, but has little relation, aside from its catalytic reaction, to UGPases of prokaryotic origin. Using protein homology modeling strategy, 3D structures for barley, poplar, and Arabidopsis UGPases have been derived, based on recently published crystal structure of human UDP-N-acetylglucosamine pyrophosphorylase. The derived 3D structures correspond to a bowl-shaped protein with the active site at a central groove, and a C-terminal domain that includes a loop (I-loop) possibly involved in dimerization. Data on a plethora of earlier described UGPase mutants from a variety of eukaryotic organisms have been revisited, and we have, in most cases, verified the role of each mutation in enzyme catalysis/regulation/structural integrity. We have also found that one of two alternatively spliced forms of poplar UGPase has a very short I-loop, suggesting differences in oligomerization ability of the two isozymes. The derivation of the structural model for plant UGPase should serve as a useful blueprint for further function/structure studies on this protein.

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

PubMed

Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

2015-08-25

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

47 CFR 51.319 - Specific unbundling requirements.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Triennial Review Order, the high frequency portion of a copper loop shall no longer be required to be... the requesting telecommunications carrier using the high frequency portion of the loop. The high... the demarcation point at the end-user customer premises, and includes the high frequency portion of...

47 CFR 51.319 - Specific unbundling requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Triennial Review Order, the high frequency portion of a copper loop shall no longer be required to be... the requesting telecommunications carrier using the high frequency portion of the loop. The high... the demarcation point at the end-user customer premises, and includes the high frequency portion of...

47 CFR 51.319 - Specific unbundling requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Triennial Review Order, the high frequency portion of a copper loop shall no longer be required to be... the requesting telecommunications carrier using the high frequency portion of the loop. The high... the demarcation point at the end-user customer premises, and includes the high frequency portion of...

Noise Computation of a Shock-Containing Supersonic Axisymmetric Jet by the CE/SE Method

NASA Technical Reports Server (NTRS)

Loh, Ching Y.; Hultgren, Lennart S.; Chang, Sin-Chung; Jorgenson, Philip C. E.

1999-01-01

The space-time conservation element solution element (CE/SE) method is employed to numerically study the near-field of a typical under-expanded jet. For the computed case-a circular jet with Mach number M(j) = 1.19-the shock-cell structure is in good agreement with experimental results. The computed noise field is in general agreement with the experiment, although further work is needed to properly close the screech feedback loop.

Combustion Stability Innovations for Liquid Rocket

DTIC Science & Technology

2010-01-31

waves within the pipe . Acoustic time for one pass = 0.003 sec. Closed end The following figure shows the second harmonic of the quarter wave mode at...waveguides at the center of the test section. The two drivers at either end can operate at sync or at a specified phase difference. The effect of close ...preserve conservation in real time. The preconditioner operates on the inner loop driving the solution to the next time level. Sufficient number of inner

Population Structure of mtDNA Variation due to Pleistocene Fluctuations in the South American Maned Wolf (Chrysocyon brachyurus, Illiger, 1815): Management Units for Conservation.

PubMed

González, Susana; Cosse, Mariana; Franco, María del Rosario; Emmons, Louise; Vynne, Carly; Duarte, José Maurício Barbanti; Beccacesi, Marcelo D; Maldonado, Jesús E

2015-01-01

The maned wolf (Chrysocyon brachyurus) is one of the largest South American canids, and conservation across this charismatic carnivore's large range is presently hampered by a lack of knowledge about possible natural subdivisions which could influence the population's viability. To elucidate the phylogeographic patterns and demographic history of the species, we used 2 mtDNA markers (D-loop and cytochrome b) from 87 individuals collected throughout their range, in Argentina, Bolivia, Brazil, and Uruguay. We found moderate levels of haplotype and nucleotide diversity, and the 14 D-loop haplotypes were closely related. Genetic structure results revealed 4 groups, and when coupled with model inferences from a coalescent analysis, suggested that maned wolves have undergone demographic fluctuations due to changes in climate and habitat during the Pleistocene glaciation period approximately 24000 years before present (YBP). This genetic signature points to an event that occurred within the timing estimated for the start of the contraction of the Cerrado around 50000 YBP. Our results reveal a genetic signature of population size expansion followed by contraction during Pleistocene interglaciations, which had similar impacts on other South American mammals. The 4 groups should for now be considered management units, within which future monitoring efforts should be conducted independently. © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

UV-IR mixing in nonassociative Snyder ϕ4 theory

NASA Astrophysics Data System (ADS)

Meljanac, Stjepan; Mignemi, Salvatore; Trampetic, Josip; You, Jiangyang

2018-03-01

Using a quantization of the nonassociative and noncommutative Snyder ϕ4 scalar field theory in a Hermitian realization, we present in this article analytical formulas for the momentum-conserving part of the one-loop two-point function of this theory in D -, 4-, and 3-dimensional Euclidean spaces, which are exact with respect to the noncommutative deformation parameter β . We prove that these integrals are regularized by the Snyder deformation. These results indicate that the Snyder deformation does partially regularize the UV divergences of the undeformed theory, as it was proposed decades ago. Furthermore, it is observed that different nonassociative ϕ4 products can generate different momentum-conserving integrals. Finally, most importantly, a logarithmic infrared divergence emerges in one of these interaction terms. We then analyze sample momentum nonconserving integral qualitatively and show that it could exhibit IR divergence too. Therefore, infrared divergences should exist, in general, in the Snyder ϕ4 theory. We consider infrared divergences at the limit p →0 as UV/IR mixings induced by nonassociativity, since they are associated to the matching UV divergence in the zero-momentum limit and appear in specific types of nonassociative ϕ4 products. We also discuss the extrapolation of the Snyder deformation parameter β to negative values as well as certain general properties of one-loop quantum corrections in Snyder ϕ4 theory at the zero-momentum limit.

The effect of partial acclimatization to high altitude on loop gain and central sleep apnoea severity.

PubMed

Andrews, Gareth; Ainslie, Philip N; Shepherd, Kelly; Dawson, Andrew; Swart, Marianne; Lucas, Samuel; Burgess, Keith R

2012-07-01

Loop gain is an engineering term that predicts the stability of a feedback control system, such as the control of breathing. Based on earlier studies at lower altitudes, it was hypothesized that acclimatization to high altitude would lead to a reduction in loop gain and thus central sleep apnoea (CSA) severity. This study used exposure to very high altitude to induce CSA in healthy subjects to investigate the effect of partial acclimatization on loop gain and CSA severity. Measurements were made on 12 subjects (age 30 ± 10 years, body mass index 22.8 ± 1.9, eight males, four females) at an altitude of 5050 m over a 2-week period upon initial arrival (days 2-4) and following partial acclimatization (days 12-14). Sleep was studied by full polysomnography, and resting arterial blood gases were measured. Loop gain was measured by the 'duty cycle' method (duration of hyperpnoea/cycle length). Partial acclimatization to high-altitude exposure was associated with both an increase in loop gain (duty cycle fell from 0.60 ± 0.05 to 0.55 ± 0.06 (P = 0.03)) and severity of CSA (apnoea-hypopnoea index increased from 76.8 ± 48.8 to 115.9 ± 20.2 (P = 0.01)), while partial arterial carbon dioxide concentration fell from 29 ± 3 to 26 ± 2 (P = 0.01). Contrary to the results at lower altitudes, at high-altitude loop gain and severity of CSA increased. © 2012 The Authors. Respirology © 2012 Asian Pacific Society of Respirology.

Genetic variation among wild lake trout populations: the 'wanted' and the 'unwanted'

USGS Publications Warehouse

Burnham-Curtis, Mary K.; Kallemeyn, Larry W.; Bronte, Charles R.; Greswell, Robert E.; Dwyer, Pat; Hamre, R.H.

1997-01-01

In this study we examine genetic variation within and among self-sustaining lake trout populations from the Great Lakes basin, the Rainy Lake basin, and Yellowstone Lake. We used RFLP analysis and direct sequencing to examine DNA sequence variation among several mitochondrial and nuclear genes, including highly conserved loci (e.g. cytochrome b, nuclear exon regions) and highly variable loci (e.g. mitochondrial d-loop and nuclear intron regions). Native Lake Superior lake trout populations show high levels of genetic diversity, while populations from the Rainy Lake basin show little or none. The lake trout population sampled from Yellowstone Lake shows moderate genetic diversity, possibly representative of a relatively large source population closely related to lake trout from Lewis Lake, Wyoming. There has been significant social and management controversy involving these lake trout populations, particularly those that are located in National Parks. In the Great Lakes and Rainy Lake basins, the controversy involves the degree to which hatchery supplementation can contribute to or negatively impact self-sustaining populations which are highly desired by recreational and commercial fisheries. In Yellowstone Lake, the lake trout are viewed as an undesirable intruder that may interfere with resident populations of highly prized native cutthroat trout.

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis.

PubMed

Han, S; Arvai, A S; Clancy, S B; Tainer, J A

2001-01-05

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework. Copyright 2001 Academic Press.

Design of Test Loops for Forced Convection Heat Transfer Studies at Supercritical State

NASA Astrophysics Data System (ADS)

Balouch, Masih N.

Worldwide research is being conducted to improve the efficiency of nuclear power plants by using supercritical water (SCW) as the working fluid. One such SCW reactor considered for future development is the CANDU-Supercritical Water Reactor (CANDU-SCWR). For safe and accurate design of the CANDU-SCWR, a detailed knowledge of forced-convection heat transfer in SCW is required. For this purpose, two supercritical fluid loops, i.e. a SCW loop and an R-134a loop are developed at Carleton University. The SCW loop is designed to operate at pressures as high as 28 MPa, temperatures up to 600 °C and mass fluxes of up to 3000 kg/m2s. The R-134a loop is designed to operate at pressures as high as 6 MPa, temperatures up to 140 °C and mass fluxes in the range of 500-6000 kg/m2s. The test loops designs allow for up to 300 kW of heating power to be imparted to the fluid. Both test loops are of the closed-loop design, where flow circulation is achieved by a centrifugal pump in the SCW loop and three parallel-connected gear pumps in the R-134a loop, respectively. The test loops are pressurized using a high-pressure nitrogen cylinder and accumulator assembly, which allows independent control of the pressure, while simultaneously dampening pump induced pressure fluctuations. Heat exchangers located upstream of the pumps control the fluid temperature in the test loops. Strategically located measuring instrumentation provides information on the flow rate, pressure and temperature in the test loops. The test loops have been designed to accommodate a variety of test-section geometries, ranging from a straight circular tube to a seven-rod bundle, achieving heat fluxes up to 2.5 MW/m2 depending on the test-section geometry. The design of both test loops allows for easy reconfiguration of the test-section orientation relative to the gravitational direction. All the test sections are of the directly-heated design, where electric current passing through the pressure retaining walls of the test sections provides the Joule heating required to heat up the fluid to supercritical conditions. A high-temperature dielectric gasket isolates the current carrying parts of the test section from the rest of the assembly. Temperature and pressure drop data are collected at the inlet and outlet, and along the heated length of the test section. The test loops and test sections are designed according to American Society of Mechanical Engineers (ASME) Pressure Piping B31.1, and Boiler and Pressure Vessel Code, Section VIII-Division 1 rules. The final test loops and test sections assemblies are certified by Technical Standards and Safety Authority (TSSA). Every attempt is made to use off-the-shelf components where possible in order to streamline the design process and reduce costs. Following a rigorous selection process, stainless steel Types 316 and 316H are selected as the construction materials for the test loops, and Inconel 625 is selected as the construction material for the test sections. This thesis describes the design of the SCW and R-134a loops along with the three test-section geometries (i.e., tubular, annular and bundle designs).

Characterization of an apple TT2-type R2R3 MYB transcription factor functionally similar to the poplar proanthocyanidin regulator PtMYB134.

PubMed

Gesell, Andreas; Yoshida, Kazuko; Tran, Lan T; Constabel, C Peter

2014-09-01

The apple MdMYB9 gene encodes a positive regulator of proanthocyanidin synthesis that activates anthocyanidin reductase promoters from apple and poplar via interaction with basic helix-loop-helix proteins. The regulation of proanthocyanidins (PAs, condensed tannins) is of great importance in food plants due to the many benefits of PAs in the human diet. Two candidate flavonoid MYB regulators, MdMYB9 and MdMYB11, were cloned from apple (Malus × domestica) based on their similarity to known MYB PA regulators. Transcript accumulation of both MdMYB9 and MdMYB11 was induced by high light and wounding, similar to the poplar (Populus spp) PA regulator PtMYB134. In transient activation assays with various basic helix-loop-helix (bHLH) co-regulators, MdMYB9 activated apple and poplar anthocyanidin reductase (ANR) promoters, while MdMYB11 showed no activity. Potential transcription factor binding elements were found within several ANR promoters, and the importance of the bHLH binding site (E-box) on ANR promoter activation was demonstrated via mutational analysis. The ability of MdMYB9 and PtMYB134 to reciprocally activate ANR promoters from both apple and poplar and to partner with heterologous bHLH co-factors from these plants confirms the high degree of conservation of PA regulatory complexes across species. The similarity in apple and poplar PA regulation suggests that regulatory genes from poplar could be effectively employed for metabolic engineering of the PA pathway in apple.

NONLINEAR AND FIBER OPTICS: Propagation of femtosecond solitons in a fiber-optic loop

NASA Astrophysics Data System (ADS)

Zakhidov, É. A.; Mirtadzhiev, F. M.; Khaĭdarov, D. V.; Kuznetsov, A. V.; Okhotnikov, A. G.

1991-03-01

An investigation was made of the propagation of fundamental femtosecond soliton pulses in a fiber-optic loop, which is an element with promising applications in logic operations. It is shown that such a loop can filter off the nonsoliton component effectively. The conditions for high-contrast self-switching of fundamental solitons in a fiber-optic loop are identified.

High-Performance Computing Data Center Cooling System Energy Efficiency |

Science.gov Websites

approaches involve a cooling distribution unit (CDU) (2), which interfaces with the facility cooling loop and to the energy recovery water (ERW) loop (5), which is a closed-loop system. There are three heat rejection options for this IT load: When possible, heat energy from the energy recovery loop is transferred

Runtime Assurance Framework Development for Highly Adaptive Flight Control Systems

DTIC Science & Technology

2015-12-01

performing a surveillance mission. The demonstration platform consisted of RTA systems for the inner- loop control, outer- loop guidance, ownship flight...For the inner- loop , the concept of employing multiple transition controllers in the reversionary control system was studied. For all feedback levels...5 RTA Protection Applied to Inner- Loop Control Systems .................................................61 5.1 General Description of Morphing Wing

PBF Reactor Building (PER620). Cubicle 10. Camera facing southeast. Loop ...

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

PBF Reactor Building (PER-620). Cubicle 10. Camera facing southeast. Loop pressurizer on right. Other equipment includes loop strained, control valves, loop piping, pressurizer interchanger, and cleanup system cooler. High-density shielding brick walls. Photographer: Kirsh. Date: November 2, 1970. INEEL negative no. 70-4908 - Idaho National Engineering Laboratory, SPERT-I & Power Burst Facility Area, Scoville, Butte County, ID

On-the-fly reduction of open loops

NASA Astrophysics Data System (ADS)

Buccioni, Federico; Pozzorini, Stefano; Zoller, Max

2018-01-01

Building on the open-loop algorithm we introduce a new method for the automated construction of one-loop amplitudes and their reduction to scalar integrals. The key idea is that the factorisation of one-loop integrands in a product of loop segments makes it possible to perform various operations on-the-fly while constructing the integrand. Reducing the integrand on-the-fly, after each segment multiplication, the construction of loop diagrams and their reduction are unified in a single numerical recursion. In this way we entirely avoid objects with high tensor rank, thereby reducing the complexity of the calculations in a drastic way. Thanks to the on-the-fly approach, which is applied also to helicity summation and for the merging of different diagrams, the speed of the original open-loop algorithm can be further augmented in a very significant way. Moreover, addressing spurious singularities of the employed reduction identities by means of simple expansions in rank-two Gram determinants, we achieve a remarkably high level of numerical stability. These features of the new algorithm, which will be made publicly available in a forthcoming release of the OpenLoops program, are particularly attractive for NLO multi-leg and NNLO real-virtual calculations.

Fast Wave Transmission Measurements on Alcator C-Mod

NASA Astrophysics Data System (ADS)

Reardon, J.; Bonoli, P. T.; Porkolab, M.; Takase, Y.; Wukitch, S. J.

1997-11-01

Data are presented from an array of single-turn loop probes newly installed on the inner wall of C-Mod, directly opposite one of the two fast-wave antennas. The 8-loop array extends 32^circ in the toroidal direction at the midplane and can distinguish electromagnetic from electrostatic modes. Data are acquired by 1GHz digitizer, spectrum analyzer, and RF detector circuit. Phase measurements during different heating scenarios show evidence of both standing and travelling waves. The measurement of toroidal mode number N_tor (conserved under the assumption of axisymmetry) is used to guide the toroidal full-wave code TORIC(Brambilla, M., IPP Report 5/66, February 1996). Amplitude measurements show modulation both by Type III ELMs and sawteeth; the observed sawtooth modulation may be interpreted as due to changes in central absorption. The amplitude of tildeB_tor measured at the inner wall is compared to the prediction of TORIC.

Rapid and sensitive detection of Lily symptomless virus by reverse transcription loop-mediated isothermal amplification.

PubMed

He, Xiangfeng; Xue, Fei; Xu, Shufa; Wang, Wenhe

2016-12-01

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of the human smoothened receptor 7TM bound to an antitumor agent

PubMed Central

Wang, Chong; Wu, Huixian; Katritch, Vsevolod; Han, Gye Won; Huang, Xi-Ping; Liu, Wei; Siu, Fai Yiu; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

2013-01-01

The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. PMID:23636324

Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor.

PubMed

Wang, Jinti; Yarov-Yarovoy, Vladimir; Kahn, Roy; Gordon, Dalia; Gurevitz, Michael; Scheuer, Todd; Catterall, William A

2011-09-13

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

PubMed

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

2016-05-10

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation

PubMed Central

Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

2016-01-01

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

DOE Office of Scientific and Technical Information (OSTI.GOV)

Galle, G.; Degert, J.; Freysz, E.

We have studied the low spin to high spin phase transition induced by nanosecond laser pulses outside and within the thermal hysteresis loop of the [Fe(Htrz){sub 2} trz](BF{sub 4}){sub 2}-H{sub 2}O spin crossover nanoparticles. We demonstrate that, whatever the temperature of the compound, the photo-switching is achieved in less than 12.5 ns. Outside the hysteresis loop, the photo-induced high spin state remains up to 100 {mu}s and then relaxes. Within the thermal hysteresis loop, the photo-induced high spin state remains as long as the temperature of the sample is kept within the thermal loop. A Raman study indicates that themore » photo-switching can be completed using single laser pulse excitation.« less

Te homogeneous precipitation in Ge dislocation loop vicinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perrin Toinin, J.; Portavoce, A., E-mail: alain.portavoce@im2np.fr; Texier, M.

2016-06-06

High resolution microscopies were used to study the interactions of Te atoms with Ge dislocation loops, after a standard n-type doping process in Ge. Te atoms neither segregate nor precipitate on dislocation loops, but form Te-Ge clusters at the same depth as dislocation loops, in contradiction with usual dopant behavior and thermodynamic expectations. Atomistic kinetic Monte Carlo simulations show that Te atoms are repulsed from dislocation loops due to elastic interactions, promoting homogeneous Te-Ge nucleation between dislocation loops. This phenomenon is enhanced by coulombic interactions between activated Te{sup 2+} or Te{sup 1+} ions.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum.

PubMed

Borisova, Anna S; Eneyskaya, Elena V; Jana, Suvamay; Badino, Silke F; Kari, Jeppe; Amore, Antonella; Karlsson, Magnus; Hansson, Henrik; Sandgren, Mats; Himmel, Michael E; Westh, Peter; Payne, Christina M; Kulminskaya, Anna A; Ståhlberg, Jerry

2018-01-01

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) Tre Cel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride ( Tat Cel7A) and Trichoderma harzianum ( Tha Cel7A) show high sequence identity with Tre Cel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The Tat Cel7A, Tha Cel7A, and Tre Cel7A enzymes were characterized for comparison of function. The catalytic domain of Tat Cel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with Tat Cel7A than either Tha Cel7A or Tre Cel7A. In synergistic saccharification of pretreated corn stover, both Tat Cel7A and Tha Cel7A were more efficient than Tre Cel7A, although Tat Cel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from Tat Cel7A with the thermostability features of Tre Cel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borisova, Anna S.; Eneyskaya, Elena V.; Jana, Suvamay

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. Themore » catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.« less

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from Trichoderma atroviride, T. reesei and T. harzianum

DOE PAGES

Borisova, Anna S.; Eneyskaya, Elena V.; Jana, Suvamay; ...

2018-01-13

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. Themore » catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.« less

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

PubMed

Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

2005-06-01

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.

Near optimum digital phase locked loops.

NASA Technical Reports Server (NTRS)

Polk, D. R.; Gupta, S. C.

1972-01-01

Near optimum digital phase locked loops are derived utilizing nonlinear estimation theory. Nonlinear approximations are employed to yield realizable loop structures. Baseband equivalent loop gains are derived which under high signal to noise ratio conditions may be calculated off-line. Additional simplifications are made which permit the application of the Kalman filter algorithms to determine the optimum loop filter. Performance is evaluated by a theoretical analysis and by simulation. Theoretical and simulated results are discussed and a comparison to analog results is made.

Man-in-the-control-loop simulation of manipulators

NASA Technical Reports Server (NTRS)

Chang, J. L.; Lin, Tsung-Chieh; Yae, K. Harold

1989-01-01

A method to achieve man-in-the-control-loop simulation is presented. Emerging real-time dynamics simulation suggests a potential for creating an interactive design workstation with a human operator in the control loop. The recursive formulation for multibody dynamics simulation is studied to determine requirements for man-in-the-control-loop simulation. High speed computer graphics techniques provides realistic visual cues for the simulator. Backhoe and robot arm simulations are implemented to demonstrate the capability of man-in-the-control-loop simulation.

Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny

PubMed Central

Messmer, Marie; Pütz, Joern; Suzuki, Takeo; Suzuki, Tsutomu; Sauter, Claude; Sissler, Marie; Catherine, Florentz

2009-01-01

Primary and secondary structures of mammalian mitochondrial (mt) tRNAs are divergent from canonical tRNA structures due to highly skewed nucleotide content and large size variability of D- and T-loops. The nonconservation of nucleotides involved in the expected network of tertiary interactions calls into question the rules governing a functional L-shaped three-dimensional (3D) structure. Here, we report the solution structure of human mt-tRNAAsp in its native post-transcriptionally modified form and as an in vitro transcript. Probing performed with nuclease S1, ribonuclease V1, dimethylsulfate, diethylpyrocarbonate and lead, revealed several secondary structures for the in vitro transcribed mt-tRNAAsp including predominantly the cloverleaf. On the contrary, the native tRNAAsp folds into a single cloverleaf structure, highlighting the contribution of the four newly identified post-transcriptional modifications to correct folding. Reactivities of nucleotides and phosphodiester bonds in the native tRNA favor existence of a full set of six classical tertiary interactions between the D-domain and the variable region, forming the core of the 3D structure. Reactivities of D- and T-loop nucleotides support an absence of interactions between these domains. According to multiple sequence alignments and search for conservation of Leontis–Westhof interactions, the tertiary network core building rules apply to all tRNAAsp from mammalian mitochondria. PMID:19767615

Recognition of A. thaliana centromeres by heterologous CENH3 requires high similarity to the endogenous protein.

PubMed

Moraes, Izabel C R; Lermontova, Inna; Schubert, Ingo

2011-02-01

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

PubMed

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches’ Broom Disease of Cacao

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baroni, Renata M.; Luo, Zhipu; Darwiche, Rabih

The pathogenic fungi Moniliophthora perniciosa causes Witches’ Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is foundmore » in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.« less

Use of randomly mutagenized genomic cDNA banks of potato spindle tuber viroid to screen for viable versions of the viroid genome.

PubMed

Więsyk, Aneta; Candresse, Thierry; Zagórski, Włodzimierz; Góra-Sochacka, Anna

2011-02-01

In an effort to study sequence space allowing the recovery of viable potato spindle tuber viroid (PSTVd) variants we have developed an in vivo selection (Selex) method to produce and bulk-inoculate by agroinfiltration large PSTVd cDNA banks in which a short stretch of the genome is mutagenized to saturation. This technique was applied to two highly conserved 6 nt-long regions of the PSTVd genome, the left terminal loop (TL bank) and part of the polypurine stretch in the upper strand of pre-melting loop 1 (PM1 bank). In each case, PSTVd accumulation was observed in a large fraction of bank-inoculated tomato plants. Characterization of the progeny molecules showed the recovery of the parental PSTVd sequence in 89 % (TL bank) and 18 % (PM1 bank) of the analysed plants. In addition, viable and genetically stable PSTVd variants with mutations outside of the known natural variability of PSTVd were recovered in both cases, although at different rates. In the case of the TL region, mutations were recovered at five of the six mutagenized positions (357, 358, 359, 1 and 3 of the genome) while for the PM1 region mutations were recovered at all six targeted positions (50-55), providing significant new insight on the plasticity of the PSTVd genome.

Structural dynamic analysis of apo and ATP-bound IRAK4 kinase

NASA Astrophysics Data System (ADS)

Gosu, Vijayakumar; Choi, Sangdun

2014-07-01

Interleukin-1 receptor-associated kinases (IRAKs) are Ser/Thr protein kinases that play an important role as signaling mediators in the signal transduction facilitated by the Toll-like receptor (TLR) and interleukin-1 receptor families. Among IRAK family members, IRAK4 is one of the drug targets for diseases related to the TLR and IL-1R signaling pathways. Experimental evidence suggests that the IRAK4 kinase domain is phosphorylated in its activation loop at T342, T345, and S346 in the fully activated state. However, the molecular interactions of subdomains within the active and inactive IRAK4 kinase domain are poorly understood. Hence, we employed a long-range molecular dynamics (MD) simulation to compare apo IRAK4 kinase domains (phosphorylated and unphosphorylated) and ATP-bound phosphorylated IRAK4 kinase domains. The MD results strongly suggested that lobe uncoupling occurs in apo unphosphorylated IRAK4 kinase via the disruption of the R334/T345 and R310/T345 interaction. In addition, apo unphosphorylated trajectory result in high mobility, particularly in the N lobe, activation segment, helix αG, and its adjoining loops. The Asp-Phe-Gly (DFG) and His-Arg-Asp (HRD) conserved kinase motif analysis showed the importance of these motifs in IRAK4 kinase activation. This study provides important information on the structural dynamics of IRAK4 kinase, which will aid in inhibitor development.

Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of anti-immunoglobulin G Affitins.

PubMed

Béhar, Ghislaine; Bellinzoni, Marco; Maillasson, Mike; Paillard-Laurance, Lauranne; Alzari, Pedro M; He, Xuemei; Mouratou, Barbara; Pecorari, Frédéric

2013-04-01

Engineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 'flat surface' library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 'flat surface & loops' library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications.

Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis.

PubMed

Huy, Nguyen Tien; Hang, Le Thi Thuy; Boamah, Daniel; Lan, Nguyen Thi Phuong; Van Thanh, Phan; Watanabe, Kiwao; Huong, Vu Thi Thu; Kikuchi, Mihoko; Ariyoshi, Koya; Morita, Kouichi; Hirayama, Kenji

2012-12-01

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Real-time fluorescence loop-mediated isothermal amplification assay for direct detection of egg drop syndrome virus.

PubMed

Zheney, Makay; Kaziyev, Zhambul; Kassenova, Gulmira; Zhao, Lingna; Liu, Wei; Liang, Lin; Li, Gang

2018-02-13

Egg drop syndrome (EDS), caused by the adenovirus "egg drop syndrome virus" (EDSV) causes severe economic losses through reduced egg production in breeder and layer flocks. The diagnosis of EDSV has been done by molecular tools since its complete genome sequence was identified. In order to enhance the capabilities of the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay, we aimed to apply the method for direct detection of the EDSV without viral DNA extraction. In order to detect the presence of the EDSV DNA, three pairs of primers were designed, from the conserved region of fiber gene of the EDSV. For our assay, test and control samples were directly used in the reaction mixture in 10-fold serial dilution. The target DNA was amplified at 65 °C, which yield positive results in a relatively short period of 40-45 min. The method reported in this study is highly sensitive as compared to polymerase chain reaction (PCR) and showed no sign of cross-reactivity or false positive results. The RealAmp accomplished specific identification of EDSV among a variety of poultry disease viruses. The direct RealAmp can be used to detect the presence of EDSV. As our result showed, the RealAmp method could be suitable for the direct detection of other DNA viruses.

TRACING THE DEVELOPMENT OF TYPEWRITING SKILLS IN AN ADAPTIVE E-LEARNING ENVIRONMENT.

PubMed

van den Bergh, Mattis; Hofman, Abe D; Schmittmann, Verena D; van der Maas, Han L J

2015-12-01

Typewriting studies which compare novice and expert typists have suggested that highly trained typing skills involve cognitive process with an inner and outer loop, which regulate keystrokes and words, respectively. The present study investigates these loops longitudinally, using multi-level modeling of 1,091,707 keystroke latencies from 62 children (M age=12.6 yr.) following an online typing course. Using finger movement repetition as indicator of the inner loop and words typed as indicator of the outer loop, practicing keystroke latencies resulted in different developmental curves for each loop. Moreover, based on plateaus in the developmental curves, the inner loop seemed to require less practice to develop than the outer loop.

The structure of high-temperature solar flare plasma in non-thermal flare models

NASA Technical Reports Server (NTRS)

Emslie, A. G.

1985-01-01

Analytic differential emission measure distributions have been derived for coronal plasma in flare loops heated both by collisions of high-energy suprathermal electrons with background plasma, and by ohmic heating by the beam-normalizing return current. For low densities, reverse current heating predominates, while for higher densities collisional heating predominates. There is thus a minimum peak temperature in an electron-heated loop. In contrast to previous approximate analyses, it is found that a stable reverse current can dominate the heating rate in a flare loop, especially in the low corona. Two 'scaling laws' are found which relate the peak temperature in the loop to the suprathermal electron flux. These laws are testable observationally and constitute a new diagnostic procedure for examining modes of energy transport in flaring loops.

Design and verification of wide-band, simultaneous, multi-frequency, tuning circuits for large moment transmitter loops

NASA Astrophysics Data System (ADS)

Dvorak, Steven L.; Sternberg, Ben K.; Feng, Wanjie

2017-03-01

In this paper we discuss the design and verification of wide-band, multi-frequency, tuning circuits for large-moment Transmitter (TX) loops. Since these multi-frequency, tuned-TX loops allow for the simultaneous transmission of multiple frequencies at high-current levels, they are ideally suited for frequency-domain geophysical systems that collect data while moving, such as helicopter mounted systems. Furthermore, since multi-frequency tuners use the same TX loop for all frequencies, instead of using separate tuned-TX loops for each frequency, they allow for the use of larger moment TX loops. In this paper we discuss the design and simulation of one- and three-frequency tuned TX loops and then present measurement results for a three-frequency, tuned-TX loop.

Responses of microbial community functional structures to pilot-scale uranium in situ bioremediation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, M.; Wu, W.-M.; Wu, L.

2010-02-15

A pilot-scale field test system with an inner loop nested within an outer loop was constructed for in situ U(VI) bioremediation at a US Department of Energy site, Oak Ridge, TN. The outer loop was used for hydrological protection of the inner loop where ethanol was injected for biostimulation of microorganisms for U(VI) reduction/immobilization. After 2 years of biostimulation with ethanol, U(VI) levels were reduced to below drinking water standard (<30 {micro}gl{sup -1}) in the inner loop monitoring wells. To elucidate the microbial community structure and functions under in situ uranium bioremediation conditions, we used a comprehensive functional gene arraymore » (GeoChip) to examine the microbial functional gene composition of the sediment samples collected from both inner and outer loop wells. Our study results showed that distinct microbial communities were established in the inner loop wells. Also, higher microbial functional gene number, diversity and abundance were observed in the inner loop wells than the outer loop wells. In addition, metal-reducing bacteria, such as Desulfovibrio, Geobacter, Anaeromyxobacter and Shewanella, and other bacteria, for example, Rhodopseudomonas and Pseudomonas, are highly abundant in the inner loop wells. Finally, the richness and abundance of microbial functional genes were highly correlated with the mean travel time of groundwater from the inner loop injection well, pH and sulfate concentration in groundwater. These results suggest that the indigenous microbial communities can be successfully stimulated for U bioremediation in the groundwater ecosystem, and their structure and performance can be manipulated or optimized by adjusting geochemical and hydrological conditions.« less

LMFBR with booster pump in pumping loop

DOEpatents

Rubinstein, H.J.

1975-10-14

A loop coolant circulation system is described for a liquid metal fast breeder reactor (LMFBR) utilizing a low head, high specific speed booster pump in the hot leg of the coolant loop with the main pump located in the cold leg of the loop, thereby providing the advantages of operating the main pump in the hot leg with the reliability of cold leg pump operation.

High-throughput sample adaptive offset hardware architecture for high-efficiency video coding

NASA Astrophysics Data System (ADS)

Zhou, Wei; Yan, Chang; Zhang, Jingzhi; Zhou, Xin

2018-03-01

A high-throughput hardware architecture for a sample adaptive offset (SAO) filter in the high-efficiency video coding video coding standard is presented. First, an implementation-friendly and simplified bitrate estimation method of rate-distortion cost calculation is proposed to reduce the computational complexity in the mode decision of SAO. Then, a high-throughput VLSI architecture for SAO is presented based on the proposed bitrate estimation method. Furthermore, multiparallel VLSI architecture for in-loop filters, which integrates both deblocking filter and SAO filter, is proposed. Six parallel strategies are applied in the proposed in-loop filters architecture to improve the system throughput and filtering speed. Experimental results show that the proposed in-loop filters architecture can achieve up to 48% higher throughput in comparison with prior work. The proposed architecture can reach a high-operating clock frequency of 297 MHz with TSMC 65-nm library and meet the real-time requirement of the in-loop filters for 8 K × 4 K video format at 132 fps.

Nucleotide Dependent Switching in Rho GTPase: Conformational Heterogeneity and Competing Molecular Interactions

PubMed Central

Kumawat, Amit; Chakrabarty, Suman; Kulkarni, Kiran

2017-01-01

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of “sub-states” such as state 1 & state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound “OFF” state have very similar conformations, whereas the GTP bound “ON” state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors. PMID:28374773

Evolution subverting essentiality: Dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus

PubMed Central

Martínez, Miguel A.; Verdaguer, Nuria; Mateu, Mauricio G.; Domingo, Esteban

1997-01-01

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature. PMID:9192645

Structures of Bacterial Biosynthetic Arginine Decarboxylases

DOE Office of Scientific and Technical Information (OSTI.GOV)

F Forouhar; S Lew; J Seetharaman

2011-12-31

Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 {angstrom} resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 {angstrom} resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a {beta}-sandwich domain, as well as two smaller helical domains. Themore » TIM-barrel and {beta}-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the {beta}-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, {pi}-stacking and van der Waals interactions.« less

Phylogenetically conserved resource partitioning in the coastal microbial loop

DOE PAGES

Bryson, Samuel; Li, Zhou; Chavez, Francisco; ...

2017-08-11

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populationsmore » did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.« less

Phylogenetically conserved resource partitioning in the coastal microbial loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bryson, Samuel; Li, Zhou; Chavez, Francisco

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populationsmore » did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.« less

Phylogenetically conserved resource partitioning in the coastal microbial loop

PubMed Central

Bryson, Samuel; Li, Zhou; Chavez, Francisco; Weber, Peter K; Pett-Ridge, Jennifer; Hettich, Robert L; Pan, Chongle; Mayali, Xavier; Mueller, Ryan S

2017-01-01

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populations did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone. PMID:28800138

Nucleotide Dependent Switching in Rho GTPase: Conformational Heterogeneity and Competing Molecular Interactions

NASA Astrophysics Data System (ADS)

Kumawat, Amit; Chakrabarty, Suman; Kulkarni, Kiran

2017-04-01

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of “sub-states” such as state 1 & state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound “OFF” state have very similar conformations, whereas the GTP bound “ON” state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors.

Establishing a Framework for the Ad/Abaxial Regulatory Network of Arabidopsis: Ascertaining Targets of Class III HOMEODOMAIN LEUCINE ZIPPER and KANADI Regulation[W

PubMed Central

Reinhart, Brenda J.; Liu, Tie; Newell, Nicole R.; Magnani, Enrico; Huang, Tengbo; Kerstetter, Randall; Michaels, Scott; Barton, M. Kathryn

2013-01-01

The broadly conserved Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) and KANADI transcription factors have opposing and transformational effects on polarity and growth in all tissues and stages of the plant's life. To obtain a comprehensive understanding of how these factors work, we have identified transcripts that change in response to induced HD-ZIPIII or KANADI function. Additional criteria used to identify high-confidence targets among this set were presence of an adjacent HD-ZIPIII binding site, expression enriched within a subdomain of the shoot apical meristem, mutant phenotype showing defect in polar leaf and/or meristem development, physical interaction between target gene product and HD-ZIPIII protein, opposite regulation by HD-ZIPIII and KANADI, and evolutionary conservation of the regulator–target relationship. We find that HD-ZIPIII and KANADI regulate tissue-specific transcription factors involved in subsidiary developmental decisions, nearly all major hormone pathways, and new actors (such as INDETERMINATE DOMAIN4) in the ad/abaxial regulatory network. Multiple feedback loops regulating HD-ZIPIII and KANADI are identified, as are mechanisms through which HD-ZIPIII and KANADI oppose each other. This work lays the foundation needed to understand the components, structure, and workings of the ad/abaxial regulatory network directing basic plant growth and development. PMID:24076978

Molecular organization of the 5S rDNA gene type II in elasmobranchs.

PubMed

Castro, Sergio I; Hleap, Jose S; Cárdenas, Heiber; Blouin, Christian

2016-01-01

The 5S rDNA gene is a non-coding RNA that can be found in 2 copies (type I and type II) in bony and cartilaginous fish. Previous studies have pointed out that type II gene is a paralog derived from type I. We analyzed the molecular organization of 5S rDNA type II in elasmobranchs. Although the structure of the 5S rDNA is supposed to be highly conserved, our results show that the secondary structure in this group possesses some variability and is different than the consensus secondary structure. One of these differences in Selachii is an internal loop at nucleotides 7 and 112. These mutations observed in the transcribed region suggest an independent origin of the gene among Batoids and Selachii. All promoters were highly conserved with the exception of BoxA, possibly due to its affinity to polymerase III. This latter enzyme recognizes a dT4 sequence as stop signal, however in Rajiformes this signal was doubled in length to dT8. This could be an adaptation toward a higher efficiency in the termination process. Our results suggest that there is no TATA box in elasmobranchs in the NTS region. We also provide some evidence suggesting that the complexity of the microsatellites present in the NTS region play an important role in the 5S rRNA gene since it is significantly correlated with the length of the NTS.