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Sample records for dimerization initiation site

  1. Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site

    PubMed Central

    Aduri, Raviprasad; Briggs, Katharine T.; Gorelick, Robert J.; Marino, John P.

    2013-01-01

    Human immunodeficiency virus genome dimerization is initiated through an RNA–RNA kissing interaction formed via the dimerization initiation site (DIS) loop sequence, which has been proposed to be converted to a more thermodynamically stable linkage by the viral p7 form of the nucleocapsid protein (NC). Here, we systematically probed the role of specific amino acids of NCp7 in its chaperone activity in the DIS conversion using 2-aminopurine (2-AP) fluorescence and nuclear magnetic resonance spectroscopy. Through comparative analysis of NCp7 mutants, the presence of positively charged residues in the N-terminus was found to be essential for both helix destabilization and strand transfer functions. It was also observed that the presence and type of the Zn finger is important for NCp7 chaperone activity, but not the order of the Zn fingers. Swapping single aromatic residues between Zn fingers had a significant effect on NCp7 activity; however, these mutants did not exhibit the same activity as mutants in which the order of the Zn fingers was changed, indicating a functional role for other flanking residues. RNA chaperone activity is further correlated with NCp7 structure and interaction with RNA through comparative analysis of nuclear magnetic resonance spectra of NCp7 variants, and complexes of these proteins with the DIS dimer. PMID:23275531

  2. Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization.

    PubMed Central

    Mougel, M; Tounekti, N; Darlix, J L; Paoletti, J; Ehresmann, B; Ehresmann, C

    1993-01-01

    Dimerization of genomic RNA is a key step in the retroviral life cycle and has been postulated to be involved in the regulation of translation, encapsidation and reverse transcription. Here, we have derived a secondary structure model of nucleotides upstream from psi and of the gag initiation region of Mo-MuLV RNA in monomeric and dimeric forms, using chemical probing, sequence comparison and computer prediction. The 5' domain is extensively base-paired and interactions take place between U5 and 5' leader sequences. The U5-PBS subdomain can fold in two mutually exclusive conformations: a very stable and extended helical structure (E form) in which 17 of the 18 nucleotides of the PBS are paired, or an irregular three-branch structure (B form) in which 10 nucleotides of the PBS are paired. The dimeric RNA adopts the B conformation. The monomeric RNA can switch from the E to the B conformation by a thermal treatment. If the E to B transition is associated to dimerization, it may facilitate annealing of the primer tRNAPro to the PBS by lowering the free energy required for melting the PBS. Furthermore, dimerization induces allosteric rearrangements around the SD site and the gag initiation region. Images PMID:8233816

  3. NMR detection of intermolecular interaction sites in the dimeric 5'-leader of the HIV-1 genome.

    PubMed

    Keane, Sarah C; Van, Verna; Frank, Heather M; Sciandra, Carly A; McCowin, Sayo; Santos, Justin; Heng, Xiao; Summers, Michael F

    2016-11-15

    HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5'-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a (2)H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5'-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5' (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a (2)H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.

  4. Dimer site-bond percolation on a triangular lattice

    NASA Astrophysics Data System (ADS)

    Ramirez, L. S.; De la Cruz Félix, N.; Centres, P. M.; Ramirez-Pastor, A. J.

    2017-02-01

    A generalization of the site-percolation problem, in which pairs of neighbor sites (site dimers) and bonds are independently and randomly occupied on a triangular lattice, has been studied by means of numerical simulations. Motivated by considerations of cluster connectivity, two distinct schemes (denoted as S{\\cap}B and S{\\cup}B ) have been considered. In S{\\cap}B (S{\\cup}B ), two points are said to be connected if a sequence of occupied sites and (or) bonds joins them. Numerical data, supplemented by analysis using finite-size scaling theory, were used to determine (i) the complete phase diagram of the system (phase boundary between the percolating and nonpercolating regions), and (ii) the values of the critical exponents (and universality) characterizing the phase transition occurring in the system.

  5. Structure of T4 pyrimidine dimer glycosylase in a reduced imine covalent complex with abasic site-containing DNA.

    PubMed

    Golan, Gali; Zharkov, Dmitry O; Grollman, Arthur P; Dodson, M L; McCullough, Amanda K; Lloyd, R Stephen; Shoham, Gil

    2006-09-15

    The base excision repair (BER) pathway for ultraviolet light (UV)-induced cyclobutane pyrimidine dimers is initiated by DNA glycosylases that also possess abasic (AP) site lyase activity. The prototypical enzyme known to catalyze these reactions is the T4 pyrimidine dimer glycosylase (T4-Pdg). The fundamental chemical reactions and the critical amino acids that lead to both glycosyl and phosphodiester bond scission are known. Catalysis proceeds via a protonated imine covalent intermediate between the alpha-amino group of the N-terminal threonine residue and the C1' of the deoxyribose sugar of the 5' pyrimidine at the dimer site. This covalent complex can be trapped as an irreversible, reduced cross-linked DNA-protein complex by incubation with a strong reducing agent. This active site trapping reaction is equally efficient on DNA substrates containing pyrimidine dimers or AP sites. Herein, we report the co-crystal structure of T4-Pdg as a reduced covalent complex with an AP site-containing duplex oligodeoxynucleotide. This high-resolution structure reveals essential precatalytic and catalytic features, including flipping of the nucleotide opposite the AP site, a sharp kink (approximately 66 degrees ) in the DNA at the dimer site and the covalent bond linking the enzyme to the DNA. Superposition of this structure with a previously published co-crystal structure of a catalytically incompetent mutant of T4-Pdg with cyclobutane dimer-containing DNA reveals new insights into the structural requirements and the mechanisms involved in DNA bending, nucleotide flipping and catalytic reaction.

  6. Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase.

    PubMed

    Ryabinina, Olga P; Minko, Irina G; Lasarev, Michael R; McCullough, Amanda K; Lloyd, R Stephen

    2011-10-10

    The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. HIV-2 genome dimerization is required for the correct processing of Gag: a second-site reversion in matrix can restore both processes in dimerization-impaired mutant viruses.

    PubMed

    L'Hernault, Anne; Weiss, Eva U; Greatorex, Jane S; Lever, Andrew M

    2012-05-01

    A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5' ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich ((392)-GGAG-(395)) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the (392)-GGAG-(395) motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein.

  8. HIV-2 Genome Dimerization Is Required for the Correct Processing of Gag: a Second-Site Reversion in Matrix Can Restore Both Processes in Dimerization-Impaired Mutant Viruses

    PubMed Central

    L'Hernault, Anne; Weiss, Eva U.; Greatorex, Jane S.

    2012-01-01

    A unique feature of retroviruses is the packaging of two copies of their genome, noncovalently linked at their 5′ ends. In vitro, dimerization of human immunodeficiency virus type 2 (HIV-2) RNA occurs by interaction of a self-complementary sequence exposed in the loop of stem-loop 1 (SL-1), also termed the dimer initiation site (DIS). However, in virions, HIV-2 genome dimerization does not depend on the DIS. Instead, a palindrome located within the packaging signal (Psi) is the essential motif for genome dimerization. We reported previously that a mutation within Psi decreasing genome dimerization and packaging also resulted in a reduced proportion of mature particles (A. L'Hernault, J. S. Greatorex, R. A. Crowther, and A. M. Lever, Retrovirology 4:90, 2007). In this study, we investigated further the relationship between HIV-2 genome dimerization, particle maturation, and infectivity by using a series of targeted mutations in SL-1. Our results show that disruption of a purine-rich (392-GGAG-395) motif within Psi causes a severe reduction in genome dimerization and a replication defect. Maintaining the extended SL-1 structure in combination with the 392-GGAG-395 motif enhanced packaging. Unlike that of HIV-1, which can replicate despite mutation of the DIS, HIV-2 replication depends critically on genome dimerization rather than just packaging efficiency. Gag processing was altered in the HIV-2 dimerization mutants, resulting in the accumulation of the MA-CA-p2 processing intermediate and suggesting a link between genome dimerization and particle assembly. Analysis of revertant SL-1 mutant viruses revealed that a compensatory mutation in matrix (70TI) could rescue viral replication and partially restore genome dimerization and Gag processing. Our results are consistent with interdependence between HIV-2 RNA dimerization and the correct proteolytic cleavage of the Gag polyprotein. PMID:22419802

  9. NMR detection of intermolecular interaction sites in the dimeric 5′-leader of the HIV-1 genome

    PubMed Central

    Keane, Sarah C.; Van, Verna; Frank, Heather M.; Sciandra, Carly A.; McCowin, Sayo; Santos, Justin; Heng, Xiao; Summers, Michael F.

    2016-01-01

    HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones. PMID:27791166

  10. Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal.

    PubMed

    Cao, Song; Chen, Shi-Jie

    2011-12-01

    We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ~3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.

  11. Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

    PubMed Central

    Cao, Song; Chen, Shi-Jie

    2011-01-01

    We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ∼3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex. PMID:22028361

  12. Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active-site formation and catalytic specificity.

    PubMed

    Itoh, Yuzuru; Bröcker, Markus J; Sekine, Shun-ichi; Söll, Dieter; Yokoyama, Shigeyuki

    2014-04-17

    The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA(Sec)). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA(Sec), formed by seryl-tRNA synthetase, to Sec-tRNA(Sec). SelA, a member of the fold-type-I pyridoxal 5'-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA(Sec) revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA(Sec). The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of "depentamerized" SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5'-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

    PubMed Central

    Seif, Elias; Niu, Meijuan; Kleiman, Lawrence

    2013-01-01

    The 5′ untranslated region (5′ UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNALys3 annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5′-UTR RNA. In this study, we show that annealing of tRNALys3 or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5′ UTR enhances its dimerization in vitro. Structural analysis of the 5′-UTR RNA using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5′ UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNALys3 to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNALys3 to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions. PMID:23960173

  14. Structural and energetic requirements for a second binding site at the dimeric β-lactoglobulin interface.

    PubMed

    Bello, Martiniano

    2016-09-01

    Through experimental and theoretical approaches, it has been shown that bovine β-lactoglobulin (βlg) uses its hydrophobic cavity or calyx as the primary binding site for hydrophobic molecules, whereas the existence of a second ligand binding site at the dimeric interface has only been structurally identified for vitamin D3 (VD3). This binding exists even in the thermally denatured state, suggesting the prevalence of this secondary site. Although crystallographic experiments have suggested that VD3 can bind to both monomeric and dimeric states without significant structural differences, theoretical and experimental reports have proposed some structural requirements. Thus, in this study, based on known experimental data, the dynamic interaction of VD3 with the monomeric or dimeric forms of βlg was investigated through a protocol combining blind docking and 2 microsecond molecular dynamics simulations coupled with binding free energy and per-residue binding free energy decomposition analyses using the Molecular Mechanics Generalized Born Surface Area approach. Binding free energy calculations allowed us to estimate the energetic differences of coupling VD3 at the calyx and the dimeric interface for the monomeric or dimeric state, revealing that the dimeric structure is required to form a stable complex with VD3 at the dimeric interface. This also has an important impact on the dimerization process, whereas although the monomeric state also forms a stable complex with VD3 at the dimeric interface, the incorporation of the entropy component contributed to producing a marginally favorable binding free energy. Finally, the per-residue decomposition analysis provided energetic information about the most relevant residues in stabilizing the different systems.

  15. Angiotensin-converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells.

    PubMed

    Kohlstedt, Karin; Gershome, Cynthia; Friedrich, Matthias; Müller-Esterl, Werner; Alhenc-Gelas, François; Busse, Rudi; Fleming, Ingrid

    2006-05-01

    The binding of angiotensin-converting enzyme (ACE) inhibitors to ACE initiates a signaling cascade that involves the phosphorylation of the enzyme on Ser1270 as well as activation of the c-Jun NH2-terminal kinase (JNK) and leads to alterations in gene expression. To clarify how ACE inhibitors activate this pathway, we determined their effect on the ability of the enzyme to dimerize and the role of ACE dimerization in the initiation of the ACE signaling cascade. In endothelial cells, ACE was detected as a monomer as well as a dimer in native gel electrophoresis and dimerization/oligomerization was confirmed using the split-ubiquitin assay in yeast. ACE inhibitors elicited a rapid, concentration-dependent increase in the dimer/monomer ratio that correlated with that of the ACE inhibitorinduced phosphorylation of ACE. Cell treatment with galactose and glucose to prevent the putative lectin-mediated self-association of ACE or with specific antibodies shielding the N terminus of ACE failed to affect either the basal or the ACE inhibitor-induced dimerization of the enzyme. In ACE-expressing Chinese hamster ovary cells, ACE inhibitors elicited ACE dimerization and phosphorylation as well as the activation of JNK with similar kinetics to those observed in endothelial cells. However, these effects were prevented by the mutation of the essential Zn2+-complexing histidines in the C-terminal active site of the enzyme. Mutation of the N-terminal active site of ACE was without effect. Together, our data suggest that ACE inhibitors can initiate the ACE signaling pathway by inducing ACE dimerization, most probably via the C-terminal active site of the enzyme.

  16. Cholesterol modulates the dimer interface of the β₂-adrenergic receptor via cholesterol occupancy sites.

    PubMed

    Prasanna, Xavier; Chattopadhyay, Amitabha; Sengupta, Durba

    2014-03-18

    The β2-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β2-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β2-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.

  17. Monitoring and targeting the initial dimerization stage of amyloid self-assembly.

    PubMed

    Bram, Yaron; Lampel, Ayala; Shaltiel-Karyo, Ronit; Ezer, Anat; Scherzer-Attali, Roni; Segal, Daniel; Gazit, Ehud

    2015-02-09

    Amyloid deposits are pathological hallmark of a large group of human degenerative disorders of unrelated etiologies. While accumulating evidence suggests that early oligomers may account for tissue degeneration, most detection tools do not allow the monitoring of early association events. Here we exploit bimolecular fluorescence complementation analysis to detect and quantify the dimerization of three major amyloidogenic polypeptides; islet amyloid polypeptide, β-amyloid and α-synuclein. The constructed systems provided direct visualization of protein-protein interactions in which only assembled dimers display strong fluorescent signal. Potential inhibitors that interfere with the initial intermolecular interactions of islet amyloid polypeptide were further identified using this system. Moreover, the identified compounds were able to inhibit the aggregation and cytotoxicity of islet amyloid polypeptide, demonstrating the importance of targeting amyloid dimer formation for future drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

    PubMed

    Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

    2017-11-01

    The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Sphingosine kinase 1 (SK1) allosteric inhibitors that target the dimerization site.

    PubMed

    Bayraktar, Ozge; Ozkirimli, Elif; Ulgen, Kutlu

    2017-08-01

    The sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) signaling pathway is a crucial target for numerous human diseases from cancer to cardiovascular diseases. However, available SK1 inhibitors that target the active site suffer from poor potency, selectivity and pharmacokinetic properties. The selectivity issue of the kinases, which share a highly-conserved ATP-pocket, can be overcome by targeting the less-conserved allosteric sites. SK1 is known to function minimally as a dimer; however, the crystal structure of the SK1 dimer has not been determined. In this study, a template-based algorithm implemented in PRISM was used to predict the SK1 dimer structure and then the possible allosteric sites at the dimer interface were determined via SiteMap. These sites were used in a virtual screening campaign that includes an integrated workflow of structure-based pharmacophore modeling, virtual screening, molecular docking, re-screening of common scaffolds to propose a series of compounds with different scaffolds as potential allosteric SK1 inhibitors. Finally, the stability of the SK1-ligand complexes was analyzed by molecular dynamics simulations. As a final outcome, ligand 7 having a 4,9-dihydro-1H-purine scaffold and ligand 12 having a 2,3,4,9-tetrahydro-1H-β-carboline scaffold were found to be potential selective inhibitors for SK1. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Hanford Site sustainable development initiatives

    SciTech Connect

    Sullivan, C.T.

    1994-05-01

    Since the days of the Manhattan Project of World War II, the economic well being of the Tri-Cities (Pasco, Kennewick, and Richland) of Washington State has been tied to the US Department of Energy missions at the nearby Hanford Site. As missions at the Site changed, so did the economic vitality of the region. The Hanford Site is now poised to complete its final mission, that of environmental restoration. When restoration is completed, the Site may be closed and the effect on the local economy will be devastating if action is not taken now. To that end, economic diversification and transition are being planned. To facilitate the process, the Hanford Site will become a sustainable development demonstration project.

  1. Structure and dimerization of translation initiation factor aIF5B in solution

    SciTech Connect

    Carø VohlanderRasmussen, Louise; Oliveira, Cristiano Luis Pinto; Byron, Olwyn; Jensen, Janni Mosgaard; Pedersen, Jan Skov; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

    2012-02-07

    Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s{sub 20,w}{sup 0} were determined to be 3.64 and 5.51 {+-} 0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5 {+-} 0.2 {angstrom} and a maximum dimension of {approx}130 {angstrom}. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.

  2. The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication.

    PubMed

    Choi, Jiwon; Kim, Bong-Suk; Zhao, Xiaoxia; Loesch-Fries, Sue

    2003-01-05

    Deletion and substitution mutations affecting the oligomerization of alfalfa mosaic virus (AMV) coat protein (CP) were studied in protoplasts to determine their effect on genome activation, an early step in AMV replication. The CP mutants that formed dimers, CPDeltaC9 and CPC-A(R)F, were highly active in initiating replication with 63-84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPDeltaC18, CPDeltaC19, CPC-WFP, and CPC-W, were much less active with 19-33% of wt CP activity. The accumulation and solubility of mutant CPs expressed from a virus-based vector in Nicotiana benthamiana were similar to that of wt CP. Analysis of CP-RNA interactions indicated that CP dimers and CP monomers interacted very differently with AMV RNA 3' ends. These results suggest that CP dimers are more efficient for replication than CP monomers because of differences in RNA binding rather than differences in expression and accumulation of the mutant CPs in infected cells.

  3. Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

    PubMed

    Gamache, Eric R; Doh, Jung H; Ritz, Justin; Laederach, Alain; Bellaousov, Stanislav; Mathews, David H; Curcio, M Joan

    2017-04-26

    The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

  4. Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

    PubMed Central

    Gamache, Eric R.; Doh, Jung H.; Ritz, Justin; Laederach, Alain; Bellaousov, Stanislav; Mathews, David H.; Curcio, M. Joan

    2017-01-01

    The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging. PMID:28445416

  5. Coexistence and competition of on-site and intersite Coulomb interactions in Mott-molecular-dimers

    NASA Astrophysics Data System (ADS)

    Juliano, R. C.; de Arruda, A. S.; Craco, L.

    2016-02-01

    We reveal the interplay between on-site (U) and intersite (V) Coulomb interactions in the extended two-site Hubbard model. Due to its atomic-like form quantum correlations intrinsic to Mott-molecular-dimers are exactly computed. Our results for physical quantities such as double occupancy and specific heat are consistent with those obtained for the one-band Hubbard model, suggesting that a two-site dimer model is able to capture the essential thermodynamic properties of strongly interacting electron systems. It is noted that intersite Coulomb interactions promote the formation of doublons, which compete with the spin-singlet state induced by the on-site Coulomb repulsion. Our results are expected to be relevant for understanding electronic and thermodynamical properties of interacting electrons in systems with strongly coupled magnetic atoms.

  6. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization

    SciTech Connect

    Gruskin, E.A.; Lloyd, R.S.

    1988-09-05

    In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

  7. Coexistence and competition of on-site and intersite Coulomb interactions in Mott-molecular-dimers

    NASA Astrophysics Data System (ADS)

    Arruda, Alberto; Juliano, Raffael; Werlang, Thiago; Craco, Luis

    2015-03-01

    Recent findings of Mott-Hubbard physics in ultracold atoms trapped in periodic potentials have reinvigorated the search for quantum simulators of fermionic and bosonic Hubbard-like models. With this in mind, we performed a systematic study of a two-site realization of the Hubbard model, i.e, in a regime where this model can exactly treated. Particularly, we reveal the interplay between on-site (U) and intersite (V) Coulomb interactions in the extended two-site Hubbard model. Due to its atomic-like form quantum correlations intrinsic to Mott-molecular-dimers are exactly computed. Our results for physical quantities such as double occupancy and specific heat are consistent with those obtained for the one-band Hubbard model, suggesting that a two-site dimer model is able to capture the essential thermodynamic properties of strongly interacting electron systems. It is shown that intersite Coulomb interactions promotes the formation of doublons, which compete with the spin-singlet state induced by the on-site Coulomb repulsion. Our results are expected to be relevant for understanding electronic and thermodynamical properties of interacting electrons in strongly coupled magnetic atoms.

  8. Finite-rate quenches of site bias in the Bose-Hubbard dimer

    NASA Astrophysics Data System (ADS)

    Venumadhav, T.; Haque, Masudul; Moessner, R.

    2010-02-01

    For a Bose-Hubbard dimer, we study quenches of the site energy imbalance, taking a highly asymmetric Hamiltonian to a fully symmetric one. The ramp is carried out over a finite time that interpolates between the instantaneous and adiabatic limits. We provide results for the excess energy of the final state compared to the ground-state energy of the final Hamiltonian as a function of the quench rate. This excess energy serves as the analog of the defect density that is considered in the Kibble-Zurek picture of ramps across phase transitions. We also examine the fate of quantum “self-trapping” when the ramp is not instantaneous.

  9. Molecular Interplay between the Dimer Interface and the Substrate-Binding Site of Human Peptidylarginine Deiminase 4

    PubMed Central

    Lee, Chien-Yun; Lin, Chu-Cheng; Liu, Yi-Liang; Liu, Guang-Yaw; Liu, Jyung-Hurng; Hung, Hui-Chih

    2017-01-01

    Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis. PMID:28209966

  10. Molecular Interplay between the Dimer Interface and the Substrate-Binding Site of Human Peptidylarginine Deiminase 4.

    PubMed

    Lee, Chien-Yun; Lin, Chu-Cheng; Liu, Yi-Liang; Liu, Guang-Yaw; Liu, Jyung-Hurng; Hung, Hui-Chih

    2017-02-17

    Our previous studies suggest that the fully active form of Peptidylarginine deiminase 4 (PAD4) should be a dimer and not a monomer. This paper provides a plausible mechanism for the control of PAD4 catalysis by molecular interplay between its dimer-interface loop (I-loop) and its substrate-binding loop (S-loop). Mutagenesis studies revealed that two hydrophobic residues, W347 and V469, are critical for substrate binding at the active site; mutating these two residues led to a severe reduction in the catalytic activity. We also identified several hydrophobic amino acid residues (L6, L279 and V283) at the dimer interface. Ultracentrifugation analysis revealed that interruption of the hydrophobicity of this region decreases dimer formation and, consequently, enzyme activity. Molecular dynamic simulations and mutagenesis studies suggested that the dimer interface and the substrate-binding site of PAD4, which consist of the I-loop and the S-loop, respectively, are responsible for substrate binding and dimer stabilization. We identified five residues with crucial roles in PAD4 catalysis and dimerization: Y435 and R441 in the I-loop, D465 and V469 in the S-loop, and W548, which stabilizes the I-loop via van der Waals interactions with C434 and Y435. The molecular interplay between the S-loop and the I-loop is crucial for PAD4 catalysis.

  11. Astronomical Site Testing Initiatives in Africa

    NASA Astrophysics Data System (ADS)

    Buckley, David A. H.; Graham, Edward; Vaughan, Richard; Belay, Solomon; Biressa, Tolu

    2015-08-01

    Two astronomical site testing initiatives are beginning in both Kenya and Ethiopia, with the aim of selecting suitable locations in those countries for modest sized (1-2m) optical telescopes.The first project, in Kenya, has initially involved a desk-top study of ~30 years of low resolution (~80 km) meteorological satellite data from the European Centre for Medium Range Weather Forecasting (so called “ERA-reanalysis” data). This was later supplemented by ~2 years of higher resolution (~12 km) United Kingdom Met Office Limited Area Model for Africa (“Africa-LAM”) data, kindly made available by the British Atmospheric Data Centre (BADC).The analysis looked at cloud cover, aerosol distribution, integrated water vapour and wind conditions, On the basis of this study, we determined a number of regions in the north of Kenya, east of the Rift Valley, which show promise as potential observatory sites. We are now in the process of installing Automatic Weather Stations (AWS) at 3 selected sites (~2000-2700 m altitude) to begin monitoring meteorological conditions over the next few years. It is eventually hoped to supplement this study with instrumentation to allow the measurement of sky brightness, local cloud cover and seeing (e.g. with a DIMM system).A similar program of astronomical site testing is due to start in 2015 in the Lalibela region of northern Ethiopia, at three potential dark sky sites with expected relatively low cloud cover, ranging in altitude from ~3600 to 4100 m.

  12. Cytosine containing dipyrimidine sites can be hotspots of cyclobutane pyrimidine dimer formation after UVB exposure.

    PubMed

    Bastien, Nathalie; Therrien, Jean-Philippe; Drouin, Régen

    2013-08-01

    Exposure to the UV component of sunlight is the principal factor leading to skin cancer development. Cyclobutane pyrimidine dimers (CPD) are considered to be the most important pre-mutagenic type of DNA damage involved in skin carcinogenesis. To better understand the biological mechanisms of UV carcinogenesis, it is critical to understand the CPD distribution between the four types of dipyrimidine sites. Most of our knowledge regarding CPD distribution comes from in vitro studies or from investigations using UVC, even though we are not naturally exposed to these UV wavelengths. We exposed normal human fibroblasts and purified DNA to UVB. Using ligation-mediated PCR, we quantified the CPD formation at 952 dipyrimidine sites among the PGK1 (phosphoglycerate kinase 1), JUN, HRAS, KRAS, NRAS and TP53 genes. In cellulo, we found a CPD distribution of 27 : 27 : 25 : 21 for TT : CC : TC : CT. This distribution is similar to that observed in vitro. In the analysed genes, we observed some extremely frequently damaged dipyrimidine sites and many of these occurred at potentially frequently mutated sites, i.e. at dipyrimidine sites containing cytosine. Also, most of the frequently damaged dipyrimidine sites in cellulo that are not frequently damaged in vitro are found on TP53 and NRAS. This indicates that many of the frequently damaged dipyrimidine sites in cellulo are on genes frequently mutated in skin cancer. All these results support the view that CPD are the main UVB-induced mutagenic photoproducts and provide evidence of the importance of CPD formation at sites containing cytosine.

  13. Identification of a phosphorylation site in cyclobutane pyrimidine dimer photolyase of rice.

    PubMed

    Teranishi, Mika; Nakamura, Kentaro; Furukawa, Haruya; Hidema, Jun

    2013-02-01

    Cyclobutane pyrimidine dimer (CPD) photolyase monomerises ultraviolet (UV) radiation-induced CPDs present in DNA, using energy from UVA and visible light. In plants, CPD photolyase activity is a crucial factor for determining UVB sensitivity. We previously demonstrated that native rice CPD photolyase is phosphorylated. To determine the phosphorylation site(s), the phosphorylation status of CPD photolyase was analyzed in rice varieties that have amino acid alterations at the potential phosphorylation sites. In wild-rice species, CPD photolyase was phosphorylated. In Poaceae species, CPD photolyase was phosphorylated in wheat but not in maize. Mutant CPD photolyase proteins, in which these putative phosphorylated residues were replaced with alanine residues, were synthesized using an insect cell-free translation system. A slow-migrating band disappeared when the serine residue at position 7 was mutated. A phospho-specific antibody was generated to determine whether this residue is phosphorylated in CPD photolyase. Only the slow-migrating band of native rice CPD photolyase was detected using this antibody, indicating that the serine residue at position 7 is a phosphorylation site in native rice CPD photolyase. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  14. Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin

    NASA Astrophysics Data System (ADS)

    Zanzoni, Serena; Ceccon, Alberto; Assfalg, Michael; Singh, Rajesh K.; Fushman, David; D'Onofrio, Mariapina

    2015-04-01

    The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with 15N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which

  15. Initiating Molecular Growth in the Interstellar Medium via Dimeric Complexes of Observed Ions and Molecules

    NASA Technical Reports Server (NTRS)

    Bera, Partha P.; Head-Gordon, Martin; Lee, Timothy J.

    2011-01-01

    A feasible initiation step for particle growth in the interstellar medium (ISM) is simulated by means of ab quantum chemistry methods. The systems studied are dimer ions formed by pairing nitrogen containing small molecules known to exist in the ISM with ions of unsaturated hydrocarbons or vice versa. Complexation energies, structures of ensuing complexes and electronic excitation spectra of the encounter complexes are estimated using various quantum chemistry methods. Moller-Plesset perturbation theory (MP2, Z-averaged perturbation theory (ZAP2), coupled cluster singles and doubles with perturbative triples corrections (CCSD(T)), and density functional theory (DFT) methods (B3LYP) were employed along with the correlation consistent cc-pVTZ and aug-cc-pVTZ basis sets. Two types of complexes are predicted. One type of complex has electrostatic binding with moderate (7-20 kcal per mol) binding energies, that are nonetheless significantly stronger than typical van der Waals interactions between molecules of this size. The other type of complex develops strong covalent bonds between the fragments. Cyclic isomers of the nitrogen containing complexes are produced very easily by ion-molecule reactions. Some of these complexes show intense ultraviolet visible spectra for electronic transitions with large oscillator strengths at the B3LYP, omegaB97, and equations of motion coupled cluster (EOM-CCSD) levels. The open shell nitrogen containing carbonaceous complexes especially exhibit a large oscillator strength electronic transition in the visible region of the electromagnetic spectrum.

  16. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  17. Structural and Electrostatic Asymmetry at the Active Site in Typical and Atypical Peroxiredoxin Dimers

    PubMed Central

    Salsbury, Freddie R.; Yuan, Ye; Knaggs, Michael H.; Poole, Leslie B.; Fetrow, Jacquelyn S.

    2012-01-01

    The peroxiredoxins (Prx) are ubiquitous peroxidases involved in important biological processes; however, details of their enzymatic mechanism remain elusive. To probe potential dynamics-function relationships, molecular dynamics simulations and electrostatic calculations were performed on the atypical 2-cysteine thiol peroxidase (Tpx) from Streptococcus pneumoniae and results compared to a previous study of a typical 2-cysteine Prx from Trypanosoma cruzi. The analyses indicate a commonality between both typical and atypical Prx: dynamic asymmetry. Asymmetry is observed in structure, fluctuations and active site electrostatics. Key residues, including Glu150 and Phe153, play roles in the developing asymmetry; furthermore, in the atypical 2-Cys Tpx, Glu150 exhibits conformation fluctuations suggesting involvement in a proton shuttle. The existence of a pathway of connected residues appears to propagate the asymmetry. The commonality of asymmetry and coupling pathways in both typical and atypical Prxs suggests a driving force towards dimer asymmetry as a common feature that plays a functional role in creating one active site with a lower cysteine pKa. PMID:22401569

  18. Site-Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single-Particle Imaging.

    PubMed

    Wissler, Jörg; Bäcker, Sandra; Feis, Alessandro; Knauer, Shirley K; Schlücker, Sebastian

    2017-08-01

    An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His6 -tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Infrared spectroscopic evidence for the initial step of dissociation of the stable benzoic acid cyclic dimer with microsolvation by a single water molecule

    NASA Astrophysics Data System (ADS)

    Katada, Marusu; Fujii, Asuka

    2017-09-01

    The hydrogen-bonded structure of the (Benzoic acid)2-(Water)1 (BA2-W1) cluster was investigated by infrared spectroscopy in the OH stretch region. The stable BA dimer has the cyclic structure with the two hydrogen bonds between the carboxyl groups. The observed infrared spectrum of BA2-W1 shows that the water molecule is inserted in between the two carboxylic groups of the cyclic dimer. This means that a single water molecule begins to dissociate the cyclic dimer. This observation can be regarded as the initial step of dissociation of the stable cyclic BA dimer in aqueous solution.

  20. Structures of dimeric dihydrodiol dehydrogenase apoenzyme and inhibitor complex: probing the subunit interface with site-directed mutagenesis.

    PubMed

    Carbone, Vincenzo; Endo, Satoshi; Sumii, Rie; Chung, Roland P-T; Matsunaga, Toshiyuki; Hara, Akira; El-Kabbani, Ossama

    2008-01-01

    Dimeric dihydrodiol dehydrogenase (DD) catalyses the nicotinamide adenine dinucleotide phosphate (NADP+)-dependent oxidation of trans-dihydrodiols of aromatic hydrocarbons to their corresponding catechols. This is the first report of the crystal structure of the dimeric enzyme determined at 2.0 A resolution. The tertiary structure is formed by a classical dinucleotide binding fold comprising of two betaalphabetaalphabeta motifs at the N-terminus and an eight-stranded, predominantly antiparallel beta-sheet at the C-terminus. The active-site of DD, occupied either by a glycerol molecule or the inhibitor 4-hydroxyacetophenone, is located in the C-terminal domain of the protein and maintained by a number of residues including Lys97, Trp125, Phe154, Leu158, Val161, Asp176, Leu177, Tyr180, Trp254, Phe279, and Asp280. The dimer interface is stabilized by a large number of intermolecular contacts mediated by the beta-sheet of each monomer, which includes an intricate hydrogen bonding network maintained in principal by Arg148 and Arg202. Site-directed mutagenesis has demonstrated that the intact dimer is not essential for catalytic activity. The similarity between the quaternary structures of mammalian DD and glucose-fructose oxidoreductase isolated from the prokaryotic organism Zymomonas mobilis suggests that both enzymes are members of a unique family of oligomeric proteins and may share a common ancestral gene. (c) 2007 Wiley-Liss, Inc.

  1. Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites

    PubMed Central

    Wu, Qian; Paul, Atanu; Su, Dan; Mehmood, Shahid; Foo, Tzeh Keong; Ochi, Takashi; Bunting, Emma L.; Xia, Bing; Robinson, Carol V.; Wang, Bin; Blundell, Tom L.

    2016-01-01

    Summary BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR. PMID:26778126

  2. Structure of BRCA1-BRCT/Abraxas Complex Reveals Phosphorylation-Dependent BRCT Dimerization at DNA Damage Sites.

    PubMed

    Wu, Qian; Paul, Atanu; Su, Dan; Mehmood, Shahid; Foo, Tzeh Keong; Ochi, Takashi; Bunting, Emma L; Xia, Bing; Robinson, Carol V; Wang, Bin; Blundell, Tom L

    2016-02-04

    BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.

  3. Initial results from MARmara SuperSITE

    NASA Astrophysics Data System (ADS)

    Meral Ozel, Nurcan; Necmioglu, Ocal; Favali, Paolo; Douglas, John; Mathieu, Pierre-Philippe; Geli, Louis; Ergintav, Semih; Oguz Ozel, Asım; Tan, Onur; Gurbuz, Cemil; Erdik, Mustafa

    2014-05-01

    shaking measurements, has been prepared by INERIS to be set up on the field to be also set up as an early warning system prototype to be progressively parameterized and tested on near to real time condition. Slip rate on the Main Marmara Fault from 3D seismic data has been estimated and extremely young age of the North Anatolian Fault in the Sea of Marmara has been determined. Seismic risk study for IGDAS Natural Gas Network including pipelines and its components has been carried out with several earthquake scenarios in Marmara Sea. An automatic shut-off algorithm has been developed for the automatic shut-off of the gas flow at the IGDAS district regulators during an extreme event. All the European and international initiatives and projects that could have links with MARsite were identified as the initial step for the integration of data management practices and coordination with ongoing research infrastructures. EPOS and EMSO are considered to be crucial links that could provide sustainability of MARsite's developments beyond the project's lifetime. Concerning EMSO, Marmara is one of the nodes of the research infrastructure, in which a permanent installation at sea is being integrated with land-based networks. In the context of EPOS, MARsite will be a thematic core service. In addition, the data collection and dissemination in MARsite is carried out according to the data management principles of EMSO and EPOS. Dissemination activities reached a certain level of maturity through the relesea of Public Annual Report, quarterly newsletter, ID card and poster, social media interaction, dedicated web sites, videos and several conferences and workhops participated, such as GEO European Projects' Workshop, Supersites Coordination Workshop and GEO-X Plenary & Geneva Ministerial Summit .

  4. Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site.

    PubMed Central

    Swanson, H I; Yang, J H

    1999-01-01

    Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ahreceptornucleartranslocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers and analyzed the ability of these dimers to interact with their derived consensus sequences and activate genes. chi(2)analysis of the selected DNA recognition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonstrated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer. These flanking sequences also regulate the ability of ARNT to competitively displace the c-Myc/Max heterodimer from a CACGTG-containing sequence. However, transient transfection analyses in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abilities to activate transcription through each other's consensus sequences. Taken together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by flanking nucleotides, transcriptional activity may also be determined by other factors, such as cellular concentrations of these proteins and their co-activators. PMID:10454619

  5. Site-specific effect of thymine dimer formation of dA sub n ter dot dT sub n tract bending and its biological implications

    SciTech Connect

    Wang, C.I.; Taylor, J.S. )

    1991-10-15

    dA{sub n}{center dot}dT{sub n} sequences, otherwise known as A tracts, are hotspots for cis-syn thymine dimer formation and deletion mutations induced by UV light. Such A tracts are also known to bend DNA, suggesting that some biological effects of UV light might be related to the distinctive structure and properties of cis-syn dimer-containing A tracts. To investigate the effect of thymine dimer formation on A-tract bending multimers of all possible dimer monoadducts of a dA{sub 6}{center dot}dT{sub 6}-containing decamer known to bend DNA were prepared along with multimers of a dimer-containing 21-mer of heterogeneous sequence. The characteristic anomalous electrophoretic behavior of the phased A-tract multimers was essentially abolished by dimer formation at the center of the A tract and was only slightly reduced by dimer formation at the ends. These effects are attributed to disruption of the A-tract structure at the site of the dimer, resulting in intact A tracts of reduced length and, hence, reduced bending. This model was suggested by the ability to formulate the estimated bend angles of the dimer-containing A tracts as approximately equal to the sum of the bend angels induced by the dimer and the remaining intact portion of the A tract. Contrary to a previous experimental study that concluded that the thymine dimer bends DNA by {approx} 30{degree}, the dimer was determined to bend DNA by only {approx} 7{degree}. Reduction of the bending of a DNA sequence by dimer formation may have a number of unpredicted and important biological consequences.

  6. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

    SciTech Connect

    Zoghbi, M. E.; Altenberg, G. A.

    2013-10-15

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.

  7. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    PubMed Central

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  8. Guidance on Initial Site Assessment at Corrective Action Sites

    EPA Pesticide Factsheets

    Guidance to be used to conduct Corrective Action site assessment efforts. Informs Resource Conservation and Recovery Act (RCRA) permit writers and enforcement officials of procedures to be used in conducting RCRA Facility Assessments.

  9. Cell cycle-independent removal of UV-induced pyrimidine dimers from the promoter and the transcription initiation domain of the human CDC2 gene.

    PubMed

    Tommasi, S; Oxyzoglou, A B; Pfeifer, G P

    2000-10-15

    To assess whether removal of UV-induced cyclobutane pyrimidine dimers (CPDs) occurs with equal efficiency at different stages of the cell cycle in a cell cycle-regulated gene, we have analyzed repair of CPDs, following a single dose of UV, in normal human fibroblasts that were synchronized in either G(0) or S phase. Based on a single nucleotide resolution analysis, we established a detailed map of DNA repair rates along the promoter region and the transcription initiation area of the human CDC2 gene. The promoter of this gene is covered by an array of sequence-specific transcription factors located between nt -280 and -9 relative to the major transcription start site. In both quiescent and S phase-synchronized fibroblasts the majority of these sequences were poorly repaired even after 24 h, probably as a result of the constitutive binding of transcription factors throughout the cell cycle. A domain of fast repair was found at sequences surrounding the transcription initiation site and continuing downstream for approximately 80 nt. CPD removal from this domain was preferential in both quiescent and proliferating fibroblasts, despite lower levels of global genome repair and a lack of CDC2 transcription in quiescent cells. We suggest that sequences involved in transcription initiation may be book-marked for efficient repair throughout the cell cycle, even when the gene is temporarily not expressed.

  10. Cell cycle-independent removal of UV-induced pyrimidine dimers from the promoter and the transcription initiation domain of the human CDC2 gene

    PubMed Central

    Tommasi, Stella; Oxyzoglou, Alexandros B.; Pfeifer, Gerd P.

    2000-01-01

    To assess whether removal of UV-induced cyclobutane pyrimidine dimers (CPDs) occurs with equal efficiency at different stages of the cell cycle in a cell cycle-regulated gene, we have analyzed repair of CPDs, following a single dose of UV, in normal human fibroblasts that were synchronized in either G0 or S phase. Based on a single nucleotide resolution analysis, we established a detailed map of DNA repair rates along the promoter region and the transcription initiation area of the human CDC2 gene. The promoter of this gene is covered by an array of sequence-specific transcription factors located between nt –280 and –9 relative to the major transcription start site. In both quiescent and S phase-synchronized fibroblasts the majority of these sequences were poorly repaired even after 24 h, probably as a result of the constitutive binding of transcription factors throughout the cell cycle. A domain of fast repair was found at sequences surrounding the transcription initiation site and continuing downstream for ∼80 nt. CPD removal from this domain was preferential in both quiescent and proliferating fibroblasts, despite lower levels of global genome repair and a lack of CDC2 transcription in quiescent cells. We suggest that sequences involved in transcription initiation may be book-marked for efficient repair throughout the cell cycle, even when the gene is temporarily not expressed. PMID:11024179

  11. Definition and prediction of the full range of transcription factor binding sites—the hepatocyte nuclear factor 1 dimeric site

    PubMed Central

    Locker, Joseph; Ghosh, David; Luc, Phuong-Van; Zheng, Jianhua

    2002-01-01

    In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding. PMID:12202766

  12. BIOREMEDIATION FIELD INITIATIVE SITE PROFILE: ESCAMBIA WOOD PRESERVING SITE - BROOKHAVEN

    EPA Science Inventory

    The Escambia Wood Preserving Site—Brookhaven in Brookhaven, Mississippi, is a former wood preserving facility that used pentachlo- rophenol (PCP) and creosote to treat wooden poles. The site contains two pressure treatment cylinders, a wastewater treatment system, five bulk pr...

  13. BIOREMEDIATION FIELD INITIATIVE SITE PROFILE: ESCAMBIA WOOD PRESERVING SITE - BROOKHAVEN

    EPA Science Inventory

    The Escambia Wood Preserving Site—Brookhaven in Brookhaven, Mississippi, is a former wood preserving facility that used pentachlo- rophenol (PCP) and creosote to treat wooden poles. The site contains two pressure treatment cylinders, a wastewater treatment system, five bulk pr...

  14. 40 CFR 280.63 - Initial site characterization.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Initial site characterization. 280.63 Section 280.63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... Hazardous Substances § 280.63 Initial site characterization. (a) Unless directed to do otherwise by the...

  15. Genome-Wide Profiling of Alternative Translation Initiation Sites.

    PubMed

    Gao, Xiangwei; Wan, Ji; Qian, Shu-Bing

    2016-01-01

    Regulation of translation initiation is a central control point in protein synthesis. Variations of start codon selection contribute to protein diversity and complexity. Systemic mapping of start codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we describe a ribosome profiling approach that enables identification of translation initiation sites on a genome-wide scale. By capturing initiating ribosomes using lactimidomycin, this approach permits qualitative and quantitative analysis of alternative translation initiation.

  16. The bZIP dimer localizes at DNA full-sites where each basic region can alternately translocate and bind to subsites at the half-site

    PubMed Central

    Chan, I-San; Al-Sarraj, Taufik; Shahravan, S. Hesam; Fedorova, Anna V.; Shin, Jumi A.

    2012-01-01

    Crystal structures of the GCN4 bZIP (basic region/leucine zipper) with the AP-1 or CRE site show how each GCN4 basic region binds to a 4-bp cognate half-site as a single DNA target; however, this may not always fully describe how bZIP proteins interact with their target sites. Previously, we showed that the GCN4 basic region interacts with all 5 bp in half-site TTGCG (termed 5H-LR), and that 5H-LR comprises two 4-bp subsites, TTGC and TGCG, which individually are also target sites of the basic region. In this work, we explored how the basic region interacts with 5H-LR when the bZIP dimer localizes to full-sites. Using AMBER molecular modeling, we simulated GCN4 bZIP complexes with full-sites containing 5H-LR to investigate in silico the interface between the basic region and 5H-LR. We also performed in vitro investigation of bZIP–DNA interactions at a number of full-sites that contain 5H-LR vs. either subsite: we analyzed results from DNase I footprinting and electrophoretic mobility shift assay (EMSA) and from EMSA titrations to quantify binding affinities. Our computational and experimental results together support a highly dynamic DNA-binding model: when a bZIP dimer localizes to its target full-site, the basic region can alternately recognize either subsite as a distinct target at 5H-LR and translocate between the subsites, potentially by sliding and hopping. This model provides added insights into how α-helical DNA-binding domains of transcription factors can localize to their gene regulatory sequences in vivo. PMID:22856882

  17. Finding the start site: redefining the human initiator element

    PubMed Central

    Kugel, Jennifer F.; Goodrich, James A.

    2017-01-01

    Transcription by RNA polymerase II (Pol II) is dictated in part by core promoter elements, which are DNA sequences flanking the transcription start site (TSS) that help direct the proper initiation of transcription. Taking advantage of recent advances in genome-wide sequencing approaches, Vo ngoc and colleagues (pp. 6–11) identified transcripts with focused sites of initiation and found that many were transcribed from promoters containing a new consensus sequence for the human initiator (Inr) core promoter element. PMID:28130343

  18. Tyrosine and tryptophan act through the same binding site at the dimer interface of yeast chorismate mutase.

    PubMed

    Schnappauf, G; Krappmann, S; Braus, G H

    1998-07-03

    Tyrosine and tryptophan are the regulators of the dimeric yeast chorismate mutase. Biochemical studies reveal two binding sites per molecule for both effectors, tyrosine or tryptophan. A single binding site is built up by helix 8 and helices 4 and 5 of two different subunits. The binding sites have been analyzed in the active enzyme by site directed mutagenesis of critical codons of the coding gene, ARO7. Gly-141 and Ser-142, which both reside on helix 8, are involved in the binding of tyrosine or tryptophan presumably by interacting specifically with the amino- and carboxylate-groups of these amino acid effectors. Interaction with Thr-145 of helix 8 is required for a strong tyrosine binding to the allosteric site. Replacement of Arg-75, which connects helices 4 and 5 or of Arg-76, which is part of helix 5 by alanine residues, resulted in unregulated enzymes. These two residues are bonded to the carboxylate group and phenolic hydroxyl group of tyrosine, respectively, but do not interact with tryptophan by hydrogen bonding in the crystal structures. Phenylalanine, which has low binding affinity slightly activated the chorismate mutase. A T145V mutant chorismate mutase, however, showed increased activation by phenylalanine. Our results support a mechanism by which tyrosine contracts the allosteric site by interacting with its phenolic hydroxyl group. Tryptophan works in an inverse way by opening the allosteric site through the steric size of its side chain.

  19. The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways*

    PubMed Central

    Scheerer, Patrick; Zhang, Fan; Kalms, Jacqueline; von Stetten, David; Krauß, Norbert; Oberpichler, Inga; Lamparter, Tilman

    2015-01-01

    Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor. PMID:25784552

  20. The class III cyclobutane pyrimidine dimer photolyase structure reveals a new antenna chromophore binding site and alternative photoreduction pathways.

    PubMed

    Scheerer, Patrick; Zhang, Fan; Kalms, Jacqueline; von Stetten, David; Krauß, Norbert; Oberpichler, Inga; Lamparter, Tilman

    2015-05-01

    Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase

    SciTech Connect

    Saporito, S.M.; Gedenk, M.; Cunningham, R.P.

    1989-05-01

    The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

  2. Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing

    SciTech Connect

    Herbst, R.; Perovic, I; Martin-Diaconescu, V; O’Brien, K; Chivers, P; Sondej Pochapsky, S; Pochapsky, T; Maroney, M

    2010-01-01

    Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys){sub 4} site to a Zn(His){sub 2}(Cys){sub 2} site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the {beta}-CH{sub 2} protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys){sub 4}) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model

  3. RNA Dimerization Promotes PKR Dimerization and Activation

    PubMed Central

    Heinicke, Laurie A.; Wong, C. Jason; Lary, Jeffrey; Nallagatla, Subba Rao; Diegelman-Parente, Amy; Zheng, Xiaofeng; Cole, James L.; Bevilacqua, Philip C.

    2009-01-01

    The double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15 bp dsRNA for one protein to bind and 30 bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eIF2α, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15 to 30 bp limits: HIV-I TAR RNA, a 23 bp hairpin with three bulges that is known to dimerize. To test whether RNA dimerization affects PKR dimerization and activation, TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15–30 bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease. PMID:19445956

  4. Persistence of magnons in a site-diluted dimerized frustrated antiferromagnet

    SciTech Connect

    Stone, Matthew B; Podlesnyak, Andrey A; Ehlers, Georg; Huq, Ashfia; Samulon, Eric C; Shapiro, Max C; Fisher, Ian R

    2011-01-01

    We present inelastic neutron scattering and thermodynamic measurements characterizing the magnetic excitations in a disordered non-magnetic substituted spin-liquid antiferromagnet. The parent compound Ba3Mn2O8 is a dimerized, quasi-two-dimensional geometrically frustrated quantum disordered antiferromagnet. We substitute this compound with non-magnetic vanadium for the S = 1 manganese atoms, Ba3(Mn1-xVx)2O8, and find that the singlet-triplet excitations which dominate the spectrum of the parent compound persist for the full range of substitution examined, x = 0.02 to 0.3. We also observe additional low-energy magnetic fluctuations which are enhanced at the greatest substitution values. These excitations may be a precursor to a low-temperature random singlet phase which may exist in Ba3(Mn1-xVx)2O8.

  5. A structural study of the K adsorption site on a Si(001)2 × 1 surface: Dimer, caves or both

    NASA Astrophysics Data System (ADS)

    Asensio, M. C.; Michel, E. G.; Alvarez, J.; Ocal, C.; Miranda, R.; Ferrer, S.

    1989-04-01

    The atomic structure of the clean Si(100) and K covered surfaces has been investigated by Auger electron diffraction (AED) monitoring the intensities along polar scans. This technique is sensitive to the asymmetric-dimer nature of the 2 × 1 reconstruction of the Si(001) surface. Data taken at room temperature for submonolayer coverages are consistent with adsorption of K on the troughs (cave position) existing between two consecutive dimer chains along the [110] direction. At 110 K both dimer and cave sites are occupied. A mild annealing to 300 K produces an overlayer redistribution in favor of the "cave" site further indicating that this site is energetically favoured as found in some recent calculations.

  6. Rapid deamination of cyclobutane pyrimidine dimer photoproducts at TCG sites in a translationally and rotationally positioned nucleosome in vivo.

    PubMed

    Cannistraro, Vincent J; Pondugula, Santhi; Song, Qian; Taylor, John-Stephen

    2015-10-30

    Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.

  7. Rapid Deamination of Cyclobutane Pyrimidine Dimer Photoproducts at TCG Sites in a Translationally and Rotationally Positioned Nucleosome in Vivo*

    PubMed Central

    Cannistraro, Vincent J.; Pondugula, Santhi; Song, Qian; Taylor, John-Stephen

    2015-01-01

    Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for TmCG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots. PMID:26354431

  8. Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding

    SciTech Connect

    Stump, D.G.; Lloyd, R.S.

    1988-03-22

    T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

  9. Finding the start site: redefining the human initiator element.

    PubMed

    Kugel, Jennifer F; Goodrich, James A

    2017-01-01

    Transcription by RNA polymerase II (Pol II) is dictated in part by core promoter elements, which are DNA sequences flanking the transcription start site (TSS) that help direct the proper initiation of transcription. Taking advantage of recent advances in genome-wide sequencing approaches, Vo ngoc and colleagues (pp. 6-11) identified transcripts with focused sites of initiation and found that many were transcribed from promoters containing a new consensus sequence for the human initiator (Inr) core promoter element. © 2017 Kugel and Goodrich; Published by Cold Spring Harbor Laboratory Press.

  10. Identification of Interaction Sites for Dimerization and Adapter Recruitment in Toll/Interleukin-1 Receptor (TIR) Domain of Toll-like Receptor 4*

    PubMed Central

    Bovijn, Celia; Ulrichts, Peter; De Smet, Anne-Sophie; Catteeuw, Dominiek; Beyaert, Rudi; Tavernier, Jan; Peelman, Frank

    2012-01-01

    Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization. PMID:22139835

  11. Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4.

    PubMed

    Bovijn, Celia; Ulrichts, Peter; De Smet, Anne-Sophie; Catteeuw, Dominiek; Beyaert, Rudi; Tavernier, Jan; Peelman, Frank

    2012-02-03

    Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.

  12. Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers.

    PubMed

    Darling, P J; Holt, J M; Ackers, G K

    2000-09-22

    The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity. Copyright 2000 Academic Press.

  13. AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium.

    PubMed

    Jiménez, Marta; André, Sabine; Siebert, Hans-C; Gabius, Hans-J; Solís, Dolores

    2006-10-01

    Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).

  14. Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site.

    PubMed

    Walsh, M A; Otwinowski, Z; Perrakis, A; Anderson, P M; Joachimiak, A

    2000-05-15

    Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. We have determined the crystal structure of cyanase at 1.65 A resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding alpha-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.

  15. Young Adult Capacity Initiative Cross-Site Analysis

    ERIC Educational Resources Information Center

    Academy for Educational Development, 2012

    2012-01-01

    This cross-site analysis presents findings about the implementation, impact, and outcomes of the Young Adult Capacity Initiative (YACI), at 13 community-based organizations in New York City. These agencies received technical assistance and small incentive grants from the Fund for the City of New York Youth Development Institute (YDI) to build…

  16. Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli

    SciTech Connect

    Koehler, D.R.; Awadallah, S.S.; Glickman, B.W. )

    1991-06-25

    An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI . On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.

  17. Initial Hazard Categorization for the Hanford Site Tank Farms

    SciTech Connect

    Savino, A.V.

    1995-10-01

    This document reports results of an Initial Hazard Categorization for the Hanford Site Tank Farms using the procedure set forth in DOE Standard 1027-92. For documentation purposes the initial Hazard Categorization calculations were made for two cases. The first case treats each tank as an individual segmented facility. The second case treats all 177 of the Hanford Site tanks as a single segmented facility. Calculations were performed using conservative estimates for the tank waste radioactive inventories. The initial hazard category is Hazard Category 2 when each tank is treated as an individual facility segment, and is also Hazard Category 2 when the 177 tanks are treated collectively as a single facility segment.

  18. Wall shear stress at the initiation site of cerebral aneurysms.

    PubMed

    Geers, A J; Morales, H G; Larrabide, I; Butakoff, C; Bijlenga, P; Frangi, A F

    2017-02-01

    Hemodynamics are believed to play an important role in the initiation of cerebral aneurysms. In particular, studies have focused on wall shear stress (WSS), which is a key regulator of vascular biology and pathology. In line with the observation that aneurysms predominantly occur at regions of high WSS, such as bifurcation apices or outer walls of vascular bends, correlations have been found between the aneurysm initiation site and high WSS. The aim of our study was to analyze the WSS field at an aneurysm initiation site that was neither a bifurcation apex nor the outer wall of a vascular bend. Ten cases with aneurysms on the A1 segment of the anterior cerebral artery were analyzed and compared with ten controls. Aneurysms were virtually removed from the vascular models of the cases to mimic the pre-aneurysm geometry. Computational fluid dynamics (CFD) simulations were created to assess the magnitude, gradient, multidirectionality, and pulsatility of the WSS. To aid the inter-subject comparison of hemodynamic variables, we mapped the branch surfaces onto a two-dimensional parametric space. This approach made it possible to view the whole branch at once for qualitative evaluation. It also allowed us to empirically define a patch for quantitative analysis, which was consistent among subjects and encapsulated the aneurysm initiation sites in our dataset. To test the sensitivity of our results, CFD simulations were repeated with a second independent observer virtually removing the aneurysms and with a 20 % higher flow rate at the inlet. We found that branches harboring aneurysms were characterized by high WSS and high WSS gradients. Among all assessed variables, the aneurysm initiation site most consistently coincided with peaks of temporal variation in the WSS magnitude.

  19. Translation from unconventional 5′ start sites drives tumour initiation

    PubMed Central

    Sendoel, Ataman; Dunn, Joshua G.; Rodriguez, Edwin H.; Naik, Shruti; Gomez, Nicholas C.; Hurwitz, Brian; Levorse, John; Dill, Brian D.; Schramek, Daniel; Molina, Henrik; Weissman, Jonathan S.; Fuchs, Elaine

    2017-01-01

    We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5′ untranslated regions in cancer, and expose new targets for therapeutic intervention. PMID:28077873

  20. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    SciTech Connect

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  1. Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy.

    PubMed

    Le Breton, Nolwenn; Adrianaivomananjaona, Tiona; Gerbaud, Guillaume; Etienne, Emilien; Bisetto, Elena; Dautant, Alain; Guigliarelli, Bruno; Haraux, Francis; Martinho, Marlène; Belle, Valérie

    2016-01-01

    The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Structure of the human dimeric ATM kinase

    PubMed Central

    Lau, Wilson C. Y.; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S. Y.

    2016-01-01

    ABSTRACT DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  3. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  4. Evidence for dimerization of dimers in K+ channel assembly.

    PubMed Central

    Tu, L; Deutsch, C

    1999-01-01

    Voltage-gated K+ channels are tetrameric, but how the four subunits assemble is not known. We analyzed inactivation kinetics and peak current levels elicited for a variety of wild-type and mutant Kv1.3 subunits, expressed singly, in combination, and as tandem constructs, to show that 1) the dominant pathway involves a dimerization of dimers, and 2) dimer-dimer interaction may involve interaction sites that differ from those involved in monomer-monomer association. Moreover, using nondenaturing gel electrophoresis, we detected dimers and tetramers, but not trimers, in the translation reaction of Kv1.3 monomers. PMID:10096897

  5. Measurement of dimeric inhibin using a modified two-site immunoradiometric assay specific for oxidized (Met O) inhibin.

    PubMed

    Knight, P G; Muttukrishna, S

    1994-06-01

    Several years ago we developed a novel two-site immunoradiometric assay (IRMA) for dimeric inhibin. However, relative to the purified 32 kDa bovine inhibin standard used at that time, the immunopotencies of crude inhibin-containing samples were much less than their biopotencies estimated by pituitary cell bioassay. In attempting to improve assay performance and resolve this discrepancy we recently discovered that introduction of a preassay oxidation step to the IRMA results in a dramatic increase in the immunopotencies of inhibin-containing test samples (e.g.: bovine, human, porcine follicular fluid (FF)) and of a new (purified in 1993) 32 kDa bovine inhibin standard. However, the oxidation step did not affect the immunopotency of our original standard (purified in 1987), indicating that this material had undergone spontaneous oxidation during long-term storage, thus accounting for its higher immunopotency in our original IRMA and providing an explanation for the discrepancy between immunoactivity and bioactivity referred to above. These findings, together with other observations on the behaviour of oxidized and non-oxidized samples of inhibin, related peptide fragments and inhibin-containing samples in the IRMA and alpha subunit radioimmunoassay (RIA), indicate that the anti-beta A82-114 monoclonal antibody (E4) used as tracer in the IRMA binds selectively to the oxidized (Met O89,91,108) form of the peptide. This property of the antibody can be exploited to advantage by incorporating simple modifications to existing inhibin/activin immunoassays to ensure that all samples and standards are fully oxidized before antibody addition.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Equilibrium unfolding studies of the rat liver methionine adenosyltransferase III, a dimeric enzyme with intersubunit active sites.

    PubMed Central

    Gasset, María; Alfonso, Carlos; Neira, José L; Rivas, Germán; Pajares, María A

    2002-01-01

    The reversible unfolding of rat liver methionine adenosyltransferase dimer by urea under equilibrium conditions has been monitored by fluorescence spectroscopy, CD, size-exclusion chromatography, analytical ultracentrifugation and enzyme activity measurements. The results obtained indicate that unfolding takes place through a three-state mechanism, involving an inactive monomeric intermediate. This intermediate has a 70% native secondary structure, binds less 8-anilinonaphthalene-1-sulphonic acid than the native dimer and has a sedimentation coefficient of 4.24+/-0.15. The variations of free energy in the absence of denaturant [DeltaG(H(2)O)] and its coefficients of urea dependence (m), calculated by the linear extrapolation model, were 36.15+/-2.3 kJ.mol(-1) and 19.87+/-0.71 kJ.mol(-1).M(-1) for the dissociation of the native dimer and 14.77+/-1.63 kJ.mol(-1) and 5.23+/-0.21 kJ.mol(-1).M(-1) for the unfolding of the monomeric intermediate respectively. Thus the global free energy change in the absence of denaturant and the m coefficient were calculated to be 65.69 kJ.mol(-1) and 30.33 kJ.mol(-1).M(-1) respectively. Analysis of the calculated thermodynamical parameters indicate the instability of the dimer in the presence of denaturant, and that the major exposure to the solvent is due to dimer dissociation. Finally, a minimum-folding mechanism for methionine adenosyltransferase III is established. PMID:11772402

  7. Photoionization-induced π↔ H site switching dynamics in phenol(+)-Rg (Rg = Ar, Kr) dimers probed by picosecond time-resolved infrared spectroscopy.

    PubMed

    Miyazaki, Mitsuhiko; Sakata, Yuri; Schütz, Markus; Dopfer, Otto; Fujii, Masaaki

    2016-09-21

    The ionization-induced π↔ H site switching reaction in phenol(+)-Rg (PhOH(+)-Rg) dimers with Rg = Ar and Kr is traced in real time by picosecond time-resolved infrared (ps-TRIR) spectroscopy. The ps-TRIR spectra show the prompt appearance of the non-vanishing free OH stretching band upon resonant photoionization of the π-bound neutral clusters, and the delayed appearance of the hydrogen-bonded (H-bonded) OH stretching band. This result directly proves that the Rg ligand switches from the π-bound site on the aromatic ring to the H-bonded site at the OH group by ionization. The subsequent H →π back reaction converges the dimer to a π↔ H equilibrium. This result is in sharp contrast to the single-step π→ H forward reaction in the PhOH(+)-Ar2 trimer with 100% yield. The reaction mechanism and yield strongly depend on intracluster vibrational energy redistribution. A classical rate equation analysis for the time evolutions of the band intensities of the two vibrations results in similar estimates for the time constants of the π→ H forward reaction of τ+ = 122 and 73 ps and the H →π back reaction of τ- = 155 and 188 ps for PhOH(+)-Ar and PhOH(+)-Kr, respectively. The one order of magnitude slower time constant in comparison to the PhOH(+)-Ar2 trimer (τ+ = 7 ps) is attributed to the decrease in density of states due to the absence of the second Ar in the dimer. The similar time constants for both PhOH(+)-Rg dimers are well rationalized by a classical interpretation based on the comparable potential energy surfaces, reaction pathways, and density of states arising from their similar intermolecular vibrational frequencies.

  8. Initial CRISM Observations of the Candidate 2007 Phoenix Landing Sites

    NASA Astrophysics Data System (ADS)

    Seelos, K. D.; Murchie, S.; Arvidson, R. E.; Seelos, F. P.

    2006-12-01

    The Mars Reconnaissance Orbiter (MRO) Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) will acquire multispectral and targeted hyperspectral visible and near infrared data of the candidate Phoenix landing sites during the first few months of primary mission operations (beginning early November). Three 150 x 75 km candidate Phoenix landing sites are located in the high northern plains of Mars within a region from 65-72° N and 120-140° E. Geomorphologic characterization of this region indicates a relatively homogeneous terrain primarily composed of multiple kilometer-scale polygonal plains with superposed degraded craters. At decameter spatial scales, the area is ubiquitously covered by patterned ground in the form of basketball terrain, stripes, and small polygons. Spectral variation of these different types of landforms and materials that are detected by CRISM at 100- or 200-meter scales (multispectral) or ~20-meter scales (targeted hyperspectral) will be analyzed and initial results presented. Implications for Phoenix landing site selection and in situ measurements will also be discussed. CRISM observations along with other MRO data will be critical to the selection of the final landing site prior to launch in August of 2007.

  9. Prodigal: prokaryotic gene recognition and translation initiation site identification

    SciTech Connect

    Chen, Gwo-Liang; LoCascio, Philip F; Land, Miriam L; Larimer, Frank W; Hauser, Loren John

    2010-01-01

    The quality of automated gene prediction in microbial organisms has improved steadily over the past decade, but there is still room for improvement. Increasing the number of correct identifications, both of genes and of the translation initiation sites for each gene, and reducing the overall number of false positives, are all desirable goals. With our years of experience in manually curating genomes for the Joint Genome Institute, we developed a new gene prediction algorithm called Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm). With Prodigal, we focused specifically on the three goals of improved gene structure prediction, improved translation initiation site recognition, and reduced false positives. We compared the results of Prodigal to existing gene-finding methods to demonstrate that it met each of these objectives. We built a fast, lightweight, open source gene prediction program called Prodigal http://compbio.ornl.gov/prodigal/. Prodigal achieved good results compared to existing methods, and we believe it will be a valuable asset to automated microbial annotation pipelines. The goals of Prodigal were to attain greater sensitivity in identifying existing genes, to predict translation initiation sites more accurately, and to minimize the number of false positive predictions. The results of Prodigal were compared to existing methods for both purely experimentally verified genes as well as curated Genbank files for a number of genomes. Prodigal's performance was found to be comparable or better to existing methods in the prediction of genes while also predicting fewer overall genes. In the prediction of translation initiation sites, Prodigal performed competitively with existing methods. Prodigal is currently already in use at many institutions, and it has been used to annotate all finished microbial genomes submitted to Genbank by DOE-JGI in 2008 and onward (a substantial percentage of the overall finished microbial genomes at NCBI). It is

  10. Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids

    PubMed Central

    Datta, Kausiki; Johnson, Neil P.; Villani, Giuseppe; Marcus, Andrew H.; von Hippel, Peter H.

    2012-01-01

    We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base–base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA ‘breathing’ at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution. PMID:22009678

  11. Prodigal: Prokaryotic Gene Recognition and Translation Initiation Site Identification

    SciTech Connect

    Hyatt, Philip Douglas; Chen, Gwo-Liang; Larimer, Frank W; LoCascio, Philip F; Hauser, Loren John; Land, Miriam L

    2010-01-01

    Background The quality of automated gene prediction in microbial organisms has improved steadily over the past decade, but there is still room for improvement. Increasing the number of correct identifications, both of genes and of the translation initiation sites for each gene, and reducing the overall number of false positives, are all desirable goals. Results With our years of experience in manually curating genomes for the Joint Genome Institute, we developed a new gene prediction algorithm called Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm). With Prodigal, we focused specifically on the three goals of improved gene structure prediction, improved translation initiation site recognition, and reduced false positives. We compared the results of Prodigal to existing gene-finding methods to demonstrate that it met each of these objectives. Conclusion We built a fast, lightweight, open source gene prediction program called Prodigal (http://compbio.ornl.gov/prodigal/). Prodigal achieved good results compared to existing methods, and we believe it will be a valuable asset to automated microbial annotation pipelines.

  12. Half-site reactivity with p-nitrophenylhydrazine and subunit separation of the dimeric copper-containing amine oxidase from Aspergillus niger.

    PubMed

    Frébort, I; Toyama, H; Matsushita, K; Adachi, O

    1995-08-01

    Structural properties of dimeric (2 x 75 kDa) copper-containing amine oxidase (EC 1.4.3.6) from Aspergillus niger were studied. The enzyme treated with SDS was dissociated into subunits which showed different mobility on polyacrylamide gel without SDS. The separated subunits had no activity but a quinone moiety was detected in both by a redox-cyclic quinone staining. After titration of the enzyme with p-nitrophenylhydrazine, which showed half-site reactivity (1 mole per dimer), and SDS treatment both p-nitro-phenylhydrazone and a remaining quinone moiety were detected in each subunit. It is suggested that the half-site reactivity with phenylhydrazine is caused by conformational changes after binding of the inhibitor to any one of the active sites leading to inaccessibility of the second active site for the inhibitor. The difference in electrophoretic mobility of the separated subunits originates probably from their structural difference likely to occur outside the active site, even if the amino acid sequences of the subunits appear to be identical.

  13. Performance improvement initiative: prevention of surgical site infection (SSI).

    PubMed

    Ng, Wai Khuan; Awad, Nawal

    2015-01-01

    Mafraq Hospital performs an average of 10,000 surgeries every year. The impact of having high volume high risk surgical procedures calls for the need to ensure safe surgery and a prevention of surgical site infection (SSI). SSI represents a significant portion of healthcare-associated infections (HAIs). The impact on morbidity, mortality, and cost of care has resulted in identifying the need to reduce SSI as a top priority to prevent healthcare associated infections. The good news is that the majority of SSIs are preventable. Mafraq Hospital performs a range of surgical procedures that covers 14 surgical specialties. The infection prevention and control team performs surveillance for SSI for all patients who undergo operative procedure included in Centers for Disease Control and Prevention (CDC) National Healthcare Safety Network (NHSN) Operative Procedure Category (40 surgical procedures). Out of the 40 CDC NHSN listed, 33 operative procedures were performed at Mafraq Hospital, of which 17 were reported with SSI for 2013 and 2014. Surgical site infection has implicated an increase average length of stay from seven to 10 additional postoperative hospital days and additional costs of AED 10,000 to AED 100,000/SSI depending on procedure and pathogen. A multidisciplinary team was formed to develop and implement measures to reduce/eliminate surgical site infection, as well as evaluate and monitor compliance. Hence a group of multidisciplinary teams were initiated to analyse the results, find out the gaps, and implement a quality improvement project to correct the deficits. Recommendations for appropriate improvement measures were formed on evidence-based international guidelines from the Institute for Healthcare Improvement (IHI) and CDC. Evidence based practice supports that many of the causes of surgical site infection can be prevented with proper medical attention and care.

  14. Collective motion of dimers.

    PubMed

    Penington, Catherine J; Korvasová, Karolína; Hughes, Barry D; Landman, Kerry A

    2012-11-01

    We consider a discrete agent-based model on a one-dimensional lattice and a two-dimensional square lattice, where each agent is a dimer occupying two sites. Agents move by vacating one occupied site in favor of a nearest-neighbor site and obey either a strict simple exclusion rule or a weaker constraint that permits partial overlaps between dimers. Using indicator variables and careful probability arguments, a discrete-time master equation for these processes is derived systematically within a mean-field approximation. In the continuum limit, nonlinear diffusion equations that describe the average agent occupancy of the dimer population are obtained. In addition, we show that multiple species of interacting subpopulations give rise to advection-diffusion equations. Averaged discrete simulation data compares very well with the solution to the continuum partial differential equation models. Since many cell types are elongated rather than circular, this work offers insight into population-level behavior of collective cellular motion.

  15. Gene and translation initiation site prediction in metagenomic sequences

    SciTech Connect

    Hyatt, Philip Douglas; LoCascio, Philip F; Hauser, Loren John; Uberbacher, Edward C

    2012-01-01

    Gene prediction in metagenomic sequences remains a difficult problem. Current sequencing technologies do not achieve sufficient coverage to assemble the individual genomes in a typical sample; consequently, sequencing runs produce a large number of short sequences whose exact origin is unknown. Since these sequences are usually smaller than the average length of a gene, algorithms must make predictions based on very little data. We present MetaProdigal, a metagenomic version of the gene prediction program Prodigal, that can identify genes in short, anonymous coding sequences with a high degree of accuracy. The novel value of the method consists of enhanced translation initiation site identification, ability to identify sequences that use alternate genetic codes and confidence values for each gene call. We compare the results of MetaProdigal with other methods and conclude with a discussion of future improvements.

  16. Accuracy improvement for identifying translation initiation sites in microbial genomes.

    PubMed

    Zhu, Huai-Qiu; Hu, Gang-Qing; Ouyang, Zheng-Qing; Wang, Jin; She, Zhen-Su

    2004-12-12

    At present the computational gene identification methods in microbial genomes have a high prediction accuracy of verified translation termination site (3' end), but a much lower accuracy of the translation initiation site (TIS, 5' end). The latter is important to the analysis and the understanding of the putative protein of a gene and the regulatory machinery of the translation. Improving the accuracy of prediction of TIS is one of the remaining open problems. In this paper, we develop a four-component statistical model to describe the TIS of prokaryotic genes. The model incorporates several features with biological meanings, including the correlation between translation termination site and TIS of genes, the sequence content around the start codon; the sequence content of the consensus signal related to ribosomal binding sites (RBSs), and the correlation between TIS and the upstream consensus signal. An entirely non-supervised training system is constructed, which takes as input a set of annotated coding open reading frames (ORFs) by any gene finder, and gives as output a set of organism-specific parameters (without any prior knowledge or empirical constants and formulas). The novel algorithm is tested on a set of reliable datasets of genes from Escherichia coli and Bacillus subtillis. MED-Start may correctly predict 95.4% of the start sites of 195 experimentally confirmed E.coli genes, 96.6% of 58 reliable B.subtillis genes. Moreover, the test results indicate that the algorithm gives higher accuracy for more reliable datasets, and is robust to the variation of gene length. MED-Start may be used as a postprocessor for a gene finder. After processing by our program, the improvement of gene start prediction of gene finder system is remarkable, e.g. the accuracy of TIS predicted by MED 1.0 increases from 61.7 to 91.5% for 854 E.coli verified genes, while that by GLIMMER 2.02 increases from 63.2 to 92.0% for the same dataset. These results show that our algorithm is

  17. Identification of the initiation site of poliovirus polyprotein synthesis

    SciTech Connect

    Dorner, A.J.; Dorner, L.F.; Larsen, G.R.; Wimmer, E.; Anderson, C.W.

    1982-06-01

    The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVPOO. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VPO, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.

  18. Higher Levels of CRP, D-dimer, IL-6, and Hyaluronic Acid Before Initiation of Antiretroviral Therapy (ART) Are Associated With Increased Risk of AIDS or Death

    PubMed Central

    Boulware, David R.; Hullsiek, Katherine Huppler; Puronen, Camille E.; Rupert, Adam; Baker, Jason V.; French, Martyn A.; Bohjanen, Paul R.; Novak, Richard M.; Neaton, James D.

    2011-01-01

    Background. Substantial morbidity occurs during the first year of antiretroviral therapy (ART) in persons with advanced human immunodeficiency virus (HIV) disease despite HIV suppression. Biomarkers may identify high-risk groups. Methods. Pre-ART and 1-month samples from an initial ART trial were evaluated for biomarkers associated with AIDS events or death within 1–12 months. Case patients (n = 63) and control patients (n = 126) were 1:2 matched on baseline CD4 cell count, hepatitis status, and randomization date. All had ≥1 log10 HIV RNA level decrease at 1 month. Results. Case patients had more frequent prior AIDS events, compared with control patients (P = .004), but similar HIV RNA levels at baseline. Pre-ART and 1-month C-reactive protein (CRP), D-dimer, and interleukin 6 (IL-6) levels and pre-ART hyaluronic acid (HA) levels were associated with new AIDS events or death (P ≤ .01). Patients who experienced immune reconstitution inflammatory syndrome (IRIS) events had higher pre-ART tumor necrosis factor α (TNF-α) and HIV RNA levels and significant 1-month increases in CRP, D-dimer, IL-6, interleukin 8, CXCL10, TNF-α, and interferon-γ levels, compared with patients who experienced non-IRIS events (P ≤ .03). Individuals with baseline CRP and HA levels above the cohort median (>2.1 mg/L and >50.0 ng/mL, respectively) had increased risk of AIDS or death (OR, 4.6 [95% CI, 2.0–10.3]; P < .001) and IRIS (OR, 8.7 [95% CI, 2.2–34.8] P = .002). Conclusions. Biomarkers of Inflammation (CRP, IL-6), coagulation (D-dimer), and tissue fibrosis (HA) measured pre-ART and at 1 month are associated with higher risk of AIDS events, IRIS, or death, warranting additional study as risk stratification strategies. PMID:21592994

  19. Localization of transcription initiation sites on the mouse mitochondrial genome

    SciTech Connect

    Fluellen, C.F.; Bhat, K.S.; Avdalovic, N.; Avadhani, M.G.

    1987-05-01

    The authors have identified the primary transcription initiation sites on the H and L strands of mouse mitochondrial (mt) genome by mapping the 5' ends of in vitro capped mt RNA, and 5' end labelling of the nascent RNA synthesized in an in vitro mt system. RNA capped with TSP GTP resolve into 4 major (25 to 150 nucleotides) and one minor (0.75 kb) bands on denaturing gels. Only the 25 nucleotide long capped RNA hybridizes to the H strand of D-loop DNA and the rest hybridize to the L-strand DNA probes. S1 protection of capped RNA and DNA hybrids, and primer extention analysis using defined DNA primers show that all of the L-strand specific primary transcripts have a common 5' end mapping at about nucleotide 16,180 +/- 5 of the genome. The 3' ends of the small RNA species map near the start of conserved sequence boxes. The 3' end of the 0.75 Kb RNA maps to the start of gene coding for tRNA/sup Phe/. The 5' end of the capped RNA hybridizing to the H strand maps at about nucleotide 16,275 to 16,280 of the genome indicating a major H strand transcription initiation at this region. The authors have also used an in vitro transcription system which involves the use of mt extract from Ehrlich ascites cells to study transcription initiation. Nascent RNA 5' end labeled with elTSP ATP and GTP closely resemble the electrophoretic pattern and S1 protection pattern obtained with the capped RNA.

  20. pH-Dependent Binding of Chloride to a Marine Alkaline Phosphatase Affects the Catalysis, Active Site Stability, and Dimer Equilibrium.

    PubMed

    Hjörleifsson, Jens G; Ásgeirsson, Bjarni

    2017-09-07

    The effect of ionic strength on enzyme activity and stability varies considerably between enzymes. Ionic strength is known to affect the catalytic activity of some alkaline phosphatases (APs), such as Escherichia coli AP, but how ions affect APs is debated. Here, we studied the effect of various ions on a cold-adapted AP from Vibrio splendidus (VAP). Previously, we have found that the active form of VAP is extremely unstable at low ionic strengths. Here we show that NaCl increased the activity and stability of VAP and that the effect was pH-dependent in the range of pH 7-10. The activity profile as a function of pH formed two maxima, indicating a possible conformational change. Bringing the pH from the neutral to the alkaline range was accompanied by a large increase in both the Ki for inorganic phosphate (product inhibition) and the KM for p-nitrophenyl phosphate. The activity transitions observed as the pH was varied correlated with structural changes as monitored by tryptophan fluorescence. Thermal and urea-induced inactivation was shown to be accompanied by neither dissociation of the active site metal ions nor dimer dissociation. This would suggest that the inactivation involved subtle changes in active site conformation. Furthermore, the VAP dimer equilibrium was studied for the first time and shown to highly favor dimerization, which was dependent on pH and NaCl concentration. Taken together, the data support a model in which anions bind to some specific acceptor in the active site of VAP, resulting in great stabilization and catalytic rate enhancement, presumably through a different mechanism.

  1. Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

    PubMed

    Wah, D A; Romero, A; Gallego del Sol, F; Cavada, B S; Ramos, M V; Grangeiro, T B; Sampaio, A H; Calvete, J J

    2001-07-20

    Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure

  2. Selective synthesis and characterization of single-site HY zeolite-supported rhodium complexes and their use as catalysts for ethylene hydrogenation and dimerization

    NASA Astrophysics Data System (ADS)

    Khivantsev, Konstantin

    Single-site Rh(CO)2, Rh(C2H4)2 and Rh(NO)2 complexes anchored on various dealuminated HY zeolites can be used as precursors for the selective surface mediated synthesis of well-defined site-isolated Rh(CO)(H)x complexes. DFT calculations and D 2 isotope exchange experiments provide strong evidence for the formation of a family of site isolated mononuclear rhodium carbonyl hydride complexes (including the first examples of RhH complexes with undissociated H2 ligands): Rh(CO)(H2), Rh(CO)(H)2, and Rh(CO)(H). The fraction of each individual complex formed varies significantly with the Si/Al ratio of the zeolite and the nature of the precursor used. HY zeolite-supported mononuclear Rh(CO)2 complexes are very active in ethylene hydrogenation and ethylene dimerization under ambient conditions. There is strong evidence for the cooperation mechanism between mononuclear rhodium complexes and Bronsted acid sites of the zeolite support in C-C bond formation process, as well as ethane formation. Finally, it is shown that the dimerization pathway selectivity can be progressively tuned (and completely switched off) by modifying the number of Bronsted acid sites on the zeolite surface. HY zeolite-supported mononuclear Rh(NO)2 complexes can be selectively formed upon exposure of Rh(CO)2/HY to the gas phase NO/He. They are structurally similar to Rh(CO)2/HY with Rh(I) retaining square planar geometry and nitrosyl ligands adopting a linear configuration. Rh(NO)2/HY30 is active in ethylene hydrogenation and ethylene dimerization under ambient conditions. This is the first unprecedented example of a supported transition-metal nitrosyl complex capable of performing a catalytic reaction. Moreover, this is the first example of a site-isolated Rh complex with ligands other than ethylene or carbonyl, which can catalyze both ethylene hydrogenation and dimerization. Unlike its dicarbonyl counterpart, dinitrosyl rhodium complex has a uniquely different reactivity towards ethylene and hydrogen

  3. Initiatives to Develop Web Sites Including Information about Brownfields Properties

    EPA Pesticide Factsheets

    This web site was created to assist in planning, designing, and operating web sites that include information about individual brownfields properties. The report is of value to parties designing or managing such sites.

  4. Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.

    PubMed

    Eiamphungporn, Warawan; Soonsanga, Sumarin; Lee, Jin-Won; Helmann, John D

    2009-03-01

    Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

  5. DNA Minor Groove Induced Dimerization of Heterocyclic Cations: Compound Structure, Binding Affinity and Specificity for a TTAA Site

    PubMed Central

    Munde, Manoj; Kumar, Arvind; Nhili, Raja; Depauw, Sabine; David-Cordonnier, Marie-Hélène; Ismail, Mohamed A.; Stephens, Chad E.; Farahat, Abdelbasset A.; Batista-Parra, Adalgisa; Boykin, David W.; Wilson, W. David

    2010-01-01

    With the increasing number and variations of genome sequences available control of gene expression with synthetic, cell permeable molecules is within reach. The variety of sequence-specific-binding agents is, however, still quite limited. Many minor groove binding agents selectivity recognize AT over GC sequences but have less ability to distinguish among different AT sequences. The goal with this paper is to develop compounds that can bind selectively to different AT sequences. A number of studies indicate that AATT and TTAA sequences have significantly different physical and interaction properties and different requirements for minor groove recognition. Although it has been difficult to get minor groove binding at TTAA, DB293, a phenyl-furan-benzimidazole-diamidine, was found to bind as a strong, cooperative dimer at TTAA but with no selectivity over AATT. In order to improve selectivity, modifications were made to each unit of DB293. Binding affinities and stoichiometries obtained from biosensor-surface plasmon resonance experiments show that DB1003, a furan-furan-benzimidazole diamidine binds strongly to TTAA as a dimer and has selectivity (KTTAA/KAATT = 6). CD and DNAse I footprinting studies confirmed the preference of this compound for TTAA. In summary (i) a favorable stacking surface provided by the pi system, (ii) H-bond donors to interact with TA base pairs at the floor of the groove provided by a benzimidazole (or indole) –NH and amidines, and (iii) appropriate curvature of the dimer complex to match the curvature of the minor groove play important roles in differentiating the TTAA and AATT minor grooves. PMID:20713062

  6. Safeguards First Principles Initiative at the Nevada Test Site

    SciTech Connect

    Geneva Johnson

    2007-07-08

    The Material Control and Accountability (MC&A) program at the Nevada Test Site (NTS) was selected as a test bed for the Safeguards First Principles Initiative (SFPI). The implementation of the SFPI is evaluated using the system effectiveness model and the program is managed under an approved MC&A Plan. The effectiveness model consists of an evaluation of the critical elements necessary to detect, deter, and/or prevent the theft or diversion of Special Nuclear Material (SNM). The modeled results indicate that the MC&A program established under this variance is still effective, without creating unacceptable risk. Extensive performance testing is conducted through the duration of the pilot to ensure the protection system is effective and no material is at an unacceptable risk. The pilot was conducted from January 1, 2007, through May 30, 2007. This paper will discuss the following activities in association with SFPI: 1. Development of Timeline 2. Crosswalk of DOE Order and SFPI 3. Peer Review 4. Deviation 5. MC&A Plan and Procedure changes 6. Changes implemented at NTS 7. Training 8. Performance Test

  7. High resolution structures of Plasmodium falciparum GST complexes provide novel insights into the dimer-tetramer transition and a novel ligand-binding site.

    PubMed

    Perbandt, Markus; Eberle, Raphael; Fischer-Riepe, Lena; Cang, Huaixing; Liebau, Eva; Betzel, Christian

    2015-09-01

    Protection from oxidative stress and efficient redox regulation are essential for malarial parasites which have to grow and multiply rapidly in pro-oxidant rich environments. Therefore, redox active proteins currently belong to the most attractive antimalarial drug targets. The glutathione S-transferase from Plasmodium falciparum (PfGST) is a redox active protein displaying a peculiar dimer-tetramer transition that causes full enzyme-inactivation. This distinct structural feature is absent in mammalian GST isoenzyme counterparts. A flexible loop between residues 113-119 has been reported to be necessary for this tetramerization process. However, here we present structural data of a modified PfGST lacking loop 113-119 at 1.9 Å resolution. Our results clearly show that this loop is not essential for the formation of stable tetramers. Moreover we present for the first time the structures of both, the inactive and tetrameric state at 1.7 Å and the active dimeric state in complex with reduced glutathione at 2.4 Å resolution. Surprisingly, the structure of the inactive tetrameric state reveals a novel non-substrate binding-site occupied by a 2-(N-morpholino) ethane sulfonic acid (MES) molecule in each monomer. Although it is known that the PfGST has the ability to bind lipophilic anionic ligands, the location of the PfGST ligand-binding site remained unclear up to now. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Heat capacity of the site-diluted spin dimer system Ba₃(Mn1-xVx)₂O₈

    DOE PAGES

    Samulon, E. C.; Shapiro, M. C.; Fisher, I. R.

    2011-08-05

    Heat-capacity and susceptibility measurements have been performed on the diluted spin dimer compound Ba₃(Mn1-xVx)₂O₈. The parent compound Ba₃Mn₂O₈ is a spin dimer system based on pairs of antiferromagnetically coupled S=1, 3d² Mn⁵⁺ ions such that the zero-field ground state is a product of singlets. Substitution of nonmagnetic S=0, 3d⁰ V⁵⁺ ions leads to an interacting network of unpaired Mn moments, the low-temperature properties of which are explored in the limit of small concentrations 0≤x≤0.05. The zero-field heat capacity of this diluted system reveals a progressive removal of magnetic entropy over an extended range of temperatures, with no evidence for amore » phase transition. The concentration dependence does not conform to expectations for a spin-glass state. Rather, the data suggest a low-temperature random singlet phase, reflecting the hierarchy of exchange energies found in this system.« less

  9. Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

    PubMed Central

    Plath, Friederike; Ringler, Philippe; Graff-Meyer, Alexandra; Stahlberg, Henning; Lauer, Matthias E.; Rufer, Arne C.; Graewert, Melissa A.; Svergun, Dmitri; Gellermann, Gerald; Finkler, Christof; Stracke, Jan O.; Koulov, Atanas; Schnaible, Volker

    2016-01-01

    ABSTRACT The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs. PMID:27031922

  10. Transductive learning as an alternative to translation initiation site identification.

    PubMed

    Nunes Pinto, Cristiano Lacerda; Nobre, Cristiane Neri; Zárate, Luis Enrique

    2017-02-02

    The correct protein coding region identification is an important and latent problem in the molecular biology field. This problem becomes a challenge due to the lack of deep knowledge about the biological systems and unfamiliarity of conservative characteristics in the messenger RNA (mRNA). Therefore, it is fundamental to research for computational methods aiming to help the patterns discovery for identification of the Translation Initiation Sites (TIS). In the field of Bioinformatics, machine learning methods have been widely applied based on the inductive inference, as Inductive Support Vector Machine (ISVM). On the other hand, not so much attention has been given to transductive inference-based machine learning methods such as Transductive Support Vector Machine (TSVM). The transductive inference performs well for problems in which the amount of unlabeled sequences is considerably greater than the labeled ones. Similarly, the problem of predicting the TIS may take advantage of transductive methods due to the fact that the amount of new sequences grows rapidly with the progress of Genome Project that allows the study of new organisms. Consequently, this work aims to investigate the transductive learning towards TIS identification and compare the results with those obtained in inductive method. The transductive inference presents better results both in F-measure and in sensitivity in comparison with the inductive method for predicting the TIS. Additionally, it presents the least failure rate for identifying the TIS, presenting a smaller number of False Negatives (FN) than the ISVM. The ISVM and TSVM methods were validated with the molecules from the most representative organisms contained in the RefSeq database: Rattus norvegicus, Mus musculus, Homo sapiens, Drosophila melanogaster and Arabidopsis thaliana. The transductive method presented F-measure and sensitivity higher than 90% and also higher than the results obtained with ISVM. The ISVM and TSVM approaches

  11. Hanford Site radioactive mixed waste thermal treatment initiative

    SciTech Connect

    Place, B.G.; Riddelle, J.G.

    1993-03-01

    This paper is a progress report of current Westinghouse Hanford Company engineering activities related to the implementation of a program for the thermal treatment of the Hanford Site radioactive mixed waste. Topics discussed include a site-specific engineering study, the review of private sector capability in thermal treatment, and thermal treatment of some of the Hanford Site radioactive mixed waste at other US Department of Energy sites.

  12. Allosterically regulated unfolding of the A'α helix exposes the dimerization site of the blue-light-sensing aureochrome-LOV domain.

    PubMed

    Herman, Elena; Kottke, Tilman

    2015-02-24

    Aureochromes have been shown to act as blue-light-regulated transcription factors in algae in the absence of phototropins. Aureochromes comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module binding the flavin chromophore and a basic region leucine zipper (bZIP) domain as an effector. The domain arrangement in aureochromes with an N-terminal effector is inversed to other LOV proteins. To clarify the role of the linking A'α helix in signaling, we have investigated the LOV domain of aureochrome1a from the diatom alga Phaeodactylum tricornutum without the N-terminal A'α helix but with the C-terminal Jα helix. Results were analyzed in comparison to those previously obtained on the LOV domain with both flanking helices and on the LOV domain with the A'α helix but without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence by a band at 1656 cm(-1) that the A'α helix unfolds in response to light. This unfolding takes place only in the presence and as a consequence of the unfolding of the Jα helix, which points to an allosteric regulation. Size exclusion chromatography shows the LOV domain to be dimeric in the absence and monomeric in the presence of the A'α helix, implying that the folded helix covers the dimerization site. Therefore, the A'α helix directly modulates the oligomerization state of the LOV domain, whereas the Jα helix acts as an allosteric regulator. Both the allosteric control and the light-induced dimerization have not been observed in phototropin-LOV2 and point to a different signaling mechanism within the full-length proteins.

  13. Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

    PubMed Central

    Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna Z.; Hatmal, Ma’mon M.; Haworth, Ian S.; Qin, Peter Z.

    2012-01-01

    The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/ RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron–Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial. PMID:22229766

  14. Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

    SciTech Connect

    Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna; Hatmal, Ma'mon M.; Haworth, Ian S.; Qin, Peter Z.

    2012-02-08

    The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

  15. Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

    PubMed

    Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

    2014-09-01

    Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

  16. Formation and dimerization of the phosphodiesterase active site of the Pseudomonas aeruginosa MorA, a bi-functional c-di-GMP regulator.

    PubMed

    Phippen, Curtis William; Mikolajek, Halina; Schlaefli, Henry George; Keevil, Charles William; Webb, Jeremy Stephen; Tews, Ivo

    2014-12-20

    Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections. Copyright © 2014. Published by Elsevier B.V.

  17. Tumor-Specific D-Dimer Concentration Ranges and Influencing Factors: A Cross-Sectional Study

    PubMed Central

    Lei, Dansheng; Yuan, Feng; Pei, Feng; Zhang, Huifeng; Yu, Anming; Wang, Kun; Chen, Hu; Chen, Liang; Wu, Xianglei; Tong, Xianli; Wang, Yefu

    2016-01-01

    D-dimer level in cancer patients is associated with risk of venous thromboembolism and deep venous thrombosis. Most cancer patients have “abnormal” D-dimer levels based on the current normal reference range. To investigate tumor-specific D-dimer reference range, we compared D-dimer levels for nine different tumour types with healthy controls by using simultaneous quantile regression and constructing a median, 5th percentile, and 95th percentile model of normal tumour D-dimer concentration. Associations with tumour primary site, stage, pathological type, and treatment were also explored. Additionally, 190 patients were tracked to reveal the relevance of initial D-dimer levels to cancer prognosis. D-dimer ranges (median, 5th, 95th) in various cancers (mg/L) were: liver 1.12, 0.27, 5.25; pancreatic 0.96, 0.23, 4.81; breast 0.44, 0.2, 2.17; gastric 0.65, 0.22, 5.03; colorectal 0.73, 0.22, 4.45; lung 0.7, 0.25, 4.0; gynaecological 0.61, 0.22, 3.98; oesophageal 0.23, 0.7, 3.45; and head and neck 0.22, 0.44, 2.19. All were significantly higher than that of healthy controls (0.18, 0.07, 0.57). D-dimer peaked 1–2 days postoperatively but had decreased to the normal range by 1 week. Additionally, cancer patients with high initial D-dimer were shown a tendency of poor prognosis in survival rate. In conclusion, D-dimer levels in cancer depend on patient age, tumour primary site, and tumour stage. Thrombosis prevention is necessary if D-dimer has not decreased to the tumor-specific baseline a week after surgery. PMID:27835633

  18. Constitutive Dimerization of Glycoprotein VI (GPVI) in Resting Platelets Is Essential for Binding to Collagen and Activation in Flowing Blood*

    PubMed Central

    Jung, Stephanie M.; Moroi, Masaaki; Soejima, Kenji; Nakagaki, Tomohiro; Miura, Yoshiki; Berndt, Michael C.; Gardiner, Elizabeth E.; Howes, Joanna-Marie; Pugh, Nicholas; Bihan, Dominique; Watson, Steve P.; Farndale, Richard W.

    2012-01-01

    The platelet collagen receptor glycoprotein VI (GPVI) has been suggested to function as a dimer, with increased affinity for collagen. Dissociation constants (Kd) obtained by measuring recombinant GPVI binding to collagenous substrates showed that GPVI dimers bind with high affinity to tandem GPO (Gly-Pro-Hyp) sequences in collagen, whereas the markedly lower affinity of the monomer for all substrates implies that it is not the collagen-binding form of GPVI. Dimer binding required a high density of immobilized triple-helical (GPO)10-containing peptide, suggesting that the dimer binds multiple, discrete peptide helices. Differential inhibition of dimer binding by dimer-specific antibodies, m-Fab-F and 204-11 Fab, suggests that m-Fab-F binds at the collagen-binding site of the dimer, and 204-11 Fab binds to a discrete site. Flow cytometric quantitation indicated that GPVI dimers account for ∼29% of total GPVI in resting platelets, whereas activation by either collagen-related peptide or thrombin increases the number of dimers to ∼39 and ∼44%, respectively. m-Fab-F inhibits both GPVI-dependent static platelet adhesion to collagen and thrombus formation on collagen under low and high shear, indicating that pre-existing dimeric GPVI is required for the initial interaction with collagen because affinity of the monomer is too low to support binding and that interaction through the dimer is essential for platelet activation. These GPVI dimers in resting circulating platelets will enable them to bind injury-exposed subendothelial collagen to initiate platelet activation. The GPVI-specific agonist collagen-related peptide or thrombin further increases the number of dimers, thereby providing a feedback mechanism for reinforcing binding to collagen and platelet activation. PMID:22773837

  19. Synergistic binding of the phosphorylated S233- and S259-binding sites of C-RAF to one 14-3-3ζ dimer.

    PubMed

    Molzan, Manuela; Ottmann, Christian

    2012-11-02

    C-RAF kinase is a central component of the Ras-RAF-MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase)-ERK (extracellular signal-regulated kinase) pathway, which has been shown to be activated in 30% of human tumors. 14-3-3 proteins inactivate C-RAF by binding to the two N-terminal phosphorylation-dependent binding sites surrounding S233 and S259. 14-3-3 proteins can bind two target sequences located on one polypeptide chain simultaneously, thereby increasing binding affinity compared to single-site binding and possibly allowing regulated 14-3-3 binding through gatekeeper phosphorylation. To date, it was unclear whether 14-3-3 proteins can bind the two N-terminal phosphorylation-dependent binding sites of C-RAF simultaneously. Fluorescence polarization using phosphorylated peptides demonstrated that S233 is the low-affinity and S259 is the high-affinity binding site, while simultaneous engagement of both sites by 14-3-3ζ enhances affinity compared to single-site binding. Determination of a 1:1 stoichiometry for the di-phosphorylated peptide binding to one 14-3-3ζ dimer with isothermal titration calorimetry was supported by the crystal structure of the 14-3-3ζ/C-RAFpS233,pS259 complex. Cellular localization studies validate the significance of these sites for cytoplasmic retention of C-RAF, suggesting an extended mechanism of RAF regulation by 14-3-3 proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Early Results in Capella's Prior Learning Assessment Experimental Site Initiative

    ERIC Educational Resources Information Center

    Klein, Jillian

    2017-01-01

    In July 2014, the U.S. Department of Education announced a new round of experimental sites focusing on competency-based education. Capella University was selected to participate in three of the Department of Education's competency-based education (CBE) experiments and began by implementing the prior learning assessment experiment, which allows…

  1. Initiating Events for Multi-Reactor Plant Sites

    SciTech Connect

    Muhlheim, Michael David; Flanagan, George F.; Poore, III, Willis P.

    2014-09-01

    Inherent in the design of modular reactors is the increased likelihood of events that initiate at a single reactor affecting another reactor. Because of the increased level of interactions between reactors, it is apparent that the Probabilistic Risk Assessments (PRAs) for modular reactor designs need to specifically address the increased interactions and dependencies.

  2. Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected

    PubMed Central

    Gómez-Cuadrado, A; Martín, M; Noël, M; Ruiz-Carrillo, A

    1995-01-01

    Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 503-amino-acid-long acidic protein which is 99.0% identical to the recently reported human NRF-1/alpha-Pal factor and highly related to the invertebrate transcription factors P3A2 and erected wing gene product (EWG). We present evidence that IBR and IBF are most likely identical proteins, differing in their degree of glycosylation. We have analyzed several molecular aspects of IBR/F and shown that the factor associates as stable homodimers and that the dimer is the relevant DNA-binding species. The evolutionarily conserved N-terminal half of IBR/F harbors the DNA-binding/dimerization domain (outer limits, 127 to 283), one or several casein kinase II sites (37 to 67), and a bipartite nuclear localization signal (89 to 106) which appears to be necessary for nuclear targeting. Binding site selection revealed that the alternating RCGCRYGCGY consensus constitutes high-affinity IBR/F binding sites and that the direct-repeat palindrome TGCGCATGCGCA is the optimal site. A survey of genes potentially regulated by this family of factors primarily revealed genes involved in growth-related metabolism. PMID:8524232

  3. Integrable oscillator type and Schrödinger type dimers

    NASA Astrophysics Data System (ADS)

    Khare, Avinash; Saxena, Avadh

    2017-02-01

    A PT-symmetric dimer is a two-site nonlinear oscillator dimer or a two-site nonlinear Schrödinger dimer where one site loses and the other site gains energy at the same rate. We present a wide class of integrable oscillator type dimers whose Hamiltonian is of arbitrary even order. Further, we also present a wide class of integrable nonlinear Schrödinger type dimers where again the Hamiltonian is of arbitrary even order. Finally, we consider a recently discussed complex dimer model and point out a few integrable cases in that model.

  4. The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: an analytical ultracentrifuge study.

    PubMed

    Catacchio, B; D'Orazio, M; Battistoni, A; Chiancone, E

    2008-03-01

    To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer-dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.

  5. DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA.

    PubMed

    Wittschieben, Birgitte Ø; Iwai, Shigenori; Wood, Richard D

    2005-12-02

    The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.

  6. Synergistic modulation of cyclobutane pyrimidine dimer photoproduct formation and deamination at a TmCG site over a full helical DNA turn in a nucleosome core particle.

    PubMed

    Song, Qian; Cannistraro, Vincent J; Taylor, John-Stephen

    2014-12-01

    Sunlight-induced C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C or 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by DNA polymerase η and defines a probable mechanism for the origin of UV-induced C to T mutations. We have now determined the photoproduct formation and deamination rates for 10 consecutive T=(m)CG CPDs over a full helical turn at the dyad axis of a nucleosome and find that whereas photoproduct formation and deamination is greatly inhibited for the CPDs closest to the histone surface, it is greatly enhanced for the outermost CPDs. Replacing the G in a T=(m)CG CPD with A greatly decreased the deamination rate. These results show that rotational position and flanking sequence in a nucleosome can significantly and synergistically modulate CPD formation and deamination that contribute to C to T mutations associated with skin cancer induction and may have influenced the evolution of the human genome. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Residues at a Single Site Differentiate Animal Cryptochromes from Cyclobutane Pyrimidine Dimer Photolyases by Affecting the Proteins' Preferences for Reduced FAD.

    PubMed

    Xu, Lei; Wen, Bin; Wang, Yuan; Tian, Changqing; Wu, Mingcai; Zhu, Guoping

    2017-06-19

    Cryptochromes (CRYs) and photolyases belong to the cryptochrome/photolyase family (CPF). Reduced FAD is essential for photolyases to photorepair UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts in DNA. In Drosophila CRY (dCRY, a type I animal CRY), FAD is converted to the anionic radical but not to the reduced state upon illumination, which might induce a conformational change in the protein to relay the light signal downstream. To explore the foundation of these differences, multiple sequence alignment of 650 CPF protein sequences was performed. We identified a site facing FAD (Ala377 in Escherichia coli CPD photolyase and Val415 in dCRY), hereafter referred to as "site 377", that was distinctly conserved across these sequences: CPD photolyases often had Ala, Ser, or Asn at this site, whereas animal CRYs had Ile, Leu, or Val. The binding affinity for reduced FAD, but not the photorepair activity of E. coli photolyase, was dramatically impaired when replacing Ala377 with any of the three CRY residues. Conversely, in V415S and V415N mutants of dCRY, FAD was photoreduced to its fully reduced state after prolonged illumination, and light-dependent conformational changes of these mutants were severely inhibited. We speculate that the residues at site 377 play a key role in the different preferences of CPF proteins for reduced FAD, which differentiate animal CRYs from CPD photolyases. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Mars Science Laboratory: Mission, Landing Site, and Initial Results

    NASA Astrophysics Data System (ADS)

    Grotzinger, John; Blake, D.; Crisp, J.; Edgett, K.; Gellert, R.; Gomez-Elvira, J.; Hassler, D.; Mahaffy, P.; Malin, M.; Meyer, M.; Mitrofanov, I.; Vasavada, A.; Wiens, R.

    2012-10-01

    Scheduled to land on August 5, 2012, the Mars Science Laboratory rover, Curiosity, will conduct an investigation of modern and ancient environments. Recent mission results will be discussed. Curiosity has a lifetime of at least one Mars year ( 23 months), and drive capability of at least 20 km. The MSL science payload was specifically assembled to assess habitability and includes a gas chromatograph-mass spectrometer and gas analyzer that will search for organic carbon in rocks, regolith fines, and the atmosphere; an x-ray diffractometer that will determine mineralogical diversity; focusable cameras that can image landscapes and rock/regolith textures in natural color; an alpha-particle x-ray spectrometer for in situ determination of rock and soil chemistry; a laser-induced breakdown spectrometer to remotely sense the chemical composition of rocks and minerals; an active neutron spectrometer designed to search for water in rocks/regolith; a weather station to measure modern-day environmental variables; and a sensor designed for continuous monitoring of background solar and cosmic radiation. The 155-km diameter Gale Crater was chosen as Curiosity’s field site based on several attributes: an interior mound of ancient flat-lying strata extending almost 5 km above the elevation of the landing site; the lower few hundred meters of the mound show a progression with relative age from clay-bearing to sulfate-bearing strata, separated by an unconformity from overlying likely anhydrous strata; the landing ellipse is characterized by a mixture of alluvial fan and high thermal inertia/high albedo stratified deposits; and a number of stratigraphically/geomorphically distinct fluvial features. Gale’s regional context and strong evidence for a progression through multiple potentially habitable environments, represented by a stratigraphic record of extraordinary extent, insure preservation of a rich record of the environmental history of early Mars.

  9. Emergence of dominant initiation sites for interictal spikes in rat neocortex

    PubMed Central

    Vitantonio, Daniel; Xu, Weifeng; Geng, Xinling; Wolff, Brian S.; Takagaki, Kentaroh; Motamedi, Gholam K.

    2015-01-01

    Neuronal populations with unbalanced inhibition can generate interictal spikes (ISs), where each IS starts from a small initiation site and then spreads activation across a larger area. We used in vivo voltage-sensitive dye imaging to map the initiation site of ISs in rat visual cortex disinhibited by epidural application of bicuculline methiodide. Immediately after the application of bicuculline, the IS initiation sites were widely distributed over the entire disinhibited area. After ∼10 min, a small number of sites became “dominant” and initiated the majority of the ISs throughout the course of imaging. Such domination also occurred in cortical slices, which lack long-range connections between the cortex and subcortical structures. This domination of IS initiation sites may allow timing-related plasticity mechanisms to provide a spatial organization where connections projecting outward from the dominant initiation site become strengthened. Understanding the spatiotemporal organization of IS initiation sites may contribute to our understanding of epileptogenesis in its very early stages, because a dominant IS initiation site with strengthened outward connectivity may ultimately develop into a seizure focus. PMID:26445866

  10. Surgical site infection prevention initiative - patient attitude and compliance.

    PubMed

    Ramos, Nicholas; Skeete, Faith; Haas, Janet P; Hutzler, Lorraine; Slover, James; Phillips, Michael; Bosco, Joseph

    2011-01-01

    Although the effect of Staphylococcus aureus (SA) decolonization on surgical site infection (SSI) rates has been studied, patient tolerance and acceptance of these regimens has not been assessed. Surgical patients at our hospital's Pre-Admission Testing Clinic (PAT) receive SA reduction protocols instructing the preoperative use of chlorhexidine gluconate (CHG) soap and intranasal mupirocin ointment (MO). Certain insurers do not cover MO costs resulting in out of pocket (OOP) expenses for some patients. This study assessed patient attitudes and compliance with our hospital's SA decolonization regimen. One-hundred-forty-six patients received surveys. Descriptive statistics were used for analysis. Of respondents fitting inclusion criteria, 81% followed the MO protocol (MO users) while 89% followed the CHG protocol (CHG users). Fifty-four percent of MO users reported OOP expenses and 13% reported a hard or very hard financial burden. Ninety-three percent of CHG users reported the protocol was easy or very easy to follow. Eighty-one percent of patients receiving the SA protocol were fully compliant despite cost or difficulty obtaining MO. Given these barriers and some difficulty with CHG application, we hypothesize compliance may be improved if MO is provided to patients without OOP expenses and if the CHG application method is simplified.

  11. Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

    PubMed

    Capozzo, Alejandra V; Wilda, Maximiliano; Bucafusco, Danilo; de los Ángeles Lavoria, María; Franco-Mahecha, Olga L; Mansilla, Florencia C; Pérez-Filgueira, Daniel M; Grigera, Pablo R

    2011-11-01

    Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.

  12. Identification of a Hanford Waste Site for Initial Deployment of the In Situ Gaseous Reduction Approach

    SciTech Connect

    Thornton, Edward C.; Cantrell, Kirk J.; Faurote, James M.; Gilmore, Tyler J.; Olsen, Khris B.; Schalla, Ronald

    2000-11-28

    In Situ Gaseous Reduction is a technology currently being developed by DOE for the remediation of soil waste sites contaminated with hexavalent chromium. This document presents the results of recent characterization activities undertaken at several of the soil waste sites at Hanford that contain siginficant levels of hexavalent chromium contamination. The objective of this study is to select a site for initial deployment of the technology at the Hanford Site.

  13. Mic10, a Core Subunit of the Mitochondrial Contact Site and Cristae Organizing System, Interacts with the Dimeric F1Fo-ATP Synthase.

    PubMed

    Rampelt, Heike; Bohnert, Maria; Zerbes, Ralf M; Horvath, Susanne E; Warscheid, Bettina; Pfanner, Nikolaus; van der Laan, Martin

    2017-04-21

    The mitochondrial contact site and cristae organizing system (MICOS) is crucial for maintaining the architecture of the mitochondrial inner membrane. MICOS is enriched at crista junctions that connect the two inner membrane domains: inner boundary membrane and cristae membrane. MICOS promotes the formation of crista junctions, whereas the oligomeric F1Fo-ATP synthase is crucial for shaping cristae rims, indicating antagonistic functions of these machineries in organizing inner membrane architecture. We report that the MICOS core subunit Mic10, but not Mic60, binds to the F1Fo-ATP synthase. Mic10 selectively associates with the dimeric form of the ATP synthase and supports the formation of ATP synthase oligomers. Our results suggest that Mic10 plays a dual role in mitochondrial inner membrane architecture. In addition to its central function in sculpting crista junctions, a fraction of Mic10 molecules interact with the cristae rim-forming F1Fo-ATP synthase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Regulated HIV-2 RNA dimerization by means of alternative RNA conformations

    PubMed Central

    Dirac, Annette M. G.; Huthoff, Hendrik; Kjems, Jørgen; Berkhout, Ben

    2002-01-01

    The dimer initiation site (DIS) hairpin of the HIV-2 untranslated leader RNA mediates in vitro dimerization through ‘loop–loop kissing’ of a loop-exposed palindrome sequence. Premature RNA dimerization must be prevented during the retroviral life cycle. A regulatory mechanism has been proposed for the HIV-1 leader RNA that can adopt an alternative conformation in which the DIS motif is effectively masked by long-distance base pairing with upstream leader sequences. We now report that HIV-2 RNA dimerization is also regulated. Sequestering of the DIS motif by base pairing interactions with downstream leader sequences mediates a switch to a dimerization-impaired conformation. The existence of two alternative conformations of the HIV-2 leader RNA is supported by UV melting experiments. Furthermore, the equilibrium between the two conformations can be shifted by annealing of antisense oligonucleotides or by deletion of certain leader regions. These measures have a profound impact on the dimerization properties of the transcript, demonstrating a mutual exclusivity between the alternative conformation and dimerization, similar to what has been described for the HIV-1 leader. The overall resemblance in regulation of HIV-1 and HIV-2 RNA dimerization suggests that a similar mechanism may be operating in other lentiviruses and perhaps all retroviridae. PMID:12060681

  15. Mechanism of HIV-1 RNA dimerization in the central region of the genome and significance for viral evolution.

    PubMed

    Piekna-Przybylska, Dorota; Sharma, Gaurav; Bambara, Robert A

    2013-08-16

    The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

  16. The dimers of cyanamide

    NASA Astrophysics Data System (ADS)

    Moffat, J. B.

    Ab initio calculations have been performed on various dimeric forms of cyanamide. The "nondissociative" dimerization of cyanamide leads to cyclic molecules all of which are unstable with respect to cyanamide. However, the molecules produced by "dissociative" dimerization are stable relative to cyanamide. Dicyandiamide is found to be the most stable of nine dimeric configurations.

  17. STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

    PubMed Central

    Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

    2009-01-01

    Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

  18. Evaluation of the Jobs Initiative: First Annual/Cross-Site Report.

    ERIC Educational Resources Information Center

    Abt Associates, Inc., Cambridge, MA.

    In 1995, the Jobs Initiative, an 8-year, six-site demonstration project, was launched to attempt to improve access to family-supporting jobs for disadvantaged young adults in the inner city. The Jobs Initiative is providing seed money to develop and implement job strategies in six cities: Denver (Colorado); Milwaukee (Wisconsin); New Orleans…

  19. Synthesis of defined ubiquitin dimers.

    PubMed

    Eger, Silvia; Scheffner, Martin; Marx, Andreas; Rubini, Marina

    2010-11-24

    Many proteins are post-translationally modified by the attachment of poly-ubiquitin (Ub) chains. Notably, the biological function of the attached Ub chain depends on the specific lysine residue used for conjugate formation. Here, we report an easy and efficient method to synthesize site-specifically linked Ub dimers by click reaction between two artificial amino acids. In fact, we were able to synthesize all seven naturally occurring Ub connectivities, providing the first example of a method that gives access to all Ub dimers. Furthermore, these synthetic Ub dimers are recognized by the natural ubiquitination machinery and are proteolytically stable, making them optimal candidates to further investigate the function of differently linked Ub chains.

  20. Dimer crystallization of chiral proteoids.

    PubMed

    Wang, Po-Yuan; Mason, Thomas G

    2017-03-08

    Proteins can self-assemble into a variety of exquisitely organized structures through hierarchical reaction pathways. To examine how different core shapes of proteins and entropy combine to influence self-assembly, we create systems of lithographically fabricated proteomimetic colloids, or 'proteoids', and explore how Brownian monolayers of mobile proteoids, which have hard interactions, self-assemble as they are slowly crowded. Remarkably, chiral C-shaped proteoids having circular heads on only one side form enantiopure lock-and-key chiral dimers; these dimers have corrugated, shape-complementary perimeters, so they, in turn, form lock-and-key arrangements into chiral dimer crystals. Time-lapse video microscopy reveals the expulsion of monomers from the growing dimer crystals through tautomerization translocation reactions which expedite the crystallization kinetics. By lithographically mutating proteoids, we also tune the types and structures of the resulting dimer crystals. Thus, rational design of sub-particle features in hard-core colloidal shapes can be used to sterically select desired self-assembly pathways without introducing any site-specific attractions, thereby generating a striking degree of hierarchical self-ordering, reminiscent of protein crystallization.

  1. Translation of encephalomyocarditis virus RNA: parameters influencing the selection of the internal initiation site.

    PubMed Central

    Kaminski, A; Belsham, G J; Jackson, R J

    1994-01-01

    The initiation of encephalomyocarditis virus RNA translation is by internal ribosome entry almost exclusively at the 11th AUG codon from the 5'-end, which is the central of the three AUG codons in the sequence..[sequence: see text].., and is located some 25 nt downstream from an oligopyrimidine tract conserved amongst related viruses. As the sequences between the oligopyrimidine tract and AUG-10/11 are poorly conserved and thus possibly serve only as a spacer, the influence of this spacer length on initiation frequency at the three AUG codons was examined in vitro and in vivo. Deletion of 11 residues resulted in initiation almost exclusively at AUG-12 but at significantly reduced overall efficiency. Insertion of eight residues caused a 15-fold increase in initiation frequency at AUG-10 and a decrease at AUG-11. Longer insertions reduced overall efficiency without changing the initiation site preferences. With the wild-type spacing, complete substitution of the oligopyrimidine tract by purines caused a 30-35% decrease in initiation efficiency, and partial substitution only a 10-15% decrease. Thus the internal initiation mechanism selects the initiation site partly on the basis of its distance from upstream elements, of which the oligopyrimidine tract is not the most critical, but for reasons not yet understood a preference for AUG-11 is superimposed on this selection. Images PMID:8157006

  2. The Site of Spontaneous Ectopic Spike Initiation Facilitates Signal Integration in a Sensory Neuron.

    PubMed

    Städele, Carola; Stein, Wolfgang

    2016-06-22

    Essential to understanding the process of neuronal signal integration is the knowledge of where within a neuron action potentials (APs) are generated. Recent studies support the idea that the precise location where APs are initiated and the properties of spike initiation zones define the cell's information processing capabilities. Notably, the location of spike initiation can be modified homeostatically within neurons to adjust neuronal activity. Here we show that this potential mechanism for neuronal plasticity can also be exploited in a rapid and dynamic fashion. We tested whether dislocation of the spike initiation zone affects signal integration by studying ectopic spike initiation in the anterior gastric receptor neuron (AGR) of the stomatogastric nervous system of Cancer borealis Like many other vertebrate and invertebrate neurons, AGR can generate ectopic APs in regions distinct from the axon initial segment. Using voltage-sensitive dyes and electrophysiology, we determined that AGR's ectopic spike activity was consistently initiated in the neuropil region of the stomatogastric ganglion motor circuits. At least one neurite branched off the AGR axon in this area; and indeed, we found that AGR's ectopic spike activity was influenced by local motor neurons. This sensorimotor interaction was state-dependent in that focal axon modulation with the biogenic amine octopamine, abolished signal integration at the primary spike initiation zone by dislocating spike initiation to a distant region of the axon. We demonstrate that the site of ectopic spike initiation is important for signal integration and that axonal neuromodulation allows for a dynamic adjustment of signal integration. Although it is known that action potentials are initiated at specific sites in the axon, it remains to be determined how the precise location of action potential initiation affects neuronal activity and signal integration. We addressed this issue by studying ectopic spiking in the axon of

  3. Universality in fermionic dimer-dimer scattering

    NASA Astrophysics Data System (ADS)

    Deltuva, A.

    2017-08-01

    Collisions of two fermionic dimers near the unitary limit are studied using exact four-particle equations for transition operators in momentum space. Universal properties of dimer-dimer phase shifts and effective range expansion (ERE) parameters are determined. The inclusion of the fourth-order momentum term in the ERE significantly extends its validity to higher collision energies. The dimer-dimer scattering length and effective range are determined in the unitary limit as well as their corrections arising due to the finite range of the two-fermion interaction. These results are of considerably higher accuracy as compared to previous works, but confirm most of the previous results except for the lattice effective field theory calculations.

  4. Neuronal Competition for Action Potential Initiation Sites in a Circuit Controlling Simple Learning

    PubMed Central

    Cruz, Georgina E.; Sahley, Christie L.; Muller, Kenneth J.

    2007-01-01

    The spatial and temporal patterns of action potential initiations were studied in a behaving leech preparation to determine the basis of increased firing that accompanies sensitization, a form of non-associative learning requiring the S-interneurons. Little is known at the network level about mechanisms of behavioral sensitization. The S-interneurons, one in each ganglion and linked by electrical synapses with both neighbors to form a chain, are interposed between sensory and motor neurons. In sensitized preparations the strength of shortening is related to S-cell firing, which itself is the result of impulses initiating in several S-cells. Because the S-cells, as independent initiation sites, all contribute to activity in the chain, it was hypothesized that during sensitization, increased multi-site activity increased the chain's firing rate. However, it was found that during sensitization, the single site with the largest initiation rate, the S-cell in the stimulated segment, suppressed initiations in adjacent ganglia. Experiments showed this was both because (1) it received the earliest, greatest input and (2) the delayed synaptic input to the adjacent S-cells coincided with the action potential refractory period. A compartmental model of the S-cell and its inputs showed that a simple, intrinsic mechanism of inexcitability after each action potential may account for suppression of impulse initiations. Thus, a non-synaptic competition between neurons alters synaptic integration in the chain. In one mode, inputs to different sites sum independently, whereas in another, synaptic input to a single site precisely specifies the overall pattern of activity. PMID:17644266

  5. Neuronal adaptation involves rapid expansion of the action potential initiation site

    PubMed Central

    Scott, Ricardo S.; Henneberger, Christian; Padmashri, Ragunathan; Anders, Stefanie; Jensen, Thomas P.; Rusakov, Dmitri A.

    2014-01-01

    Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma–axon recordings combined with axonal Na+ and Ca2+ imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na+ channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale. PMID:24851940

  6. Plant Response to Soils, Site Preparation, and Initial Pine Planting Density

    Treesearch

    Henry A. Pearson; Gale L. Wolters; Ronald E. Thill; Alton Martin; V. Clark Baldwin

    1995-01-01

    This study described the effects of soils, site preparation, and initial pine regeneration spacings on tree growth and the associated understory woody and herbaceous plant succession. Although Sawyer soils appeared more productive than Ruston soils before the harvest and regeneration treatments, woody and herbaceous plant differences were not apparent between the...

  7. Interim Report for Bioventing Field Initiative at Site UST 173, Robins Air Force Base, Georgia

    DTIC Science & Technology

    2007-11-02

    This report describes the activities conducted at Robins Air Force Base (AFB), Georgia, Site UST 173 as part of the Bioventing Field Initiative for...which includes a soil gas survey, air permeability test, in situ respiration tests, and installation of bioventing systems. The specific objectives of this task are described in the following section.

  8. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Finality of referenced NRC approvals; partial initial decision on site suitability. 52.83 Section 52.83 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Combined Licenses § 52.83 Finality of referenced NRC...

  9. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Finality of referenced NRC approvals; partial initial decision on site suitability. 52.83 Section 52.83 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Combined Licenses § 52.83 Finality of referenced NRC...

  10. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Finality of referenced NRC approvals; partial initial decision on site suitability. 52.83 Section 52.83 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Combined Licenses § 52.83 Finality of referenced NRC...

  11. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Finality of referenced NRC approvals; partial initial decision on site suitability. 52.83 Section 52.83 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Combined Licenses § 52.83 Finality of referenced NRC...

  12. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Finality of referenced NRC approvals; partial initial decision on site suitability. 52.83 Section 52.83 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSES, CERTIFICATIONS, AND APPROVALS FOR NUCLEAR POWER PLANTS Combined Licenses § 52.83 Finality of referenced NRC...

  13. Identification and sequence of the initiation site for rat 45S ribosomal RNA synthesis.

    PubMed Central

    Harrington, C A; Chikaraishi, D M

    1983-01-01

    The transcription initiation site for rat 45S precursor ribosomal RNA synthesis was determined by nuclease protection mapping with two single-strand endonucleases. S1 and mung bean, and one single-strand exonuclease, ExoVII. These experiments were performed with end-labeled ribosomal DNA from double-stranded pBR322 recombinants and from single-stranded M13 recombinants. Results from experiments using both kinds of DNA and all three enzymes showed that the 5' end of 45S RNA mapped to a unique site 125 bases upstream from the Hind III site in the ribosomal DNA gene. The DNA surrounding this site (designated +1) was sequenced from -281 to +641. The entire sequence of this region shows extensive homology to the comparable region of mouse. This includes three stretches of T residues in the non-coding strand between +300 and +630. Two sets of direct repeats adjacent to these T-rich regions are observed. Comparison of the mouse and human ribosomal DNA transcription initiation sites with the rat sequence reported in this paper demonstrates a conserved sequence at +2 to +16, CTGACACGCTGTCCT. This suggests that this region may be important for the initiation of transcription on mammalian ribosomal DNAs. Images PMID:6304628

  14. Alternative transcription initiation sites and polyadenylation sites are recruited during Mu suppression at the rf2a locus of maize.

    PubMed Central

    Cui, Xiangqin; Hsia, An-Ping; Liu, Feng; Ashlock, Daniel A; Wise, Roger P; Schnable, Patrick S

    2003-01-01

    Even in the absence of excisional loss of the associated Mu transposons, some Mu-induced mutant alleles of maize can lose their capacity to condition a mutant phenotype. Three of five Mu-derived rf2a alleles are susceptible to such Mu suppression. The suppressible rf2a-m9437 allele has a novel Mu transposon insertion (Mu10) in its 5' untranslated region (UTR). The suppressible rf2a-m9390 allele has a Mu1 insertion in its 5' UTR. During suppression, alternative transcription initiation sites flanking the Mu1 transposon yield functional transcripts. The suppressible rf2a-m8110 allele has an rcy/Mu7 insertion in its 3' UTR. Suppression of this allele occurs via a previously unreported mechanism; sequences in the terminal inverted repeats of rcy/Mu7 function as alternative polyadenylation sites such that the suppressed rf2a-m8110 allele yields functional rf2a transcripts. No significant differences were observed in the nucleotide compositions of these alternative polyadenylation sites as compared with 94 other polyadenylation sites from maize genes. PMID:12618406

  15. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

    Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. Images PMID:3006062

  16. NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)]4.

    PubMed

    Pradeep, Tarikere Palakshan; Barthwal, Ritu

    2016-01-01

    The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

  17. The two-state dimer receptor model: a general model for receptor dimers.

    PubMed

    Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

    2006-06-01

    Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

  18. Spin Dimer Analysis for Antiferromagnetic Spin Exchange Interactions of Magnetic Solids with Several Unpaired Electrons per Spin Site: Trends in the Spin Exchange Parameters of the Compounds Consisting of MnF

    SciTech Connect

    Koo, H.-J.; Whangbo, M.-H.; Coste, S.; Jobic, S.

    2001-02-01

    For magnetic solids with several unpaired spins per spin site, the average spin orbital interaction energies < {Delta}e > and the average spin orbital interaction energy squares < ({Delta}e){sup 2} > were defined as a qualitative measure for the strengths of their antiferromagnetic spin exchange interactions. The trends in the antiferromagnetic spin exchange interactions of the magnetic solids containing MnF{sub 5} chains and CrX{sub 2} (X=O, S) layers were examined in terms of the < {Delta}e > and < ({Delta}e){sup 2} > values calculated for their spin dimers.

  19. Quality improvement initiative: Preventative Surgical Site Infection Protocol in Vascular Surgery.

    PubMed

    Parizh, David; Ascher, Enrico; Raza Rizvi, Syed Ali; Hingorani, Anil; Amaturo, Michael; Johnson, Eric

    2017-01-01

    Objective A quality improvement initiative was employed to decrease single institution surgical site infection rate in open lower extremity revascularization procedures. In an attempt to lower patient morbidity, we developed and implemented the Preventative Surgical Site Infection Protocol in Vascular Surgery. Surgical site infections lead to prolonged hospital stays, adjunctive procedure, and additive costs. We employed targeted interventions to address the common risk factors that predispose patients to post-operative complications. Methods Retrospective review was performed between 2012 and 2016 for all surgical site infections after revascularization procedures of the lower extremity. A quality improvement protocol was initiated in January 2015. Primary outcome was the assessment of surgical site infection rate reduction in the pre-protocol vs. post-protocol era. Secondary outcomes evaluated patient demographics, closure method, perioperative antibiotic coverage, and management outcomes. Results Implementation of the protocol decreased the surgical site infection rate from 6.4% to 1.6% p = 0.0137). Patient demographics and comorbidities were assessed and failed to demonstrate a statistically significant difference among the infection and no-infection groups. Wound closure with monocryl suture vs. staple proved to be associated with decreased surgical site infection rate ( p < 0.005). Conclusions Preventative measures, in the form of a standardized protocol, to decrease surgical site infections in the vascular surgery population are effective and necessary. Our data suggest that there may be benefit in the incorporation of MRSA and Gram-negative coverage as part of the Surgical Care Improvement Project perioperative guidelines.

  20. Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.

    PubMed Central

    Yoon, Y; Sanchez, J A; Brun, C; Huberman, J A

    1995-01-01

    New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit. PMID:7739533

  1. Effects of Modification of the Transcription Initiation Site Context on Citrus Tristeza Virus Subgenomic RNA Synthesis†

    PubMed Central

    Ayllón, María A.; Gowda, Siddarame; Satyanarayana, Tatineni; Karasev, Alexander V.; Adkins, Scott; Mawassi, Munir; Guerri, José; Moreno, Pedro; Dawson, William O.

    2003-01-01

    Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3′-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5′ termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5′ or 3′ of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism. PMID:12915539

  2. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair.

    PubMed

    Du, Fengxia; Zhang, Minjie; Li, Xiaohua; Yang, Caiyun; Meng, Hao; Wang, Dong; Chang, Shuang; Xu, Ye; Price, Brendan; Sun, Yingli

    2014-10-03

    The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.

  3. Evolutionary Origins of Two-Barrel RNA Polymerases and Site-Specific Transcription Initiation.

    PubMed

    Fouqueau, Thomas; Blombach, Fabian; Werner, Finn

    2017-09-08

    Evolution-related multisubunit RNA polymerases (RNAPs) carry out RNA synthesis in all domains life. Although their catalytic cores and fundamental mechanisms of transcription elongation are conserved, the initiation stage of the transcription cycle differs substantially in bacteria, archaea, and eukaryotes in terms of the requirements for accessory factors and details of the molecular mechanisms. This review focuses on recent insights into the evolution of the transcription apparatus with regard to (a) the surprisingly pervasive double-Ψ β-barrel active-site configuration among different nucleic acid polymerase families, (b) the origin and phylogenetic distribution of TBP, TFB, and TFE transcription factors, and (c) the functional relationship between transcription and translation initiation mechanisms in terms of transcription start site selection and RNA structure.

  4. Perform Initial Measurements to Investigate Microwave Detection for Location of Hemorrhage Sites Within the Body

    DTIC Science & Technology

    1998-08-01

    ability of electromagnetic waves in the RF and microwave region to detect regions of blood pooling in the body. The purpose is to demonstrate the...exposure to electromagnetic waves. The data presented is based upon research and approximations to the actual problem. RF or microwave energy interactions...DAMD17-96-C-6074 TITLE: Perform Initial Measurements to Investigate Microwave Detection for Location of Hemorrhage Sites Within the Body PRINCIPAL

  5. Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites.

    PubMed

    Borde, Valérie; Robine, Nicolas; Lin, Waka; Bonfils, Sandrine; Géli, Vincent; Nicolas, Alain

    2009-01-21

    The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

  6. Transcriptional and structural impact of TATA-initiation site spacing in mammalian core promoters

    PubMed Central

    Ponjavic, Jasmina; Lenhard, Boris; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Sandelin, Albin

    2006-01-01

    Background The TATA box, one of the most well studied core promoter elements, is associated with induced, context-specific expression. The lack of precise transcription start site (TSS) locations linked with expression information has impeded genome-wide characterization of the interaction between TATA and the pre-initiation complex. Results Using a comprehensive set of 5.66 × 106 sequenced 5' cDNA ends from diverse tissues mapped to the mouse genome, we found that the TATA-TSS distance is correlated with the tissue specificity of the downstream transcript. To achieve tissue-specific regulation, the TATA box position relative to the TSS is constrained to a narrow window (-32 to -29), where positions -31 and -30 are the optimal positions for achieving high tissue specificity. Slightly larger spacings can be accommodated only when there is no optimally spaced initiation signal; in contrast, the TATA box like motifs found downstream of position -28 are generally nonfunctional. The strength of the TATA binding protein-DNA interaction plays a subordinate role to spacing in terms of tissue specificity. Furthermore, promoters with different TATA-TSS spacings have distinct features in terms of consensus sequence around the initiation site and distribution of alternative TSSs. Unexpectedly, promoters that have two dominant, consecutive TSSs are TATA depleted and have a novel GGG initiation site consensus. Conclusion In this report we present the most comprehensive characterization of TATA-TSS spacing and functionality to date. The coupling of spacing to tissue specificity at the transcriptome level provides important clues as to the function of core promoters and the choice of TSS by the pre-initiation complex. PMID:16916456

  7. Combined Use of Residual Dipolar Couplings and Solution X-ray Scattering To Rapidly Probe Rigid-Body Conformational Transitions in a Non-phosphorylatable Active-Site Mutant of the 128 kDa Enzyme I Dimer

    SciTech Connect

    Takayama, Yuki; Schwieters, Charles D.; Grishaev, Alexander; Ghirlando, Rodolfo; Clore, G. Marius

    2012-10-23

    The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN{sup {alpha}/{beta}} subdomain and an aspartate (Asp129) on the EIN{sup {alpha}} subdomain results in a small ({approx}9{sup o}) reorientation of the EIN{sup {alpha}} and EIN{sup {alpha}/{beta}} subdomains that is in turn propagated to a larger reorientation ({approx}26{sup o}) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

  8. Combined use of residual dipolar couplings and solution X-ray scattering to rapidly probe rigid-body conformational transitions in a non-phosphorylatable active-site mutant of the 128 kDa enzyme I dimer.

    PubMed

    Takayama, Yuki; Schwieters, Charles D; Grishaev, Alexander; Ghirlando, Rodolfo; Clore, G Marius

    2011-01-26

    The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN(α/β) subdomain and an aspartate (Asp129) on the EIN(α) subdomain results in a small (∼9°) reorientation of the EIN(α) and EIN(α/β) subdomains that is in turn propagated to a larger reorientation (∼26°) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

  9. Quantitative analysis of mammalian translation initiation sites by FACS-seq.

    PubMed

    Noderer, William L; Flockhart, Ross J; Bhaduri, Aparna; Diaz de Arce, Alexander J; Zhang, Jiajing; Khavari, Paul A; Wang, Clifford L

    2014-08-28

    An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the -6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  10. Quantitative analysis of mammalian translation initiation sites by FACS-seq

    PubMed Central

    Noderer, William L; Flockhart, Ross J; Bhaduri, Aparna; Diaz de Arce, Alexander J; Zhang, Jiajing; Khavari, Paul A; Wang, Clifford L

    2014-01-01

    An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing (FACS-seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence. PMID:25170020

  11. Heat capacity of the site-diluted spin dimer system Ba₃(Mn1-xVx)₂O₈

    SciTech Connect

    Samulon, E. C.; Shapiro, M. C.; Fisher, I. R.

    2011-08-05

    Heat-capacity and susceptibility measurements have been performed on the diluted spin dimer compound Ba₃(Mn1-xVx)₂O₈. The parent compound Ba₃Mn₂O₈ is a spin dimer system based on pairs of antiferromagnetically coupled S=1, 3d² Mn⁵⁺ ions such that the zero-field ground state is a product of singlets. Substitution of nonmagnetic S=0, 3d⁰ V⁵⁺ ions leads to an interacting network of unpaired Mn moments, the low-temperature properties of which are explored in the limit of small concentrations 0≤x≤0.05. The zero-field heat capacity of this diluted system reveals a progressive removal of magnetic entropy over an extended range of temperatures, with no evidence for a phase transition. The concentration dependence does not conform to expectations for a spin-glass state. Rather, the data suggest a low-temperature random singlet phase, reflecting the hierarchy of exchange energies found in this system.

  12. The nucleotide sequence of the putative transcription initiation site of a cloned ribosomal RNA gene of the mouse.

    PubMed Central

    Urano, Y; Kominami, R; Mishima, Y; Muramatsu, M

    1980-01-01

    Approximately one kilobase pairs surrounding and upstream the transcription initiation site of a cloned ribosomal DNA (rDNA) of the mouse were sequenced. The putative transcription initiation site was determined by two independent methods: one nuclease S1 protection and the other reverse transcriptase elongation mapping using isolated 45S ribosomal RNA precursor (45S RNA) and appropriate restriction fragments of rDNA. Both methods gave an identical result; 45S RNA had a structure starting from ACTCTTAG---. Characteristically, mouse rDNA had many T clusters (greater than or equal to 5) upstream the initiation site, the longest being 21 consecutive T's. A pentadecanucleotide, TGCCTCCCGAGTGCA, appeared twice within 260 nucleotides upstream the putative initiation site. No such characteristic sequences were found downstream this site. Little similarity was found in the upstream of the transcription initiation site between the mouse, Xenopus laevis and Saccharomyces cerevisiae rDNA. Images PMID:6162156

  13. Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers.

    PubMed

    Jang, Hyunbum; Muratcioglu, Serena; Gursoy, Attila; Keskin, Ozlem; Nussinov, Ruth

    2016-06-15

    Are the dimer structures of active Ras isoforms similar? This question is significant since Ras can activate its effectors as a monomer; however, as a dimer, it promotes Raf's activation and MAPK (mitogen-activated protein kinase) cell signalling. In the present study, we model possible catalytic domain dimer interfaces of membrane-anchored GTP-bound K-Ras4B and H-Ras, and compare their conformations. The active helical dimers formed by the allosteric lobe are isoform-specific: K-Ras4B-GTP favours the α3 and α4 interface; H-Ras-GTP favours α4 and α5. Both isoforms also populate a stable β-sheet dimer interface formed by the effector lobe; a less stable β-sandwich interface is sustained by salt bridges of the β-sheet side chains. Raf's high-affinity β-sheet interaction is promoted by the active helical interface. Collectively, Ras isoforms' dimer conformations are not uniform; instead, the isoform-specific dimers reflect the favoured interactions of the HVRs (hypervariable regions) with cell membrane microdomains, biasing the effector-binding site orientations, thus isoform binding selectivity.

  14. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

    PubMed

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-05-07

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy.

  15. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

    PubMed Central

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-01-01

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy. PMID:25950479

  16. A New GLORIA (Global Research Initiative in Alpine Environments Site in Southwestern Montana

    NASA Astrophysics Data System (ADS)

    Apple, M. E.; Warden, J. E.; Apple, C. J.; Pullman, T. Y.; Gallagher, J. H.

    2008-12-01

    Global climate change is predicted to have a major impact on the alpine environments and plants of western North America. Alpine plant species and treelines may migrate upwards due to warmer temperatures. Species composition, vegetation cover, and the phenology of photosynthesis, flowering, pollination, and seed dispersal may change. The Global Research Initiative in Alpine Environments (GLORIA) is a network of alpine sites established with the goal of understanding the interactions between climate change and alpine plants. The Continental Divide traverses Southwestern Montana, where the flora contains representative species from both sides of the divide. In the summer of 2008, we established a GLORIA site in southwestern Montana east of the Continental Divide with the objective of determining whether the temperature changes at the site, and if so, how temperature changes influence alpine plants. We are monitoring soil temperature along with species composition and percent cover of alpine plants at four sub-summits along an ascending altitudinal gradient. We placed the treeline, lower alpine, and upper alpine sites on Mt. Fleecer (45°49'36.06"N, 112°48'08.18"W, 2886.2 m (9469 ft)) and the highest sub-summit on Keokirk Mountain, (45°35'37.94"N, 112°57'03.89"W, 2987.3 m (9801 ft)) in the Pioneer Range. Interesting species on these mountains include Lewisia pygmaea, the Pygmy Bitterroot, Silene acaulis, the Moss Campion, Eritrichium nanum, the Alpine Forget-Me-Not, Lloydia serotina, the Alpine Lily, and Pinus albicaulis, the Whitebark Pine. This new site will remain in place indefinitely. Baseline and subsequent data from this site will be linked with the global network of GLORIA sites with which we will assess changes in alpine flora.

  17. Factors that Determine the Efficiency of HIV-1 Strand Transfer Initiated at a Specific Site

    PubMed Central

    Rigby, Sean T.; Van Nostrand, Keith P.; Rose, April E.; Gorelick, Robert J.; Mathews, David H.; Bambara, Robert A.

    2010-01-01

    Summary HIV-1 employs strand transfer for recombination between the two viral genomes. We previously provided evidence that strand transfer proceeds by an invasion-mediated mechanism, in which a DNA segment on the original RNA template is invaded by a second RNA template at a gap site. The initial RNA-DNA hybrid then expands until the DNA is fully transferred. Ribonuclease H (RNase H) cleavages and nucleocapsid protein (NC) were required for long distance propagation of the hybrid. The evaluation was performed on a unique substrate with a short gap serving as a pre-created invasion site (PCIS). In our current work, this substrate provided the opportunity to test what factors influence a specific invasion site to support transfer, and distinguish factors that influence invasion site creation from those that impact later steps. RNase H can act in a polymerization-dependent or -independent mode. Polymerization-dependent and -independent RNase H were found to be important to create efficiently-used invasion sites in the primer-donor complex, with or without NC. Propagation and terminus transfer steps, emanating from a PCIS in the presence of NC, were stimulated by polymerization-dependent but not -independent RNase H. RNase H can carry out primary and secondary cleavages during synthesis. While both modes of cleavage promoted invasion, only primary cleavage promoted propagation in the presence of NC in our system. These observations suggest that once invasion is initiated at a short gap, it can propagate through an adjacent region interrupted only by nicks, with help by NC. We considered the possibility that propagation solely by strand exchange was a significant contributor to transfers. However, it did not promote transfer, even if synthetic progress of the RT was intentionally slowed, which is consistent with strand exchange by random walk in which rate declines precipitously with distance. PMID:19853618

  18. [Homologue pairing: initiation sites and effects on crossing over and chromosome disjunction in Drosophila melanogaster].

    PubMed

    Chubykin, V L

    1996-01-01

    The role of homologue pairing and chromocentral association of chromosomes in recombination and segregation during cell division is discussed. Peculiarities of mitotic and meiotic chromosome pairing in Drosophila males and females are considered. On the basis of our own and published data, the presence and localization of sites of homologue pairing initiation in euchromatin are substantiated. The effects of transfer of initiation sites along a chromosome (exemplified by inversions) on chromosome pairing (asynapsis), crossing over (intrachromosomal, interchromosomal, and centromeric effects), and segregation are discussed. To record the effects of pairing sites on crossing over, a method of comparing crossing-over frequencies in an inverted region with those in a region of the same size and position with regard to the centromere on cytological maps was proposed. Chromosomes orient toward opposite division poles during paracentromeric heterochromatin pairing. This occurs after successful euchromatin pairing, during which the chromocentral circular structure is reorganized. If heterochromatin pairing is disrupted because of structural or locus mutations, nonexchange bivalents segregate randomly. In this case, chromosome coordination may occur due to proximal chiasmata or chromocentral associations between homologues.

  19. Initial Presentation Sites as Predictors of Herpes Zoster Complications: A Nationwide Cohort Study

    PubMed Central

    Wang, Wen-Yi; Liu, Sing-Huh; Lin, Meng-Yin; Lin, Che-Chen

    2016-01-01

    Herpes zoster (HZ) is associated with complications such as postherpetic neuralgia (PHN) and HZ ophthalmicus (HZO). However, few studies have focused on identifying patients having a high risk of PHN and HZO according to the initial presentation sites. The current study investigated these factors in a nationwide population-based cohort derived from Taiwan’s Longitudinal Health Insurance Database. The results indicate that the initial presentation sites can predict the complication site of HZ. In this study, elderly patients were found to be more susceptible to HZ and were the first to present with neurological signs (HZN). Furthermore, compared with patients with HZO and other signs (HZT), those with HZN had a higher comorbidity risk. Patients with HZN showed a significantly higher visceral complication risk than did those with HZO (adjusted hazard ratio [HR] = 1.47, 95% confidence interval [CI] = 1.27–1.71). In addition, patients with HZT showed lower risks of ocular and neurological complications than did those with HZN after stratification by age and sex (adjusted HR = 0.46, 95% CI = 0.31–0.68 and HR = 0.73, 95% CI = 0.59–0.91, respectively). PMID:27711168

  20. Initiation of DNA replication from non-canonical sites on an origin-depleted chromosome.

    PubMed

    Bogenschutz, Naomi L; Rodriguez, Jairo; Tsukiyama, Toshio

    2014-01-01

    Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven well-characterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.

  1. Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase.

    PubMed

    Ospanov, Ruslan; Kochetov, German; Kurganov, Boris

    2006-11-20

    The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.

  2. Global Research Initiative in Alpine Environments: A New GLORIA Site in Southwestern Montana

    NASA Astrophysics Data System (ADS)

    Apple, M. E.; Pullman, T. Y.; Mitman, G. G.

    2007-12-01

    Global climate change is expected to have pronounced effects on the alpine environments and thus the alpine plants of western North America. Predicted responses include an upward migration of treelines, altered species compositions, changes in the percentage of land covered by vegetation, and a change in the phenology of alpine plants. To determine the effects of climate change on the alpine flora of southwestern Montana, we are installing a GLORIA (Global Research Initiative in Alpine Environments) site in order to monitor temperature, species composition, and percent cover of vascular plants, lichens, and mosses along an ascending altitudinal gradient. We are including lichens and mosses because of their importance as ecological indicator species. The abundance and spatial distribution of lichens and mosses provides essential baseline data for long-term monitoring of local and global impacts on the environment. Mt. Fleecer (9250 ft.), which is west of the continental divide and semi-isolated from other peaks in the Anaconda-Pintlar Range, is currently the most likely location for the southwestern Montana GLORIA site. Mt. Fleecer is accessible because it does not have the steep and hazardous glaciated talus cirques that characterize many of the neighboring, higher peaks. However, if an accessible and suitable higher summit is found, then it will be included as the highest summit in the GLORIA site. Interesting species at Mt. Fleecer include the whitebark pine, Pinus albicaulis, which is a keystone species in high mountain ecosystems of the western United States and Canada, the green gentian, Frasera speciosa, and the shooting star, Dodecatheon pulchellum. Data from this site will become part of a global network of GLORIA sites with which we will assess changes in alpine flora. Information gained from this GLORIA site can also be used as a link between studies of alpine climate change and related investigations on the timing of snowmelt and its influence on

  3. Initial source and site characterization studies for the U.C. Santa Barbara campus

    SciTech Connect

    Archuleta, R.; Nicholson, C.; Steidl, J.; Gurrola, L.; Alex, C.; Cochran, E.; Ely, G.; Tyler, T.

    1997-12-01

    The University of California Campus-Laboratory Collaboration (CLC) project is an integrated 3 year effort involving Lawrence Livermore National Laboratory (LLNL) and four UC campuses - Los Angeles (UCLA), Riverside (UCR), Santa Barbara (UCSB), and San Diego (UCSD) - plus additional collaborators at San Diego State University (SDSU), at Los Alamos National Laboratory and in industry. The primary purpose of the project is to estimate potential ground motions from large earthquakes and to predict site-specific ground motions for one critical structure on each campus. This project thus combines the disciplines of geology, seismology, geodesy, soil dynamics, and earthquake engineering into a fully integrated approach. Once completed, the CLC project will provide a template to evaluate other buildings at each of the four UC campuses, as well as provide a methodology for evaluating seismic hazards at other critical sites in California, including other UC locations at risk from large earthquakes. Another important objective of the CLC project is the education of students and other professional in the application of this integrated, multidisciplinary, state-of-the-art approach to the assessment of earthquake hazard. For each campus targeted by the CLC project, the seismic hazard study will consist of four phases: Phase I - Initial source and site characterization, Phase II - Drilling, logging, seismic monitoring, and laboratory dynamic soil testing, Phase III - Modeling of predicted site-specific earthquake ground motions, and Phase IV - Calculations of 3D building response. This report cover Phase I for the UCSB campus and incudes results up through March 1997.

  4. Mechanisms Governing the Selection of Translation Initiation Sites on Foot-and-Mouth Disease Virus RNA ▿

    PubMed Central

    Pöyry, Tuija A. A.; Jackson, Richard J.

    2011-01-01

    Translation initiation dependent on the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) occurs at two sites (Lab and Lb), 84 nucleotides (nt) apart. In vitro translation of an mRNA comprising the IRES and Lab-Lb intervening segment fused to a chloramphenicol acetyltransferase (CAT) reporter has been used to study the parameters influencing the ratio of the two products and the combined product yield as measures of relative initiation site usage and productive ribosome recruitment, respectively. With wild-type mRNA, ∼40% of initiation occurred at the Lab site, which was increased to 90% by optimization of its context, but decreased to 20% by mutations that reduced downstream secondary structure, with no change in recruitment in either case. Inserting 5 nt into the pyrimidine-rich tract located just upstream of the Lab site increased initiation at this site by 75% and ribosome recruitment by 50%. Mutating the Lab site to RCG or RUN codons decreased recruitment by 20 to 30% but stimulated Lb initiation by 20 to 40%. An antisense oligodeoxynucleotide annealing across the Lab site inhibited initiation at both sites. These and related results lead to the following conclusions. Recruitment by the wild-type IRES is limited by its short oligopyrimidine tract. At least 90% of internal ribosome entry occurs at the Lab AUG, but initiation at this site is restricted by its poor context, despite a counteracting effect of downstream secondary structure. Initiation at the Lb site is by ribosomes that access it by linear scanning from the original entry site, and not by an independent entry process. PMID:21813609

  5. Molecular Mechanisms in the Repair of the Cyclobutane Pyrimidine Dimer

    NASA Astrophysics Data System (ADS)

    Hassanali, Ali A.; Zhong, Dongping; Singer, Sherwin J.

    2009-06-01

    Exposure to far UV radiation induces DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Cyclobutane dimer lesions can be repaired by the enzyme photolyase, in which the absorption of a blue light photon initiates a sequence of photochemical events leading to the injection of an electron at the site of the CPD lesion in DNA. The electron catalyzes the repair of the cyclobutane dimer, splitting the CPD to is original pyrimidine units, and is subsequently recaptured by the photolyase protein. In this work we investigate the molecular mechanism of the repair of the cyclobutane dimer radical anion in aqueous solution using ab initio MD simulations. Umbrella sampling is used to determine a two-dimensional free energy surface as a function of the C5-C5-4 and C6-C6-4 distances. The neutral dimer is unable to surmount a large free energy barrier for repair. Upon addition of an electron, the splitting of the C5-C5-4 coordinate is virtually barrier less. Transition state theory predicts that the splitting of the C6-C6-4 bond is complete on a picosecond timescale. The free energy surface suggests that the splitting of the two bonds is asynchronously concerted. Our work is the first to explicitly include the electronic degrees of freedom for both the cyclobutane dimer and the surrounding water pocket. The ab initio simulations show that at least 30% of the electron density is delocalized onto the surrounding solvent during the splitting process. Simulations on the neutral surface show that back electron transfer from the dimer is critical for the completion of splitting: splitting of the C5-C5' and C6-C6' bonds can be reversed or enhanced depending on when electron return occurs. To maximize splitting yield, the back electron transfer should occur beyond the transition state along the splitting coordinate. Non-equilibrium trajectories are also conducted that begin with the electron added to a neutral unrepaired solvated CPD. Our results indicate that there are two

  6. Photochemical dimerization of organic compounds

    DOEpatents

    Crabtree, Robert H.; Brown, Stephen H.; Muedas, Cesar A.; Ferguson, Richard R.

    1992-01-01

    At least one of selectivity and reaction rate of photosensitized vapor phase dimerizations, including dehydrodimerizations, hydrodimerizations and cross-dimerizations of saturated and unsaturated organic compounds is improved by conducting the dimerization in the presence of hydrogen or nitrous oxide.

  7. Biomarker and histopathologic responses in flatfish following initial site remediation in Eagle Harbor, WA

    SciTech Connect

    Myers, M.S.; Anulacion, B.F.; French, B.; Hom, T.; Collier, T.K.

    1995-12-31

    Eagle Harbor is designated as an EPA Superfund site due to high sediment concentrations of creosote-derived polycyclic aromatic hydrocarbons (PAHs) released chronically from a nearby creosoting facility. Previous (1984--86) field and laboratory studies with adult English sole (Pleuronectes vetulus) from this site demonstrated high prevalences of toxicopathic liver lesions including neoplasms in resident sole, and inducibility of several neoplasia-related lesion types by injections of a PAH-rich fraction extracted from Eagle Harbor sediment. Further studies (1986--88) expanded the target species to also include starry flounder (Platichthys stellatus) and rock sole (Lepidopsetta bilineata), and incorporated biomarkers of PAH exposure and effect, including hepatic CYP1A expression and biliary fluorescent aromatic compounds to estimate PAH exposure and metabolism, and bulky hydrophobic DNA adducts to estimate PAHs bound to hepatic DNA. Hepatic lesion prevalences and biomarker values in these three species from Eagle Harbor were among the highest found at Puget Sound sites. In the initial phase of site remediation, a cap of uncontaminated sediment was placed over the most contaminated portions of Eagle Harbor from September `93 to March `94, to provide improved benthic habitat and sequester PAH-contaminated sediments. Lesion prevalences and biomarker values in these three flatfish species just before capping began were generally reduced compared to historical data, possibly as a result of creosoting facility closure and site-based source controls. Similar data from fish collected immediately after and at 3, 6, and 12 months after cap completion are presented to determine the efficacy of the capping in ameliorating PAH exposure and associated effects in resident flatfish species.

  8. Dominant role of the 5' TAR bulge in dimerization of HIV-1 genomic RNA, but no evidence of TAR-TAR kissing during in vivo virus assembly.

    PubMed

    Jalalirad, Mohammad; Saadatmand, Jenan; Laughrea, Michael

    2012-05-08

    The 5' untranslated region of HIV-1 genomic RNA (gRNA) contains two stem-loop structures that appear to be equally important for gRNA dimerization: the 57-nucleotide 5' TAR, at the very 5' end, and the 35-nucleotide SL1 (nucleotides 243-277). SL1 is well-known for containing the dimerization initiation site (DIS) in its apical loop. The DIS is a six-nucleotide palindrome. Here, we investigated the mechanism of TAR-directed gRNA dimerization. We found that the trinucleotide bulge (UCU24) of the 5' TAR has dominant impacts on both formation of HIV-1 RNA dimers and maturation of the formed dimers. The ΔUCU trinucleotide deletion strongly inhibited the first process and blocked the other, thus impairing gRNA dimerization as severely as deletion of the entire 5' TAR, and more severely than deletion of the DIS, inactivation of the viral protease, or most severe mutations in the nucleocapsid protein. The apical loop of TAR contains a 10-nucleotide palindrome that has been postulated to stimulate gRNA dimerization by a TAR-TAR kissing mechanism analogous to the one used by SL1 to stimulate dimerization. Using mutations that strongly destabilize formation of the TAR palindrome duplex, as well as compensatory mutations that restore duplex formation to a wild-type-like level, we found no evidence of TAR-TAR kissing, even though mutations nullifying the kissing potential of the TAR palindrome could impair dimerization by a mechanism other than hindering of SL1. However, nullifying the kissing potential of TAR had much less severe effects than ΔUCU. By not uncovering a dimerization mechanism intrinsic to TAR, our data suggest that TAR mutations exert their effect 3' of TAR, yet not on SL1, because TAR and SL1 mutations have synergistic effects on gRNA dimerization.

  9. Functional analysis of the internal translation initiation site of foot-and-mouth disease virus.

    PubMed Central

    Kühn, R; Luz, N; Beck, E

    1990-01-01

    Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites. Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential. A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly. The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E. V. Pilipenko, V. M. Blinov, B. K. Chernov, T. M. Dmitrieva, and V. I. Agol, Nucleic Acids Res. 17:5701-5711, 1989). They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation. Images PMID:2168956

  10. A Model Analysis of Mechanisms for Radial Microtubular Patterns at Root Hair Initiation Sites

    PubMed Central

    Krupinski, Pawel; Bozorg, Behruz; Larsson, André; Pietra, Stefano; Grebe, Markus; Jönsson, Henrik

    2016-01-01

    Plant cells have two main modes of growth generating anisotropic structures. Diffuse growth where whole cell walls extend in specific directions, guided by anisotropically positioned cellulose fibers, and tip growth, with inhomogeneous addition of new cell wall material at the tip of the structure. Cells are known to regulate these processes via molecular signals and the cytoskeleton. Mechanical stress has been proposed to provide an input to the positioning of the cellulose fibers via cortical microtubules in diffuse growth. In particular, a stress feedback model predicts a circumferential pattern of fibers surrounding apical tissues and growing primordia, guided by the anisotropic curvature in such tissues. In contrast, during the initiation of tip growing root hairs, a star-like radial pattern has recently been observed. Here, we use detailed finite element models to analyze how a change in mechanical properties at the root hair initiation site can lead to star-like stress patterns in order to understand whether a stress-based feedback model can also explain the microtubule patterns seen during root hair initiation. We show that two independent mechanisms, individually or combined, can be sufficient to generate radial patterns. In the first, new material is added locally at the position of the root hair. In the second, increased tension in the initiation area provides a mechanism. Finally, we describe how a molecular model of Rho-of-plant (ROP) GTPases activation driven by auxin can position a patch of activated ROP protein basally along a 2D root epidermal cell plasma membrane, paving the way for models where mechanical and molecular mechanisms cooperate in the initial placement and outgrowth of root hairs. PMID:27840629

  11. A Model Analysis of Mechanisms for Radial Microtubular Patterns at Root Hair Initiation Sites.

    PubMed

    Krupinski, Pawel; Bozorg, Behruz; Larsson, André; Pietra, Stefano; Grebe, Markus; Jönsson, Henrik

    2016-01-01

    Plant cells have two main modes of growth generating anisotropic structures. Diffuse growth where whole cell walls extend in specific directions, guided by anisotropically positioned cellulose fibers, and tip growth, with inhomogeneous addition of new cell wall material at the tip of the structure. Cells are known to regulate these processes via molecular signals and the cytoskeleton. Mechanical stress has been proposed to provide an input to the positioning of the cellulose fibers via cortical microtubules in diffuse growth. In particular, a stress feedback model predicts a circumferential pattern of fibers surrounding apical tissues and growing primordia, guided by the anisotropic curvature in such tissues. In contrast, during the initiation of tip growing root hairs, a star-like radial pattern has recently been observed. Here, we use detailed finite element models to analyze how a change in mechanical properties at the root hair initiation site can lead to star-like stress patterns in order to understand whether a stress-based feedback model can also explain the microtubule patterns seen during root hair initiation. We show that two independent mechanisms, individually or combined, can be sufficient to generate radial patterns. In the first, new material is added locally at the position of the root hair. In the second, increased tension in the initiation area provides a mechanism. Finally, we describe how a molecular model of Rho-of-plant (ROP) GTPases activation driven by auxin can position a patch of activated ROP protein basally along a 2D root epidermal cell plasma membrane, paving the way for models where mechanical and molecular mechanisms cooperate in the initial placement and outgrowth of root hairs.

  12. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed Central

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-01-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994). PMID:7651413

  13. Site-directed photo-cross-linking of rRNA transcription initiation complexes.

    PubMed

    Gong, X; Radebaugh, C A; Geiss, G K; Simon, M N; Paule, M R

    1995-09-01

    Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

  14. Site of initial diabetes education does not affect metabolic outcomes in children with T1DM.

    PubMed

    Tonyushkina, Ksenia N; Visintainer, Paul F; Jasinski, Christopher F; Wadzinski, Thomas L; Allen, Holley F

    2014-03-01

    To determine the difference in metabolic outcomes at 1 and 2 yr post type 1 diabetes mellitus (T1DM) diagnosis in children depending on the site of initial diabetes education: inpatient, vs. outpatient, vs. mixed locations. A retrospective chart review was performed for all patients with new onset antibody positive T1DM, aged 1-18 yr old, diagnosed in 2004-2009, and followed for at least 1 yr in a diabetes program at a tertiary academic health care center. Patients were divided into three groups based on the site of initial diabetes education: inpatient, outpatient, and mixed locations. The primary outcome was A1c at 1 and 2 yr. We enrolled 238 children (133 boys), mean (± SD) age 9.9 (± 4.1). A1c levels did not differ among inpatient, outpatient, and mixed location groups at 1 and 2 yr post diagnosis (p = 0.85 and p = 0.69, respectively) and the long-acting insulin doses were similar at 1 and 2 yr (p = 0.18 and p = 0.15, respectively). There was no difference in the number of acute diabetes complications between the groups. At 1 yr, 21.8% of outpatient-educated children were on insulin pump therapy in contrast to 14.7% of inpatient and 2.7% of mixed educated groups (p = 0.04). Families of children with new onset T1DM can be successfully and safely educated in a clinic setting. An 'education' admission for a medically stable patient is not necessary most of the time, however, clinical judgment and careful assessment of the family's coping and learning capabilities are important when determining the site of education. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Changes in chromatin structure at recombination initiation sites during yeast meiosis.

    PubMed Central

    Ohta, K; Shibata, T; Nicolas, A

    1994-01-01

    Transient double-strand breaks (DSBs) occur during Saccharomyces cerevisiae meiosis at recombination hot spots and are thought to initiate most, if not all, homologous recombination between chromosomes. To uncover the regulatory mechanisms active in DSB formation, we have monitored the change in local chromatin structure at the ARG4 and CYS3 recombination hot spots over the course of meiosis. Micrococcal nuclease (MNase) digestion of isolated meiotic chromatin followed by indirect end-labeling revealed that the DSB sites in both loci are hypersensitive to MNase and that their sensitivity increases 2- to 4-fold prior to the appearance of meiotic DSBs and recombination products. Other sensitive sites are not significantly altered. The study of hyper- and hypo-recombinogenic constructs at the ARG4 locus, also revealed that the MNase sensitivity at the DSB site correlates with both the extent of DSBs and the rate of gene conversion. These results suggest that the local chromatin structure and its modification in early meiosis play an important role in the positioning and frequency of meiotic DSBs, leading to meiotic recombination. Images PMID:7988571

  16. Assessment of Effectiveness of Geologic Isolation Systems: REFERENCE SITE INITIAL ASSESSMENT FOR A SALT DOME REPOSITORY

    SciTech Connect

    Harwell, M. A.; Brandstetter, A.; Benson, G. L.; Bradley, D. J.; Serne, R. J.; Soldat, J. K; Cole, C. R.; Deutsch, W. J.; Gupta, S. K.; Harwell, C. C.; Napier, B. A.; Reisenauer, A. E.; Prater, L. S.; Simmons, C. S.; Strenge, D. L.; Washburn, J. F.; Zellmer, J. T.

    1982-06-01

    As a methodology demonstration for the Office of Nuclear Waste Isolation (ONWI), the Assessment of Effectiveness of Geologic Isolation Systems (AEGIS) Program conducted an initial reference site analysis of the long-term effectiveness of a salt dome repository. The Hainesville Salt Dome in Texas was chosen to be representative of the Gulf Coast interior salt domes; however, the Hainesville Site has been eliminated as a possible nuclear waste repository site. The data used for this exercise are not adequate for an actual assessment, nor have all the parametric analyses been made that would adequately characterize the response of the geosystem surrounding the repository. Additionally, because this was the first exercise of the complete AEGIS and WASTE Rock Interaction Technology (WRIT) methodology, this report provides the initial opportunity for the methodology, specifically applied to a site, to be reviewed by the community outside the AEGIS. The scenario evaluation, as a part of the methodology demonstration, involved consideration of a large variety of potentially disruptive phenomena, which alone or in concert could lead to a breach in a salt dome repository and to a subsequent transport of the radionuclides to the environment. Without waste- and repository-induced effects, no plausible natural geologic events or processes which would compromise the repository integrity could be envisioned over the one-million-year time frame after closure. Near-field (waste- and repository-induced) effects were excluded from consideration in this analysis, but they can be added in future analyses when that methodology development is more complete. The potential for consequential human intrusion into salt domes within a million-year time frame led to the consideration of a solution mining intrusion scenario. The AEGIS staff developed a specific human intrusion scenario at 100 years and 1000 years post-closure, which is one of a whole suite of possible scenarios. This scenario

  17. Assessment of Effectiveness of Geologic Isolation Systems: REFERENCE SITE INITIAL ASSESSMENT FOR A SALT DOME REPOSITORY

    SciTech Connect

    Harwell, M. A.; Brandstetter, A.; Benson, G. L.; Raymond, J. R.; Brandley, D. J.; Serne, R. J.; Soldat, J. K.; Cole, C. R.; Deutsch, W. J.; Gupta, S. K.; Harwell, C. C.; Napier, B. A.; Reisenauer, A. E.; Prater, L. S.; Simmons, C. S.; Strenge, D. L.; Washburn, J. F.; Zellmer, J. T.

    1982-06-01

    As a methodology demonstration for the Office of Nuclear Waste Isolation (ONWI), the Assessment of Effectiveness of Geologic Isolation Systems (AEGIS) Program conducted an initial reference site analysis of the long-term effectiveness of a salt dome repository. The Hainesville Salt Dome in Texas was chosen to be representative of the Gulf Coast interior salt domes; however, the Hainesville Site has been eliminated as a possible nuclear waste repository site. The data used for this exercise are not adequate for an actual assessment, nor have all the parametric analyses been made that would adequately characterize the response of the geosystem surrounding the repository. Additionally, because this was the first exercise of the complete AEGIS and WASTE Rock Interaction Technology (WRIT) methodology, this report provides the initial opportunity for the methodology, specifically applied to a site, to be reviewed by the community outside the AEGIS. The scenario evaluation, as a part of the methodology demonstration, involved consideration of a large variety of potentially disruptive phenomena, which alone or in concert could lead to a breach in a salt dome repository and to a subsequent transport of the radionuclides to the environment. Without waste- and repository-induced effects, no plausible natural geologic events or processes which would compromise the repository integrity could be envisioned over the one-million-year time frame after closure. Near-field (waste- and repository-induced) effects were excluded from consideration in this analysis, but they can be added in future analyses when that methodology development is more complete. The potential for consequential human intrusion into salt domes within a million-year time frame led to the consideration of a solution mining intrusion scenario. The AEGIS staff developed a specific human intrusion scenario at 100 years and 1000 years post-closure, which is one of a whole suite of possible scenarios. This scenario

  18. GnRH agonist reduces estrogen receptor dimerization in GT1-7 cells: evidence for cross-talk between membrane-initiated estrogen and GnRH signaling.

    PubMed

    Chason, Rebecca J; Kang, Jung-Hoon; Gerkowicz, Sabrina A; Dufau, Maria L; Catt, Kevin J; Segars, James H

    2015-03-15

    17β-estradiol (E2), a key participant on the initiation of the LH surge, exerts both positive and negative feedback on GnRH neurons. We sought to investigate potential interactions between estrogen receptors alpha (ERα) and beta (ERβ) and gonadotropin releasing hormone receptor (GnRH-R) in GT1-7 cells. Radioligand binding studies demonstrated a significant decrease in saturation E2 binding in cells treated with GnRH agonist. Conversely, there was a significant reduction in GnRH binding in GT1-7 cells treated with E2. In BRET(1) experiments, ERα-ERα dimerization was suppressed in GT1-7 cells treated with GnRH agonist (p < 0.05). There was no evidence of direct interaction between ERs and GnRH-R. This study provides the first evidence of reduced ERα homodimerization by GnRH agonist. Collectively, these findings demonstrate significant cross-talk between membrane-initiated GnRH and E2 signaling in GT1-7 cells.

  19. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    SciTech Connect

    Du, Fengxia; Zhang, Minjie; Li, Xiaohua; Yang, Caiyun; Meng, Hao; Wang, Dong; Chang, Shuang; Xu, Ye; Price, Brendan; Sun, Yingli

    2014-10-03

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.

  20. How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

    PubMed

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

  1. How MCM loading and spreading specify eukaryotic DNA replication initiation sites

    PubMed Central

    Hyrien, Olivier

    2016-01-01

    DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes. PMID:27635237

  2. Protein dimerization. Inside job.

    PubMed

    Metzger, H

    1994-04-01

    In a sophisticated combination of genetic engineering and organic synthesis, a general method for dimerizing recombinant intracellular proteins has been devised; the usefulness of the method should now be testable.

  3. Possible Role of Dimerization in Human Immunodeficiency Virus Type 1 Genome RNA Packaging

    PubMed Central

    Sakuragi, Jun-Ichi; Ueda, Shigeharu; Iwamoto, Aikichi; Shioda, Tatsuo

    2003-01-01

    The dimer initiation site/dimer linkage sequence (DIS/DLS) region in the human immunodeficiency virus type 1 (HIV-1) RNA genome is suggested to play important roles in various steps of the virus life cycle. However, due to the presence of a putative DIS/DLS region located within the encapsidation signal region (E/psi), it is difficult to perform a mutational analysis of DIS/DLS without affecting the packaging of RNA into virions. Recently, we demonstrated that duplication of the DIS/DLS region in viral RNA caused the production of partially monomeric RNAs in virions, indicating that the region indeed mediated RNA-RNA interaction. We utilized this system to assess the precise location of DIS/DLS in the 5′ region of the HIV-1 genome with minimum effect on RNA packaging. We found that the entire lower stem of the U5/L stem-loop was required for packaging, whereas the region important for dimer formation was only 10 bases long within the lower stem of the U5/L stem-loop. The R/U5 stem-loop was required for RNA packaging but was completely dispensable for dimer formation. The SL1 lower stem was important for both dimerization and packaging, but surprisingly, deletion of the palindromic sequence at the top of the loop only partially affected dimerization. These results clearly indicated that the E/psi of HIV-1 is much larger than the DIS/DLS and that the primary DIS/DLS is completely included in the E/psi. Therefore, it is suggested that RNA dimerization is a part of RNA packaging, which requires multiple steps. PMID:12634365

  4. Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures

    PubMed Central

    Kague, Erika; Roy, Paula; Asselin, Garrett; Hu, Gui; Stanley, Alexandra; Albertson, Craig; Simonet, Jacqueline; Fisher, Shannon

    2017-01-01

    During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures. PMID:26992365

  5. Uterine cervical cancer with brain metastasis as the initial site of presentation.

    PubMed

    Sato, Yumi; Tanaka, Kei; Kobayashi, Yoichi; Shibuya, Hiromi; Nishigaya, Yoshiko; Momomura, Mai; Matsumoto, Hironori; Iwashita, Mitsutoshi

    2015-07-01

    Brain metastasis from uterine cervical cancer is rare, with an incidence of 0.5%, and usually occurs late in the course of the disease. We report a case of uterine cervical cancer with brain metastasis as the initial site of presentation. A 50-year-old woman with headache, vertigo, amnesia and loss of appetite was admitted for persistent vomiting. Contrast enhanced computed tomography showed a solitary right frontal cerebral lesion with ring enhancement and uterine cervical tumor. She was diagnosed with uterine cervical squamous cell carcinoma with parametrium invasion and no other distant affected organs were detected. The cerebral lesion was surgically removed and pathologically proved to be metastasis of uterine cervical squamous cell carcinoma. The patient underwent concurrent chemoradiotherapy, followed by cerebral radiation therapy, but multiple metastases to the liver and lung developed and the patient died 7 months after diagnosis of brain metastasis.

  6. Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures.

    PubMed

    Kague, Erika; Roy, Paula; Asselin, Garrett; Hu, Gui; Simonet, Jacqueline; Stanley, Alexandra; Albertson, Craig; Fisher, Shannon

    2016-05-15

    During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

  7. Control of Gene Expression by RNA Binding Protein Action on Alternative Translation Initiation Sites

    PubMed Central

    Re, Angela; Waldron, Levi; Quattrone, Alessandro

    2016-01-01

    Transcript levels do not faithfully predict protein levels, due to post-transcriptional regulation of gene expression mediated by RNA binding proteins (RBPs) and non-coding RNAs. We developed a multivariate linear regression model integrating RBP levels and predicted RBP-mRNA regulatory interactions from matched transcript and protein datasets. RBPs significantly improved the accuracy in predicting protein abundance of a portion of the total modeled mRNAs in three panels of tissues and cells and for different methods employed in the detection of mRNA and protein. The presence of upstream translation initiation sites (uTISs) at the mRNA 5’ untranslated regions was strongly associated with improvement in predictive accuracy. On the basis of these observations, we propose that the recently discovered widespread uTISs in the human genome can be a previously unappreciated substrate of translational control mediated by RBPs. PMID:27923063

  8. Initial formulation results for in situ grouting of a waste trench at ORNL Site No. 6

    SciTech Connect

    Tallent, O.K.; McDaniel, E.W.; Spence, R.D.; Godsey, T.T.

    1987-01-01

    An investigation is being conducted by the Chemical Technology Division to assist the Environmental Sciences Division in developing a grout formulation for use in testing in situ grouting in a waste trench at ORNL Site 6. This final report satisfies the milestone of Subtack 12 entitled, ''Low Level Waste (LLW) Trench Grouting Assessment,'' which was initially issued as RAP-86-7, December 31, 1985. Grouts prepared from dry-solid blends containing Type I Portland cement, ASTM Class C or Class F fly ash, and bentonite, mixed water at ratios of 10 to 15 lb/gal, were evaluated. The grouts prepared with ASTM Class C fly ash exhibited significantly better properties than those prepared with ASTM Class F fly ash. The grouts containing ASTM Class C fly ash satisfy tentative performance criteria for the project. 8 refs., 7 tabs.

  9. Dimers in nucleating vapors

    NASA Astrophysics Data System (ADS)

    Lushnikov, A. A.; Kulmala, M.

    1998-09-01

    The dimer stage of nucleation may affect considerably the rate of the nucleation process at high supersaturation of the nucleating vapor. Assuming that the dimer formation limits the nucleation rate, the kinetics of the particle formation-growth process is studied starting with the definition of dimers as bound states of two associating molecules. The partition function of dimer states is calculated by summing the Boltzmann factor over all classical bound states, and the equilibrium population of dimers is found for two types of intermolecular forces: the Lennard-Jones (LJ) and rectangular well+hard core (RW) potentials. The principle of detailed balance is used for calculating the evaporation rate of dimers. The kinetics of the particle formation-growth process is then investigated under the assumption that the trimers are stable with respect to evaporation and that the condensation rate is a power function of the particle mass. If the power exponent λ=n/(n+1) (n is a non-negative integer), the kinetics of the process is described by a finite set of moments of particle mass distribution. When the characteristic time of the particle formation by nucleation is much shorter than that of the condensational growth, n+2 universal functions of a nondimensional time define the kinetic process. These functions are calculated for λ=2/3 (gas-to-particle conversion in the free molecular regime) and λ=1/2 (formation of islands on surfaces).

  10. U.S. Department of Energy's site screening, site selection, and initial characterization for storage of CO2 in deep geological formations

    USGS Publications Warehouse

    Rodosta, T.D.; Litynski, J.T.; Plasynski, S.I.; Hickman, S.; Frailey, S.; Myer, L.

    2011-01-01

    The U.S. Department of Energy (DOE) is the lead Federal agency for the development and deployment of carbon sequestration technologies. As part of its mission to facilitate technology transfer and develop guidelines from lessons learned, DOE is developing a series of best practice manuals (BPMs) for carbon capture and storage (CCS). The "Site Screening, Site Selection, and Initial Characterization for Storage of CO2 in Deep Geological Formations" BPM is a compilation of best practices and includes flowchart diagrams illustrating the general decision making process for Site Screening, Site Selection, and Initial Characterization. The BPM integrates the knowledge gained from various programmatic efforts, with particular emphasis on the Characterization Phase through pilot-scale CO2 injection testing of the Validation Phase of the Regional Carbon Sequestration Partnership (RCSP) Initiative. Key geologic and surface elements that suitable candidate storage sites should possess are identified, along with example Site Screening, Site Selection, and Initial Characterization protocols for large-scale geologic storage projects located across diverse geologic and regional settings. This manual has been written as a working document, establishing a framework and methodology for proper site selection for CO2 geologic storage. This will be useful for future CO2 emitters, transporters, and storage providers. It will also be of use in informing local, regional, state, and national governmental agencies of best practices in proper sequestration site selection. Furthermore, it will educate the inquisitive general public on options and processes for geologic CO2 storage. In addition to providing best practices, the manual presents a geologic storage resource and capacity classification system. The system provides a "standard" to communicate storage and capacity estimates, uncertainty and project development risk, data guidelines and analyses for adequate site characterization, and

  11. Correlation between pairing initiation sites, recombination nodules and meiotic recombination in Sordaria macrospora.

    PubMed

    Zickler, D; Moreau, P J; Huynh, A D; Slezec, A M

    1992-09-01

    The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of "recombination nodules." Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants.

  12. Correlation between Pairing Initiation Sites, Recombination Nodules and Meiotic Recombination in Sordaria Macrospora

    PubMed Central

    Zickler, D.; Moreau, PJF.; Huynh, A. D.; Slezec, A. M.

    1992-01-01

    The decrease of meiotic exchanges (crossing over and conversion) in two mutants of Sordaria macrospora correlated strongly with a reduction of chiasmata and of both types of ``recombination nodules.'' Serial section reconstruction electron microscopy was used to compare the synapsis pattern of meiotic prophase I in wild type and mutants. First, synapsis occurred but the number of synaptonemal complex initiation sites was reduced in both mutants. Second, this reduction was accompanied by, or resulted in, modifications of the pattern of synapsis. Genetic and synaptonemal complex maps were compared in three regions along one chromosome arm divided into well marked intervals. Reciprocal exchange frequencies and number of recombination nodules correlated in wild type in the three analyzed intervals, but disparity was found between the location of recombination nodules and exchanges in the mutants. Despite the twofold exchange decrease, sections of the genome such as the short arm of chromosome 2 and telomere regions were sheltered from nodule decrease and from pairing modifications. This indicated a certain amount of diversity in the control of these features and suggested that exchange frequency was dependent not only on the amount of effective pairing but also on the localization of the pairing sites, as revealed by the synaptonemal complex progression in the mutants. PMID:1398050

  13. Implementation of geriatric acute care best practices: initial results of the NICHE SITE self-evaluation.

    PubMed

    Boltz, Marie; Capezuti, Elizabeth; Shuluk, Joseph; Brouwer, Julianna; Carolan, Deirdre; Conway, Shirley; DeRosa, Sue; LaReau, Rita; Lyons, Denise; Nickoley, Sue; Smith, Tyleen; Galvin, James E

    2013-12-01

    Nurses Improving Care of Healthsystem Elders (NICHE) provides hospitals with tools and resources to implement an initiative to improve health outcomes in older adults and their families. Beginning in 2011, members have engaged in a process of program self-evaluation, designed to evaluate internal progress toward developing, sustaining, and disseminating NICHE. This manuscript describes the NICHE Site Self-evaluation and reports the inaugural self-evaluation data in 180 North American hospitals. NICHE members evaluate their program utilizing the following dimensions of a geriatric acute care program: guiding principles, organizational structures, leadership, geriatric staff competence, interdisciplinary resources and processes, patient- and family-centered approaches, environment of care, and quality metrics. The majority of NICHE sites were at the progressive implementation level (n = 100, 55.6%), having implemented interdisciplinary geriatric education and the geriatric resource nurse (GRN) model on at least one unit; 29% have implemented the GRN model on multiple units, including specialty areas. Bed size, teaching status, and Magnet status were not associated with level of implementation, suggesting that NICHE implementation can be successful in a variety of settings and communities. © 2013 Wiley Publishing Asia Pty Ltd.

  14. Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination.

    PubMed

    Juszkiewicz, Szymon; Hegde, Ramanujan S

    2017-02-16

    Diverse cellular stressors have been observed to trigger site-specific ubiquitination on several ribosomal proteins. However, the ubiquitin ligases, biochemical consequences, and physiologic pathways linked to these modifications are not known. Here, we show in mammalian cells that the ubiquitin ligase ZNF598 is required for ribosomes to terminally stall during translation of poly(A) sequences. ZNF598-mediated stalling initiated the ribosome-associated quality control (RQC) pathway for degradation of nascent truncated proteins. Biochemical ubiquitination reactions identified two sites of mono-ubiquitination on the 40S protein eS10 as the primary ribosomal target of ZNF598. Cells lacking ZNF598 activity or containing ubiquitination-resistant eS10 ribosomes failed to stall efficiently on poly(A) sequences. In the absence of stalling, read-through of poly(A) produces a poly-lysine tag, which might alter the localization and solubility of the associated protein. Thus, ribosome ubiquitination can modulate translation elongation and impacts co-translational quality control to minimize production of aberrant proteins.

  15. Initial Implementation of the Hanford Site-Wide Groundwater Flow and Uncertainty Analysis Framework

    NASA Astrophysics Data System (ADS)

    Cole, C. R.; Vermeul, V. R.; Freedman, V. L.; Bergeron, M. P.

    2002-05-01

    Since Hanford operations began in 1943, large volumes of wastewater have been discharged into the subsurface, creating groundwater mounds (> 20m) and regional-scale contaminant plumes that will require monitoring at least through site closure. Since the cessation of wastewater disposal activities in 1988, many of the ~700 monitoring wells that previously documented mounding and contaminant movement are currently going dry. An initial implementation of the Hanford Site uncertainty methodology presented in this paper and a companion poster, investigates which of the ~700 monitoring wells are likely to go dry between now and 2050. The long-term goals of the Pacific Northwest National Laboratory effort at Hanford include the development and implementation of an uncertainty methodology with the site-wide groundwater flow and transport model. Results are presented for two different conceptual models of the base of the unconfined aquifer. Model parameter uncertainty was determined through transient inverse modeling (1943-1996) using UCODE and ~76,000 historical observations of head. Since an analysis of model linearity using Beale's measure indicated that the model was sufficiently linear, the uncertainty in predicted future water levels was determined using linear confidence and prediction intervals. Both a steady-state and a transient case (1996-2050) were investigated in order to determine which of the current monitoring wells are likely to go dry. Results demonstrated that the uncertainty methodology can be used to evaluate the potential loss of existing monitoring wells in strategic locations, and to assist in the development of a long-term strategy for their replacement. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy under Contract DE-AC06-76RL01830.

  16. Comparative analysis of contextual bias around the translation initiation sites in plant genomes.

    PubMed

    Gupta, Paras; Rangan, Latha; Ramesh, T Venkata; Gupta, Mudit

    2016-09-07

    Nucleotide distribution around translation initiation site (TIS) is thought to play an important role in determining translation efficiency. Kozak in vertebrates and later Joshi et al. in plants identified context sequence having a key role in translation efficiency, but a great variation regarding this context sequence has been observed among different taxa. The present study aims to refine the context sequence around initiation codon in plants and addresses the sampling error problem by using complete genomes of 7 monocots and 7 dicots separately. Besides positions -3 and +4, significant conservation at -2 and +5 positions was also found and nucleotide bias at the latter two positions was shown to directly influence translation efficiency in the taxon studied. About 1.8% (monocots) and 2.4% (dicots) of the total sequences fit the context sequence from positions -3 to +5, which might be indicative of lower number of housekeeping genes in the transcriptome. A three base periodicity was observed in 5' UTR and CDS of monocots and only in CDS of dicots as confirmed against random occurrence and annotation errors. Deterministic enrichment of GCNAUGGC in monocots, AANAUGGC in dicots and GCNAUGGC in plants around TIS was also established (where AUG denotes the start codon), which can serve as an arbiter of putative TIS with efficient translation in plants.

  17. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites.

    PubMed

    Ammerman, Michelle L; Presnyak, Vladimir; Fisk, John C; Foda, Bardees M; Read, Laurie K

    2010-11-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.

  18. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

    PubMed Central

    Ammerman, Michelle L.; Presnyak, Vladimir; Fisk, John C.; Foda, Bardees M.; Read, Laurie K.

    2010-01-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs. PMID:20855539

  19. Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony

    PubMed Central

    Jha, Jyoti K.

    2016-01-01

    The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites. The altered SeqA binding, however, does not affect initiation synchrony. Synchrony is also unaffected when individual SeqA sites are mutated. An apparent exception was one mutant where the mutation also changed an overlapping DnaA site. In this mutant, the observed asynchrony could be from altered DnaA binding, as selectively mutating this SeqA site did not cause asynchrony. These results reveal robust initiation synchrony against alterations of individual SeqA binding sites. The redundancy apparently ensures SeqA function in controlling replication in E. coli. PMID:27930658

  20. Structural probing of a pathogenic tRNA dimer

    PubMed Central

    ROY, MARC D.; WITTENHAGEN, LISA M.; KELLEY, SHANA O.

    2005-01-01

    The A3243G mutation within the human mitochondrial (hs mt) tRNALeu(UUR) gene is associated with maternally inherited deafness and diabetes (MIDD) and other mitochondrial encephalopathies. One of the most pronounced structural effects of this mutation is the disruption of the native structure through stabilization of a high-affinity dimeric complex. We conducted a series of studies that address the structural properties of this tRNA dimer, and we assessed its formation under physiological conditions. Enzymatic probing was used to directly define the dimeric interface for the complex, and a discrete region of the D-stem and loop of hs mt tRNALeu(UUR) was identified. The dependence of dimerization on magnesium ions and temperature was also tested. The formation of the tRNA dimer is influenced by temperature, with dimerization becoming more efficient at physiological temperature. Complexation of the mutant tRNA is also affected by the amount of magnesium present, and occurs at concentrations present intracellularly. Terbium probing experiments revealed a specific metal ion-binding site localized at the site of the A3243G mutation that is unique to the dimer structure. This metal ion-binding site presents a striking parallel to dimeric complexes of viral RNAs, which use the same hexanucleotide sequence for complexation and feature a similarly positioned metal ion-binding site within the dimeric structure. Taken together, these results indicate that the unique dimeric complex formed by the hs mt tRNALeu(UUR) A3243G mutant exhibits interesting similarities to biological RNA dimers, and may play a role in the loss of function caused by this mutation in vivo. PMID:15701731

  1. INITIAL SINGLE SHELL TANK (SST) SYSTEM PERFORMANCE ASSESSMENT OF THE HANFORD SITE

    SciTech Connect

    JARAYSI, M.N.

    2007-01-08

    The ''Initial Single-Shell Tank System Performance Assessment for the Hanford Site [1] (SST PA) presents the analysis of the long-term impacts of residual wastes assumed to remain after retrieval of tank waste and closure of the SST farms at the US Department of Energy (DOE) Hanford Site. The SST PA supports key elements of the closure process agreed upon in 2004 by DOE, the Washington State Department of Ecology (Ecology), and the US Environmental Protection Agency (EPA). The SST PA element is defined in Appendix I of the ''Hanford Federal Facility Agreement and Consent Order'' (HFFACO) (Ecology et al. 1989) [2], the document that establishes the overall closure process for the SST and double-shell tank (DST) systems. The approach incorporated in the SST PA integrates substantive features of both hazardous and radioactive waste management regulations into a single analysis. The defense-in-depth approach used in this analysis defined two major engineering barriers (a surface barrier and the grouted tank structure) and one natural barrier (the vadose zone) that will be relied on to control waste release into the accessible environment and attain expected performance metrics. The analysis evaluates specific barrier characteristics and other site features that influence contaminant migration by the various pathways. A ''reference'' case and a suite of sensitivity/uncertainty cases are considered. The ''reference case'' evaluates environmental impacts assuming central tendency estimates of site conditions. ''Reference'' case analysis results show residual tank waste impacts on nearby groundwater, air resources; or inadvertent intruders to be well below most important performance objectives. Conversely, past releases to the soil, from previous tank farm operations, are shown to have groundwater impacts that re significantly above most performance objectives. Sensitivity/uncertainty cases examine single and multiple parameter variability along with plausible alternatives

  2. Initial Single-Shell Tank System Performance Assessment for the Hanford Site

    SciTech Connect

    Jaraysi, M.N.; Kristofzski, J.G.; Connelly, M.P.; Wood, M.I.; Knepp, A.J.; Quintero, R.A.

    2007-07-01

    The Initial Single-Shell Tank System Performance Assessment for the Hanford Site (SST PA) presents the analysis of the long-term impacts of residual wastes assumed to remain after retrieval of tank wastes and closure of the SST farms at the U.S. Department of Energy (DOE) Hanford Site. The SST PA supports key elements of the closure process agreed upon in 2004 by DOE, the Washington State Department of Ecology (Ecology), and the U.S. Environmental Protection Agency (EPA). The SST PA element is defined in Appendix I of the Hanford Federal Facility Agreement and Consent Order (HFFACO) (Ecology et al. 1989), the document that establishes the overall closure process for the SST and double-shell tank (DST) systems. The approach incorporated in the SST PA integrates substantive features of both hazardous and radioactive waste management regulations into a single analysis. The defense-in-depth approach used in this analysis defined two major engineering barriers (a surface barrier and the grouted tank structure) and one natural barrier (the vadose zone) that will be relied on to control waste release into the accessible environment and attain expected performance metrics. The analysis evaluates specific barrier characteristics and other site features that influence contaminant migration by the various pathways. A 'reference' case and a suite of sensitivity/uncertainty cases are considered. The 'reference case' evaluates environmental impacts assuming central tendency estimates of site conditions. 'Reference' case analysis results show residual tank waste impacts on nearby groundwater, air resources; or inadvertent intruders to be well below most important performance objectives. Conversely, past releases to the soil, from previous tank farm operations, are shown to have groundwater impacts that are significantly above most performance objectives. Sensitivity/uncertainty cases examine single and multiple parameter variability along with plausible alternatives to 'reference

  3. Internal energy selection in vacuum ultraviolet photoionization of ethanol and ethanol dimers.

    PubMed

    Bodi, Andras

    2013-10-14

    Internal energy selected ethanol monomer and ethanol dimer ions were prepared by threshold photoionization of a supersonic molecular beam seeded with ethanol. The dissociative photoionization processes of the monomer, the lowest-energy CH3-loss channel of the dimer, and the fragmentation of larger clusters were found to be disjunct from the ionization onset to about 12 eV, which made it possible to determine the 0 K appearance energy of C-C bond breaking in the H-donor unit of the ethanol dimer cation as 9.719 ± 0.004 eV. This reaction energy is used together with ab initio calculations in a thermochemical cycle to determine the binding energy change from the neutral ethanol dimer to a protonated ethanol-formaldehyde adduct. The cycle also shows general agreement between experiment, theory, and previously published enthalpies of formation. The role of the initial ionization site, or rather the initial photoion state, is also discussed based on the dimer breakdown diagram and excited state calculations. There is no evidence for isolated state behavior, and the ethanol dimer dissociative photoionization processes appear to be governed by statistical theory and the ground electronic state of the ion. In the monomer breakdown diagram, the smoothly changing branching ratio between H and CH3 loss is at odds with rate theory predictions, and shows that none of the currently employed few-parameter rate models, appropriate for experimental rate curve fitting, yields a correct description for this process in the experimental energy range.

  4. Initial basalt target site selection evaluation for the Mars penetrator drop test

    NASA Technical Reports Server (NTRS)

    Bunch, T. E.; Quaide, W. L.; Polkowski, G.

    1976-01-01

    Potential basalt target sites for an air drop penetrator test were described and the criteria involved in site selection were discussed. A summary of the background field geology and recommendations for optimum sites are also presented.

  5. Initial Field Trials of the Site Characterization and Analysis Penetrometer System (SCAPS). Reconnaissance of Jacksonville Naval Air Station Waste Oil and Solvents Disposal Site

    DTIC Science & Technology

    1993-12-01

    Engineers Waterways Experiment Station DTIC Initial Field Trials of the Site ELECTF Characterization and Analysis JAN2 5 1994D Penetrometer System...038Prepared f NlFclitie 24En g m Prepared for Naval Facilities Engineering Command The contents of this report are not to be used for advertising. publication...Characterization and Analysis Penetrometer Sysstem (SCAPS) Reconnaissance of Jacksonville Naval Air Station Waste Oil and Solvents Disposal Site by Stafford S

  6. Initial results from seismic monitoring at the Aquistore CO2 storage site, Saskatchewan, Canada

    DOE PAGES

    White, D. J.; Roach, L. A.N.; Roberts, B.; ...

    2014-12-31

    from 2012 shows excellent repeatability (NRMS less than 10%) which will provide enhanced monitoring sensitivity to smaller amounts of CO2. The permanent array also provides continuous passive monitoring for injection-related microseismicity. Passive monitoring has been ongoing since the summer of 2012 in order to establish levels of background seismicity before CO2 injection starts in 2014. Microseismic monitoring was augmented in 2013 by the installation of 3 broadband seismograph stations surrounding the Aquistore site. These surface installations should provide a detection capability of seismic events with magnitudes as low as ~0. Downhole seismic methods are also being utilized for CO2 monitoring at the Aquistore site. Baseline crosswell tomographic images depict details (meters-scale) of the reservoir in the 150-m interval between the observation and injection wells. This level of resolution is designed to track the CO2 migration between the wells during the initial injection period. A baseline 3D vertical seismic profile (VSP) was acquired in the fall of 2013 to provide seismic images with resolution on a scale between that provided by the surface seismic array and the downhole tomography. The 3D VSP was recorded simultaneously using both a conventional array of downhole geophones (60-levels) and an optical fibre system. The latter utilized an optical fiber cable deployed on the outside of the monitor well casing and cemented in place. A direct comparison of these two methodologies will determine the suitability of using the fiber cable for ongoing time-lapse VSP monitoring.« less

  7. Glycine Transporter Dimers

    PubMed Central

    Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette; Dutertre, Sébastien; Hastrup, Hanne; Jha, Alok; Gether, Ulrik; Sitte, Harald H.; Betz, Heinrich; Eulenburg, Volker

    2015-01-01

    Different Na+/Cl−-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuTAa, as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs. PMID:18252709

  8. Establishing a health demographic surveillance site in Bhaktapur district, Nepal: initial experiences and findings

    PubMed Central

    2012-01-01

    Background A health demographic surveillance system (HDSS) provides longitudinal data regarding health and demography in countries with coverage error and poor quality data on vital registration systems due to lack of public awareness, inadequate legal basis and limited use of data in health planning. The health system in Nepal, a low-income country, does not focus primarily on health registration, and does not conduct regular health data collection. This study aimed to initiate and establish the first HDSS in Nepal. Results We conducted a baseline survey in Jhaukhel and Duwakot, two villages in Bhaktapur district. The study surveyed 2,712 households comprising a total population of 13,669. The sex ratio in the study area was 101 males per 100 females and the average household size was 5. The crude birth and death rates were 9.7 and 3.9/1,000 population/year, respectively. About 11% of births occurred at home, and we found no mortality in infants and children less than 5 years of age. Various health problems were found commonly and some of them include respiratory problems (41.9%); headache, vertigo and dizziness (16.7%); bone and joint pain (14.4%); gastrointestinal problems (13.9%); heart disease, including hypertension (8.8%); accidents and injuries (2.9%); and diabetes mellitus (2.6%). The prevalence of non-communicable disease (NCD) was 4.3% (95% CI: 3.83; 4.86) among individuals older than 30 years. Age-adjusted odds ratios showed that risk factors, such as sex, ethnic group, occupation and education, associated with NCD. Conclusion Our baseline survey demonstrated that it is possible to collect accurate and reliable data in a village setting in Nepal, and this study successfully established an HDSS site. We determined that both maternal and child health are better in the surveillance site compared to the entire country. Risk factors associated with NCDs dominated morbidity and mortality patterns. PMID:22950751

  9. A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells

    PubMed Central

    Zhang, Ya; Huang, Liang; Fu, Haiqing; Smith, Owen K.; Lin, Chii Mei; Utani, Koichi; Rao, Mishal; Reinhold, William C.; Redon, Christophe E.; Ryan, Michael; Kim, RyangGuk; You, Yang; Hanna, Harlington; Boisclair, Yves; Long, Qiaoming; Aladjem, Mirit I.

    2016-01-01

    Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins. PMID:27272143

  10. Cooperativity between remote sites of ectopic spiking allows afterdischarge to be initiated and maintained at different locations

    PubMed Central

    Coggan, Jay S; Sejnowski, Terrence J; Prescott, Steven A

    2015-01-01

    Many symptoms of nerve damage arise from ectopic spiking caused by hyperexcitability. Ectopic spiking can originate at the site of axonal damage and elsewhere within affected neurons. This raises the question of whether localized damage elicits cell-wide changes in excitability and/or if localized changes in excitability can drive abnormal spiking at remote locations. Computer modeling revealed an example of the latter involving afterdischarge (AD) – stimulus-evoked spiking that outlasts stimulation. We found that AD originating in a hyperexcitable region of axon could shift to the soma where it was maintained. This repositioning of ectopic spike initiation was independent of distance between the two sites but relied on the rate and number of ectopic spikes originating from the first site. Nonlinear dynamical analysis of a reduced model demonstrated that properties which rendered the axonal site prone to initiating AD discouraged it from maintaining AD, whereas the soma had the inverse properties thus enabling the two sites to interact cooperatively. A first phase of AD originating in the axon could, by providing sufficient drive to trigger somatic AD, give way to a second phase of AD originating in the soma such that spiking continued when axonal AD failed. Ectopic spikes originating from the soma during phase 2 AD propagated successfully through the defunct site of axonal spike initiation. This novel mechanism whereby ectopic spiking at one site facilitates ectopic spiking at another site is likely to contribute to the chronification of hyperexcitability in conditions such as neuropathic pain. PMID:25929191

  11. Superbackscattering nanoparticle dimers.

    PubMed

    Liberal, Iñigo; Ederra, Iñigo; Gonzalo, Ramón; Ziolkowski, Richard W

    2015-07-10

    The theory and design of superbackscattering nanoparticle dimers are presented. We analytically derive the optimal configurations and the upper bound of their backscattering cross-sections. In particular, it is demonstrated that electrically small nanoparticle dimers can enhance the backscattering by a factor of 6.25 with respect to single dipolar particles. We demonstrate that optimal designs approaching this theoretical limit can be found by using a simple circuit model. The study of practical implementations based on plasmonic and high-permittivity particles has been also addressed. Moreover, the numerical examples reveal that the dimers can attain close to a fourfold enhancement of the single nanoparticle response even in the presence of high losses.

  12. Initiating a participatory action research process in the Agincourt health and socio–demographic surveillance site

    PubMed Central

    Wariri, Oghenebrume; D’Ambruoso, Lucia; Twine, Rhian; Ngobeni, Sizzy; van der Merwe, Maria; Spies, Barry; Kahn, Kathleen; Tollman, Stephen; Wagner, Ryan G; Byass, Peter

    2017-01-01

    Background Despite progressive health policy, disease burdens in South Africa remain patterned by deeply entrenched social inequalities. Accounting for the relationships between context, health and risk can provide important information for equitable service delivery. The aims of the research were to initiate a participatory research process with communities in a low income setting and produce evidence of practical relevance. Methods We initiated a participatory action research (PAR) process in the Agincourt health and socio–demographic surveillance site (HDSS) in rural north–east South Africa. Three village–based discussion groups were convened and consulted about conditions to examine, one of which was under–5 mortality. A series of discussions followed in which routine HDSS data were presented and participants’ subjective perspectives were elicited and systematized into collective forms of knowledge using ranking, diagramming and participatory photography. The process concluded with a priority setting exercise. Visual and narrative data were thematically analyzed to complement the participants’ analysis. Results A range of social and structural root causes of under–5 mortality were identified: poverty, unemployment, inadequate housing, unsafe environments and shortages of clean water. Despite these constraints, single mothers were often viewed as negligent. A series of mid–level contributory factors in clinics were also identified: overcrowding, poor staffing, delays in treatment and shortages of medications. In a similar sense, pronounced blame and negativity were directed toward clinic nurses in spite of the systems constraints identified. Actions to address these issues were prioritized as: expanding clinics, improving accountability and responsiveness of health workers, improving employment, providing clean water, and expanding community engagement for health promotion. Conclusions We initiated a PAR process to gain local knowledge and

  13. Initiating a participatory action research process in the Agincourt health and socio-demographic surveillance site.

    PubMed

    Wariri, Oghenebrume; D'Ambruoso, Lucia; Twine, Rhian; Ngobeni, Sizzy; van der Merwe, Maria; Spies, Barry; Kahn, Kathleen; Tollman, Stephen; Wagner, Ryan G; Byass, Peter

    2017-06-01

    Despite progressive health policy, disease burdens in South Africa remain patterned by deeply entrenched social inequalities. Accounting for the relationships between context, health and risk can provide important information for equitable service delivery. The aims of the research were to initiate a participatory research process with communities in a low income setting and produce evidence of practical relevance. We initiated a participatory action research (PAR) process in the Agincourt health and socio-demographic surveillance site (HDSS) in rural north-east South Africa. Three village-based discussion groups were convened and consulted about conditions to examine, one of which was under-5 mortality. A series of discussions followed in which routine HDSS data were presented and participants' subjective perspectives were elicited and systematized into collective forms of knowledge using ranking, diagramming and participatory photography. The process concluded with a priority setting exercise. Visual and narrative data were thematically analyzed to complement the participants' analysis. A range of social and structural root causes of under-5 mortality were identified: poverty, unemployment, inadequate housing, unsafe environments and shortages of clean water. Despite these constraints, single mothers were often viewed as negligent. A series of mid-level contributory factors in clinics were also identified: overcrowding, poor staffing, delays in treatment and shortages of medications. In a similar sense, pronounced blame and negativity were directed toward clinic nurses in spite of the systems constraints identified. Actions to address these issues were prioritized as: expanding clinics, improving accountability and responsiveness of health workers, improving employment, providing clean water, and expanding community engagement for health promotion. We initiated a PAR process to gain local knowledge and prioritize actions. The process was acceptable to those

  14. Succession on regraded placer mine spoil in Alaska, USA, in relation to initial site characteristics

    USGS Publications Warehouse

    Densmore, R.V.

    1994-01-01

    This study evaluated the rate and pattern of natural succession on regraded placer mine spoil in relation to initial substrate characteristics. The study site was the Glen Creek watershed of the Kantishna mining area of Denali National Park and Preserve, Alaska. After regrading, twelve 0.01-ha plots were established and substrate characteristics were measured. Natural plant succession was evaluated after five growing seasons. Three successional patterns were identified on the basis of plant community characteristics using cluster analysis, and were related to substrate characteristics. First, a riparian plant community with vigorous Salix alaxensis and Alnus crispa grew rapidly on topsoil that had been spread over the regraded spoil. Second, a similar plant community with less vigorous S. alaxensis developed more slowly on unprocessed spoil and spoil amended with a small amount of topsoil. Third, processed spoil remained almost bare of vegetation, although S. alaxensis was able to establish and persist in a stunted growth form. In contrast, Alnus crispa had difficulty establishing on processed spoil, but the few established seedlings grew well. Several substrate variables, including the proportion of silt and clay vs. sand, total nitrogen, and water retention capacity, were good predictors of the rate and pattern of succession. Total nitrogen was the best single predictor for the number of vigorous S. alaxensis.

  15. Identification of the transcriptional initiation site of ribosomal RNA genes in the crustacean Artemia.

    PubMed Central

    Gil, I; Gallego, M E; Renart, J; Cruces, J

    1987-01-01

    The proximal part of the Intergenic Spacer, as well as most of the External Transcribed Spacer of the ribosomal RNA type I genes from the crustacean Artemia have been sequenced. We have identified in the Intergenic Spacer five repeats of around 600 bp in length and, possibly, two imperfect or truncated repeats, derived from the principal ones. These sequences are separated by 485 bp from the 17S rRNA coding sequence. We have also identified the start point of transcription by S1 nuclease analysis. This start point is found 248 bp inside the first repeat. The sequence around the start point shows homology with that described for other members of the same phylum, mostly insects. The most conserved regions are from -1 to +25, and the G residue at position -16. At least the three 600-bp repeats upstream from that containing the promoter also contain the start point sequence, and could therefore act as initiation sites for snPIRNA and/or as enhancer sequences for ribosomal RNA gene transcription. Images PMID:3627976

  16. Succession on regraded placer mine spoil in Alaska, USA, in relation to initial site characteristics

    USGS Publications Warehouse

    Densmore, R.V.

    1994-01-01

    This study evaluated the rate and pattern of natural succession on regraded placer mine spoil in relation to initial substrate characteristics. The study site was the Glen Creek watershed of the Kantishna mining area of Denali National Park and Preserve, Alaska. After regrading, twelve 0.01-ha plots were established and substrate characteristics were measured. Natural plant succession was evaluated after five growing seasons. Three successional patterns were identified on the basis of plant community characteristics using cluster analysis, and were related to substrate characteristics. First, a riparian plant community with vigorous Salix alaxensis and Alnus crispa grew rapidly on topsoil that had been spread over the regraded spoil. Second, a similar plant community with less vigorous S. alaxensis developed more slowly on unprocessed spoil and spoil amended with a small amount of topsoil. Third, processed spoil remained almost bare of vegetation, although S. alaxensis was able to establish and persist in a stunted growth form. In contrast, Alnus crispa had difficulty establishing on processed spoil, but the few established seedlings grew well. Several substrate variables, including the proportion of silt and clay vs. sand, total nitrogen, and water retention capacity, were good predictors of the rate and pattern of succession. Total nitrogen was the best single predictor for the number of vigorous S. alaxensis.

  17. Initialization of a spin qubit in a site-controlled nanowire quantum dot

    NASA Astrophysics Data System (ADS)

    Lagoudakis, Konstantinos G.; McMahon, Peter L.; Fischer, Kevin A.; Puri, Shruti; Müller, Kai; Dalacu, Dan; Poole, Philip J.; Reimer, Michael E.; Zwiller, Val; Yamamoto, Yoshihisa; Vučković, Jelena

    2016-05-01

    A fault-tolerant quantum repeater or quantum computer using solid-state spin-based quantum bits will likely require a physical implementation with many spins arranged in a grid. Self-assembled quantum dots (QDs) have been established as attractive candidates for building spin-based quantum information processing devices, but such QDs are randomly positioned, which makes them unsuitable for constructing large-scale processors. Recent efforts have shown that QDs embedded in nanowires can be deterministically positioned in regular arrays, can store single charges, and have excellent optical properties, but so far there have been no demonstrations of spin qubit operations using nanowire QDs. Here we demonstrate optical pumping of individual spins trapped in site-controlled nanowire QDs, resulting in high-fidelity spin-qubit initialization. This represents the next step towards establishing spins in nanowire QDs as quantum memories suitable for use in a large-scale, fault-tolerant quantum computer or repeater based on all-optical control of the spin qubits.

  18. Initial Experience with Electronic Tracking of Specific Tumor Sites in Men Undergoing Active Surveillance of Prostate Cancer

    PubMed Central

    Sonn, Geoffrey A.; Filson, Christopher P.; Chang, Edward; Natarajan, Shyam; Margolis, Daniel J.; Macairan, Malu; Lieu, Patricia; Huang, Jiaoti; Dorey, Frederick J.; Reiter, Robert E.; Marks, Leonard S.

    2014-01-01

    Objectives Targeted biopsy, using magnetic resonance (MR) – ultrasound (US) fusion, may allow tracking of specific cancer sites in the prostate. We aimed to evaluate initial use of the technique to follow tumor sites in men on active surveillance of prostate cancer. Methods and Materials Fifty-three men with prostate cancer (all T1c) underwent re-biopsy of 74 positive biopsy sites, which were tracked and targeted using the Artemis MR-US fusion device (Eigen, Grass Valley, CA, USA) from March 2010 through January 2013. The initial biopsy included 12 cores from a standard template (mapped by software) and directed biopsies from regions of interest seen on MRI. In the repeat biopsy, samples were taken from sites containing cancer at the initial biopsy. Outcomes of interest at second MR-US biopsy included (a) presence of any cancer and (b) presence of clinically significant cancer. Results All cancers on initial biopsy were either Gleason score 3+3=6 (N=63) or 3+4=7 (N=11). At initial biopsy, 23 cancers were within an MRI target, and 51 were found on systematic biopsy. Cancer detection rate on repeat biopsy (29/74, 39%) was independent of Gleason score on initial biopsy (p=NS) but directly related to initial cancer core length (CCL) (p<0.02). Repeat sampling of cancerous sites within MRI targets was more likely to show cancer than re-sampling of tumorous systematic sites (61% vs. 29%, p=0.005). When initial CCL was ≥4 mm within an MRI target, over 80% (5/6) of follow-up tracking biopsies were positive. An increase of Gleason score was uncommon (9/74, 12%). Conclusions Monitoring of specific prostate cancer-containing sites may be achieved in some men using an electronic tracking system. The chances of finding tumor on repeat specific-site sampling was directly related to the length of tumor in the initial biopsy core and presence of tumor within an MRI target; upgrading of Gleason score was uncommon. Further research is required to evaluate the potential utility of

  19. Structure of Active, Dimeric Human Telomerase

    PubMed Central

    Sauerwald, Anselm; Sandin, Sara; Cristofari, Gaël; Scheres, Sjors H.W.; Lingner, Joachim; Rhodes, Daniela

    2013-01-01

    Telomerase contains a large RNA subunit TER and a protein catalytic subunit TERT. Whether telomerase functions as monomer or dimer has been a matter of debate. Here we report biochemical and labeling data that show that in vivo assembled human telomerase contains two TERT subunits and binds two telomeric DNA substrates. Importantly, catalytic activity requires both TERT active sites to be functional, demonstrating that human telomerase functions as a dimer. We also present the three-dimensional structure of active, full-length human telomerase dimer, determined by single-particle electron microscopy in negative stain. Telomerase has a bilobal architecture, with the two monomers linked by a flexible interface. The monomer reconstruction at 23Å resolution, and fitting of the atomic structure of the beetle TERT subunit reveals the spatial relationship between RNA and protein subunits, providing insights into the telomerase architecture. PMID:23474713

  20. A Novel Quality Measure and Correction Procedure for the Annotation of Microbial Translation Initiation Sites

    PubMed Central

    Overmars, Lex; Siezen, Roland J.; Francke, Christof

    2015-01-01

    The identification of translation initiation sites (TISs) constitutes an important aspect of sequence-based genome analysis. An erroneous TIS annotation can impair the identification of regulatory elements and N-terminal signal peptides, and also may flaw the determination of descent, for any particular gene. We have formulated a reference-free method to score the TIS annotation quality. The method is based on a comparison of the observed and expected distribution of all TISs in a particular genome given prior gene-calling. We have assessed the TIS annotations for all available NCBI RefSeq microbial genomes and found that approximately 87% is of appropriate quality, whereas 13% needs substantial improvement. We have analyzed a number of factors that could affect TIS annotation quality such as GC-content, taxonomy, the fraction of genes with a Shine-Dalgarno sequence and the year of publication. The analysis showed that only the first factor has a clear effect. We have then formulated a straightforward Principle Component Analysis-based TIS identification strategy to self-organize and score potential TISs. The strategy is independent of reference data and a priori calculations. A representative set of 277 genomes was subjected to the analysis and we found a clear increase in TIS annotation quality for the genomes with a low quality score. The PCA-based annotation was also compared with annotation with the current tool of reference, Prodigal. The comparison for the model genome of Escherichia coli K12 showed that both methods supplement each other and that prediction agreement can be used as an indicator of a correct TIS annotation. Importantly, the data suggest that the addition of a PCA-based strategy to a Prodigal prediction can be used to ‘flag’ TIS annotations for re-evaluation and in addition can be used to evaluate a given annotation in case a Prodigal annotation is lacking. PMID:26204119

  1. Laparoendoscopic single site surgery for extravesical repair of vesicovaginal fistula using conventional instruments: Our initial experience

    PubMed Central

    Mahadevappa, Nagabhushana; Gudage, Swathi; Senguttavan, Karthikeyan V.; Mallya, Ashwin; Dharwadkar, Sachin

    2016-01-01

    Objective: Vesicovaginal fistula (VVF) is a major complication with psychosocial ramifications. In literature, few VVF cases have been managed by laparoendoscopic single site surgery (LESS) and for the 1st time we report VVF repair by LESS using conventional laparoscopic instruments. We present our initial experience and to assess its feasibility, safety and outcome. Patients and Methods: From March 2012 to September 2015, LESS VVF repair was done for ten patients aged between 30 and 65 (45.6 ± 10.15) years, who presented with supratrigonal VVF. LESS was performed by modified O’Conor technique using regular trocars with conventional instruments. Data were collected regarding feasibility, intra- or post-operative pain, analgesic requirement, complication, and recovery. Results: All 10 cases were completed successfully, without conversion to a standard laparoscopic or open approach. The mean operative time was 182.5 ± 32.25 (150–250) min. The mean blood loss was 100 mL. The respective mean visual analog score for pain on day 1, 2, and 3 was 9.2 ± 1, 5 ± 1, and 1.4 ± 2.3. The analgesic requirement in the form of intravenous tramadol on days 1, 2, and 3 was 160 ± 51.6, 80 ± 63.2, and 30 ± 48.3, mgs respectively. No major intra- or post-operative complications were observed. The mean hospital stay was 2.6 ± 0.7 (2–4) days. Conclusion: In select patients, LESS extravesical repair of VVF using conventional laparoscopic instruments is safe, feasible with all the advantages of single port surgery at no added cost. Additional experience and comparative studies with conventional laparoscopy are warranted. PMID:27453652

  2. PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

    PubMed Central

    Reuter, Kerstin; Helms, Volkhard

    2016-01-01

    Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

  3. A Link between Dimerization and Autophosphorylation of the Response Regulator PhoB*

    PubMed Central

    Creager-Allen, Rachel L.; Silversmith, Ruth E.; Bourret, Robert B.

    2013-01-01

    Response regulator proteins within two-component signal transduction systems are activated by phosphorylation and can catalyze their own covalent phosphorylation using small molecule phosphodonors. To date, comprehensive kinetic characterization of response regulator autophosphorylation is limited to CheY, which follows a simple model of phosphodonor binding followed by phosphorylation. We characterized autophosphorylation of the response regulator PhoB, known to dimerize upon phosphorylation. In contrast to CheY, PhoB time traces exhibited an initial lag phase and gave apparent pseudo-first order rate constants that increased with protein concentration. Furthermore, plots of the apparent autophosphorylation rate constant versus phosphodonor concentration were sigmoidal, as were PhoB binding isotherms for the phosphoryl group analog BeF3−. Successful mathematical modeling of the kinetic data necessitated inclusion of the formation of a PhoB heterodimer (one phosphorylated and one unphosphorylated monomer) with an enhanced rate of phosphorylation. Specifically, dimerization constants for the PhoB heterodimer and homodimer (two phosphorylated monomers) were similar, but the rate constant for heterodimer phosphorylation was ∼10-fold higher than for the monomer. In a test of the model, disruption of the known PhoBN dimerization interface by mutation led to markedly slower and noncooperative autophosphorylation kinetics. Furthermore, phosphotransfer from the sensor kinase PhoR was enhanced by dimer formation. Phosphorylation-mediated dimerization allows many response regulators to bind to tandem DNA-binding sites and regulate transcription. Our data challenge the notion that response regulator dimers primarily form between two phosphorylated monomers and raise the possibility that response regulator heterodimers containing one phosphoryl group may participate in gene regulation. PMID:23760278

  4. Quantum Dimer Model: Phase Diagrams

    NASA Astrophysics Data System (ADS)

    Goldstein, Garry; Chamon, Claudio; Castelnovo, Claudio

    We present new theoretical analysis of the Quantum Dimer Model. We study dimer models on square, cubic and triangular lattices and we reproduce their phase diagrams (which were previously known only numerically). We show that there are several types of dimer liquids and solids. We present preliminary analysis of several other models including doped dimers and planar spin ice, and some results on the Kagome and hexagonal lattices.

  5. Action potentials in retinal ganglion cells are initiated at the site of maximal curvature of the extracellular potential.

    PubMed

    Eickenscheidt, Max; Zeck, Günther

    2014-06-01

    The initiation of an action potential by extracellular stimulation occurs after local depolarization of the neuronal membrane above threshold. Although the technique shows remarkable clinical success, the site of action and the relevant stimulation parameters are not completely understood. Here we identify the site of action potential initiation in rabbit retinal ganglion cells (RGCs) interfaced to an array of extracellular capacitive stimulation electrodes. We determine which feature of the extracellular potential governs action potential initiation by simultaneous stimulation and recording RGCs interfaced in epiretinal configuration. Stimulation electrodes were combined to areas of different size and were presented at different positions with respect to the RGC. Based on stimulation by electrodes beneath the RGC soma and simultaneous sub-millisecond latency measurement we infer axonal initiation at the site of maximal curvature of the extracellular potential. Stimulation by electrodes at different positions along the axon reveals a nearly constant threshold current density except for a narrow region close to the cell soma. These findings are explained by the concept of the activating function modified to consider a region of lower excitability close to the cell soma. We present a framework how to estimate the site of action potential initiation and the stimulus required to cross threshold in neurons tightly interfaced to capacitive stimulation electrodes. Our results underscore the necessity of rigorous electrical characterization of the stimulation electrodes and of the interfaced neural tissue.

  6. Action potentials in retinal ganglion cells are initiated at the site of maximal curvature of the extracellular potential

    NASA Astrophysics Data System (ADS)

    Eickenscheidt, Max; Zeck, Günther

    2014-06-01

    Objective. The initiation of an action potential by extracellular stimulation occurs after local depolarization of the neuronal membrane above threshold. Although the technique shows remarkable clinical success, the site of action and the relevant stimulation parameters are not completely understood. Approach. Here we identify the site of action potential initiation in rabbit retinal ganglion cells (RGCs) interfaced to an array of extracellular capacitive stimulation electrodes. We determine which feature of the extracellular potential governs action potential initiation by simultaneous stimulation and recording RGCs interfaced in epiretinal configuration. Stimulation electrodes were combined to areas of different size and were presented at different positions with respect to the RGC. Main results. Based on stimulation by electrodes beneath the RGC soma and simultaneous sub-millisecond latency measurement we infer axonal initiation at the site of maximal curvature of the extracellular potential. Stimulation by electrodes at different positions along the axon reveals a nearly constant threshold current density except for a narrow region close to the cell soma. These findings are explained by the concept of the activating function modified to consider a region of lower excitability close to the cell soma. Significance. We present a framework how to estimate the site of action potential initiation and the stimulus required to cross threshold in neurons tightly interfaced to capacitive stimulation electrodes. Our results underscore the necessity of rigorous electrical characterization of the stimulation electrodes and of the interfaced neural tissue.

  7. A short sequence motif in the 5' leader of the HIV-1 genome modulates extended RNA dimer formation and virus replication.

    PubMed

    van Bel, Nikki; Das, Atze T; Cornelissen, Marion; Abbink, Truus E M; Berkhout, Ben

    2014-12-19

    The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.

  8. Palladium dimers adsorbed on graphene: A DFT study

    NASA Astrophysics Data System (ADS)

    Kaur, Gagandeep; Gupta, Shuchi; Dharamvir, Keya

    2015-05-01

    The 2D structure of graphene shows a great promise for enhanced catalytic activity when adsorbed with palladium. We performed a systematic density functional theory (DFT) study of the adsorption of palladium dimer (Pd2) on graphene using SIESTA package, in the generalized gradient approximation (GGA). The adsorption energy, geometry, and charge transfer of Pd2-graphene system are calculated. Both horizontal and vertical orientations of Pd2 on graphene are studied. Our calculations revealed that the minimum energy configuration for Pd dimer is parallel to the graphene sheet with its two atoms occupying centre of adjacent hexagonal rings of graphene sheet. Magnetic moment is induced for Pd dimer adsorbed on graphene in vertical orientation while horizontal orientation of Pd dimer on graphene do not exhibit magnetism. Insignificant energy differences among adsorption sites means that dimer mobility on the graphene sheet is high. There is imperceptible distortion of graphene sheet perpendicular to its plane. However, some lateral displacements are seen.

  9. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP IRON AND STEEL MILL SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    This document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  10. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP METAL FINISHING SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    The document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  11. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP AUTOMOTIVE REPAIR SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    The document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  12. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP AUTOMOTIVE REPAIR SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    The document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  13. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP METAL FINISHING SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    The document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  14. TECHNICAL APPROACHES TO CHARACTERIZING AND CLEANING UP IRON AND STEEL MILL SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    This document provides brownfields planners with an overview of the technical methods that can be used to achieve successful site assessment and cleanup which are two key components of the brownfields redevelopment process. No two brownfields sites are identical and planners will...

  15. Single-molecule force measurements of the polymerizing dimeric subunit of von Willebrand factor

    NASA Astrophysics Data System (ADS)

    Wijeratne, Sithara S.; Li, Jingqiang; Yeh, Hui-Chun; Nolasco, Leticia; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Moake, Joel L.; Dong, Jing-fei; Kiang, Ching-Hwa

    2016-01-01

    Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress (100 dynes/cm2 for 3 min at 37°C ). We conclude that under the shear conditions used (100 dynes/cm2 for 3 min at 37°C ) , VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

  16. GE23077 binds to the RNA polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.

    PubMed

    Zhang, Yu; Degen, David; Ho, Mary X; Sineva, Elena; Ebright, Katherine Y; Ebright, Yon W; Mekler, Vladimir; Vahedian-Movahed, Hanif; Feng, Yu; Yin, Ruiheng; Tuske, Steve; Irschik, Herbert; Jansen, Rolf; Maffioli, Sonia; Donadio, Stefano; Arnold, Eddy; Ebright, Richard H

    2014-04-22

    Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001.

  17. Recombination Can Initiate and Terminate at a Large Number of Sites within the Rosy Locus of Drosophila Melanogaster

    PubMed Central

    Clark, S. H.; Hilliker, A. J.; Chovnick, A.

    1988-01-01

    This report presents the results of a recombination experiment designed to question the existence of special sites for the initiation or termination of a recombination heteroduplex within the region of the rosy locus. Intragenic recombination events were monitored between two physically separated rosy mutant alleles ry(301) and ry(2) utilizing DNA restriction site polymorphisms as genetic markers. Both ry(301) and ry(2) are known from previous studies to be associated with gene conversion frequencies an order of magnitude lower than single site mutations. The mutations are associated with large, well defined insertions located as internal sites within the locus in prior intragenic mapping studies. On the molecular map, they represent large insertions approximately 2.7 kb apart in the second and third exons, respectively, of the XDH coding region. The present study monitors intragenic recombination in a mutant heterozygous genotype in which DNA homology is disrupted by these large discontinuities, greater than the region of DNA homology and flanking both sides of the locus. If initiation/or termination requires separate sites at either end of the locus, then intragenic recombination within the rosy locus of the heterozygote should be eliminated. Contrary to expectation, significant recombination between these sites is seen. PMID:2834266

  18. Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

    PubMed Central

    Langley, Alexander R.; Gräf, Stefan; Smith, James C.; Krude, Torsten

    2016-01-01

    Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. PMID:27587586

  19. Initial field trials of the site characterization and analysis penetrometer system (SCAPS). Reconnaissance of Jacksonville Naval Air Station waste oil and solvents disposal site. Final report

    SciTech Connect

    Cooper, S.S.; Douglas, D.H.; Sharp, M.K.; Olsen, R.A.; Comes, G.D.

    1993-12-01

    At the request of the Naval Facilities Engineering Command (NAVFAC), Southern Division, Charleston, SC, the U.S. Army Engineer Waterways Experiment Station (WES) conducted the initial field trial of the Site Characterization and Analysis Penetrometer System (SCAPS) at Jacksonville Naval Air Station (NAS), Jacksonville FL. This work was carried out by a field crew consisting of personnel from WES and the Naval Ocean Systems Center during the period of 16 July 1990 to 14 August 1990. The SCAPS investigation at the Jacksonville NAS has two primary objectives: (a) to provide data that could be useful in formulating remediation plans for the facility and (b) to provide for the initial field trial of the SCAPS currently under development by WES for the U.S. Army Toxic and Hazardous Materials Agency (USATHAMA), now the U.S. Army Environmental Center. The original concepts for the SCAPS was to develop an integrated site screening characterization system whose capabilities would include (a) surface mapping, (b) geophysical surveys using magnetic, induced electromagnetic, and radar instruments, (c) measurements of soil strength, soil electrical resistivity, and laser-induced soil fluorometry Cone penetrometer, Site Characterization and Analysis Laser Induced Fluorescence(LIF), Penetrometer System(SCAPS) POL Contamination, using screening instrumentation mounted in a soil penetrometer, (d) soil and fluid samplers, and (e) computerized data acquisition, interpretation, and visualization. The goal of the SCAPS program is to provide detailed, rapid, and cost-effective surface and subsurface data for input to site assessment/remediation efforts.

  20. COST ESTIMATING TOOLS AND RESOURCES FOR ADDRESSING SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    Brownfields redevelopment contributes to the revitalization of communities across the U.S. Reuse of these abandoned, contaminated sites spurs economic growth, builds community pride, protects public health, and helps maintain our nation's "greenfields," often at a relatively low ...

  1. COST ESTIMATING TOOLS AND RESOURCES FOR ADDRESSING SITES UNDER THE BROWNFIELDS INITIATIVE

    EPA Science Inventory

    Brownfields redevelopment contributes to the revitalization of communities across the U.S. Reuse of these abandoned, contaminated sites spurs economic growth, builds community pride, protects public health, and helps maintain our nation's "greenfields," often at a relatively low ...

  2. An Initial Evaluation of Siting Considerations on Current and Future Wind Deployment

    SciTech Connect

    Tegen, Suzanne; Lantz, Eric; Mai, Trieu; Heimiller, Donna; Hand, Maureen; Ibanez, Eduardo

    2016-07-01

    This report provides a deeper understanding of the wind project development process, from desktop studies to a successful project in the ground. It examines three siting consideration categories that wind project sponsors must include in the development process: wildlife (species that live in, near, or migrate through the area where wind development is possible), radar (wind turbines can cause interference with radar signals), and public engagement (representing communities and stakeholders who live near wind power projects). The research shows that although this country's abundant wind resource provides numerous options for addressing siting considerations, actually siting individual projects is becoming more difficult because of regulatory and other uncertainties. Model results are based on the premise that developers will be able to site, permit, and build successful projects, which is not always the case in reality.

  3. Mapping of the Neisseria meningitidis NadA Cell-Binding Site: Relevance of Predicted α-Helices in the NH2-Terminal and Dimeric Coiled-Coil Regions▿

    PubMed Central

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele

    2011-01-01

    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH2-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH2-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH2 globular head domain and the NH2 dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen. PMID:20971901

  4. Mapping of the Neisseria meningitidis NadA cell-binding site: relevance of predicted {alpha}-helices in the NH2-terminal and dimeric coiled-coil regions.

    PubMed

    Tavano, Regina; Capecchi, Barbara; Montanari, Paolo; Franzoso, Susanna; Marin, Oriano; Sztukowska, Maryta; Cecchini, Paola; Segat, Daniela; Scarselli, Maria; Aricò, Beatrice; Papini, Emanuele

    2011-01-01

    NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH(2)-terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH(2)-terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH(2) globular head domain and the NH(2) dimeric intrachain coiled-coil α-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.

  5. Unique properties of human β-defensin 6 (hBD6) and glycosaminoglycan complex: sandwich-like dimerization and competition with the chemokine receptor 2 (CCR2) binding site.

    PubMed

    De Paula, Viviane S; Pomin, Vitor H; Valente, Ana Paula

    2014-08-15

    Defensins are components of the innate immune system that promote the directional migration and activation of dendritic cells, thereby modulating the adaptive immune response. Because matrix glycosaminoglycan (GAG) is known to be important for these functions, we characterized the structural features of human β-defensin 6 (hBD6) and GAG interaction using a combination of structural and in silico analyses. Our results showed that GAG model compounds, a pentasaccharide (fondaparinux, FX) and an octasaccharide heparin derivative (dp8) bind to the α-helix and in the loops between the β2 and β3 strands, inducing the formation of a ternary complex with a 2:1 hBD6:FX stoichiometry. Competition experiments indicated an overlap of GAG and chemokine receptor CCR2 binding sites. An NMR-derived model of the ternary complex revealed that FX interacts with hBD6 along the dimerization interface, primarily contacting the α-helices and β2-β3 loops from each monomer. We further demonstrated that high-pressure NMR spectroscopy could capture an intermediate stage of hBD6-FX interaction, exhibiting features of a cooperative binding mechanism. Collectively, these data suggest a "sandwich-like" model in which two hBD6 molecules bind a single FX chain and provide novel structural insights into how defensin orchestrates leukocyte recruitment through GAG binding and G protein-coupled receptor activation. Despite the similarity to chemokines and hBD2, our data indicate different properties for the hBD6-GAG complex. This work adds significant information to the currently limited data available for the molecular structures and dynamics of defensin carbohydrate binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase: Characterization of the Dimer Interface

    SciTech Connect

    Yang,M.; Horii, K.; Herr, A.; Kirley, T.

    2006-01-01

    Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile{sup 170}, Ser{sup 172}, and Ser{sup 226} with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys{sup 30}, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

  7. Dynamical dimer-dimer correlation functions from exact diagonalization

    SciTech Connect

    Werner, Ralph

    2001-05-01

    A regularization method is presented to deduce dynamic correlation functions from exact diagonalization calculations. It is applied to dimer-dimer correlation functions in quantum spin chains relevant for the description of spin-Peierls systems. Exact results for the XY model are presented. The analysis draws into doubt that the dimer-dimer correlation functions show the same scale invariance as spin-spin correlation functions. The results are applied to describe the quasielastic scattering in CuGeO{sub 3} and the hardening of the Peierls-active phonons.

  8. Identification of a RNA Polymerase II Initiation Site in the Long Terminal Repeat of Moloney Murine Leukemia Viral DNA

    NASA Astrophysics Data System (ADS)

    Fuhrman, Shella A.; van Beveren, Charles; Verma, Inder M.

    1981-09-01

    We have used a soluble in vitro RNA polymerase II transcription system to define the site of initiation of Moloney murine leukemia viral RNA synthesis. Molecularly cloned integrated and unintegrated Moloney murine leukemia virus DNAs were used as templates. The 5' ends of in vitro transcripts and virion RNA of Moloney murine leukemia virus were compared by nuclease S1 protection experiments. Our results indicate that viral sequences upstream of the in vivo cap site are implicated in the transcription of viral RNA and that the 5' end of an in vitro transcript derived from an integrated Moloney murine leukemia virus clone corresponds to the 5' end of viral genomic RNA.

  9. Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

    PubMed Central

    Aregger, Michael; Cowling, Victoria H.

    2013-01-01

    Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation. PMID:23863084

  10. Initial atmospheric-dispersion modeling in support of the multiple-site incineration study

    SciTech Connect

    Holton, G. A.; Little, C. A.; O'Donnell, F. R.; Etnier, E. L.; Travis, C. C.

    1982-04-01

    Several modeling series which estimate population exposure to stack emissions from incineration of hazardous organic materials at 22 commercial incinerator sites are presented. These modeling series can be divided into three groups. One group estimates long- and short-term atmospheric concentrations and population exposures for each of 22 sites. These modeling predictions can be used to assess chronic and acute exposure. The second group consists of sensitivity analyses which show the effect of changes in stack parameters on the number of people exposed to specified concentration levels. The third group compares concentration estimates of two atmospheric dispersion computer codes. Results of each modeling series are contained in the appendices of this report.

  11. Adsorption of silver dimer on graphene - A DFT study

    SciTech Connect

    Kaur, Gagandeep; Gupta, Shuchi; Rani, Pooja; Dharamvir, Keya

    2014-04-24

    We performed a systematic density functional theory (DFT) study of the adsorption of silver dimer (Ag{sub 2}) on graphene using SIESTA (Spanish Initiative for Electronic Simulations with Thousands of Atoms) package, in the generalized gradient approximation (GGA). The adsorption energy, geometry, and charge transfer of Ag2-graphene system are calculated. The minimum energy configuration for a silver dimer is parallel to the graphene sheet with its two atoms directly above the centre of carbon-carbon bond. The negligible charge transfer between the dimer and the surface is also indicative of a weak bond. The methodology demonstrated in this paper may be applied to larger silver clusters on graphene sheet.

  12. The acrylonitrile dimer ion

    NASA Astrophysics Data System (ADS)

    Ervasti, Henri K.; Jobst, Karl J.; Burgers, Peter C.; Ruttink, Paul J. Ae; Terlouw, Johan K.

    2007-04-01

    Large energy barriers prohibit the rearrangement of solitary acrylonitrile ions, CH2CHCN+, into their more stable hydrogen-shift isomers CH2CCNH+ or CHCH-CNH+. This prompted us to examine if these isomerizations occur by self-catalysis in acrylonitrile dimer ions. Such ions, generated by chemical ionization experiments of acrylonitrile with an excess of carbon dioxide, undergo five dissociations in the [mu]s time frame, as witnessed by peaks at m/z 53, 54, 79, 80 and 105 in their metastable ion mass spectrum. Collision experiments on these product ions, deuterium labeling, and a detailed computational analysis using the CBS-QB3 model chemistry lead to the following conclusions: (i) the m/z 54 ions are ions CH2CHCNH+ generated by self-protonation in ion-dipole stabilized hydrogen-bridged dimer ions [CH2CHCN...H-C(CN)CH2]+ and [CH2CHCN...H-C(H)C(H)CN]+; the proton shifts in these ions are associated with a small reverse barrier; (ii) dissociation of the H-bridged ions into CH2CCNH+ or CHCH-CNH+ by self-catalysis is energetically feasible but kinetically improbable: experiment shows that the m/z 53 ions are CH2CHCN+ ions, generated by back dissociation; (iii) the peaks at m/z 79, 80 and 105 correspond with the losses of HCN, C2H2 and H, respectively. The calculations indicate that these ions are generated from dimer ions that have adopted the (much more stable) covalently bound "head-to-tail" structure [CH2CHCN-C(H2)C(H)CN]+; experiments indicate that the m/z 79 (C5H5N) and m/z 105 (C6H6N2) ions have linear structures but the m/z 80 (C4H4N2) ions consist of ionized pyrimidine in admixture with its stable pyrimidine-2-ylidene isomer. Acrylonitrile is a confirmed species in interstellar space and our study provides experimental and computational evidence that its dimer radical cation yields the ionized prebiotic pyrimidine molecule.

  13. Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

    PubMed Central

    Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

    2004-01-01

    In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

  14. Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli.

    PubMed

    Zhelyabovskaya, Olga B; Berlin, Yuri A; Birikh, Klara R

    2004-03-19

    In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase).

  15. Solitary Retroperitoneal Metastasis as the Initial Site of the Relapse of Osteosarcoma Revealed by FDG PET/CT.

    PubMed

    Liu, Bin; Yang, Hua; Servaes, Sabah; Zhuang, Hongming

    2015-11-01

    Adjuvant and neoadjuvant chemotherapy has altered the metastatic pattern of osteosarcoma. Overwhelming majority of the metastases from osteosarcoma are to the lungs and to the bones. Uncommon metastases to other sites can occur but usually accompany pulmonary and skeletal metastases. Here, we describe an asymptotic 14-year-old boy with solitary retroperitoneal metastasis as the initial relapse of osteosarcoma revealed by FDG PET/CT.

  16. Parental Involvement in Active Transport to School Initiatives: A Multi-Site Case Study

    ERIC Educational Resources Information Center

    Eyler, Amy; Baldwin, Julie; Carnoske, Cheryl; Nickelson, Jan; Troped, Philip; Steinman, Lesley; Pluto, Delores; Litt, Jill; Evenson, Kelly; Terpstra, Jennifer; Brownson, Ross; Schmid, Thomas

    2008-01-01

    Background: Increasing physical activity in youth is a recommended approach to curbing the childhood obesity epidemic. One way to help increase children's daily activity is to promote active transportation to and from school (ATS). Purpose: The purpose of this case study was to explore parental perception of, and participation in, ATS initiatives.…

  17. Molecular view of 43 S complex formation and start site selection in eukaryotic translation initiation.

    PubMed

    Lorsch, Jon R; Dever, Thomas E

    2010-07-09

    A central step to high fidelity protein synthesis is selection of the proper start codon. Recent structural, biochemical, and genetic analyses have provided molecular insights into the coordinated activities of the initiation factors in start codon selection. A molecular model is emerging in which start codon recognition is linked to dynamic reorganization of factors on the ribosome and structural changes in the ribosome itself.

  18. D Dimer in acute care

    PubMed Central

    Sathe, Prachee M.; Patwa, Urvil D.

    2014-01-01

    Pulmonary embolism, Deep Vein Thrombosis (DVT) and Disseminated intravascular coagulation (DIC) are important sources of mortality and morbidity in intensive care unit (ICU). And every time D-dimer remains the the commonest investigation. Many times D-dimer is erroneously considered as a diagnostic test in above mentioned conditions. Its interpretation requires cautions. To circumvent this source of error it is necessary to understand D-dimer test and its significance in various disorder. This article review some basic details of D-dimer, condition associated with its increased level and some prognostic value in intracranial hemorrhage and gastrointestinal (GI) bleed. PMID:25337485

  19. Nariva Swamp Ramsar Site, Trinidad and Tobago (West Indies) Wetland Habitat Restoration Initiative

    Treesearch

    Montserrat Carbonell; Nadra Nathai-Gyan

    2005-01-01

    Trinidad and Tobago, a twin island nation, is the most southerly of the Caribbean islands and lies just 11 km off the coast of Venezuela, near the Orinoco delta. Trinidad, the larger of the two islands, is approximately 5,000 km² and the Nariva Swamp is located on its eastern coast (fig. 1). In 1993, this site was designated as a wetland of international...

  20. Department of the Navy Explosives Safety Site Approval Process Improvement Initiative

    DTIC Science & Technology

    2010-07-01

    software deployment and sustainment within the DON for: • Automated Site Planning Tool ( ASPT )—ESSv6 • WebSAR • Explosives Safety Database The...Measures  Administrative Record for ESA  Configuration/Data Control of facility-related ES data for ASPT The NOSSA level roll-up of the...the DDESB by 30 December 2010. At this point, the following principles are guiding the development of the implementation plan:  Use DDESB ASPT

  1. Gasless laparoendoscopic single-site surgery with abdominal wall lift in general surgery: initial experience.

    PubMed

    Zhang, Guangyong; Liu, Shaozhuang; Yu, Wenbin; Wang, Lei; Liu, Nan; Li, Feng; Hu, Sanyuan

    2011-01-01

    Laparoendoscopic single-site surgery (LESS) was motivated by the desire to make minimally invasive surgery even more "minimal." We performed gasless laparoendoscopic single-site surgery (GLESS) with abdominal wall lift (AWL) for cholecystectomy and fenestration of liver cyst. This study aims to assess the safety and feasibility of the techniques. From June to December 2009, 18 cases of gasless laparoendoscopic single-site cholecystectomy (GLESC) and 4 cases of fenestration of liver cyst (GLESF) were performed in Qilu Hospital of Shandong University, Shandong, China. Subcutaneous abdominal wall lifting system, LAP protector, flexible laparoscopes, and bent and articulating instruments were used during the procedures. Clinical data regarding patient demographics, operating time, blood loss, complications, and postoperative hospital stay were collected and analyzed retrospectively in the study. 17 cases of GLESC and 4 cases of GLESF were performed successfully, and 1 case of GLESC was converted to laparoendoscopic single-site cholecystectomy using AWL combined with low-pressure pneumoperitoneum. Mean body mass index was 23.7 ± 3.1 kg/m(2) for GLESC and 22.9 ± 1.5 kg/m(2) for GLESF. Mean operating time was 64 ± 17 min for GLESC and 101 ± 10 min for GLESF. Mean blood loss was 8 ± 3 ml for GLESC and 24 ± 11 ml for GLESF. Despite minor wound complication, no postoperative complications were observed during mean follow-up of 118 and 95 days for GLESC and GLESF, respectively. GLESS with AWL is safe and feasible for cholecystectomy and fenestration of liver cyst. The techniques provide satisfactory operative field exposure and an easier access method for LESS. Instrument collisions are greatly ameliorated both extra- and intracorporeally through use of flexible laparoscopes and bent and articulating instruments. This may prove to be a better approach for LESS techniques.

  2. di-EOS - "distributed EOS": Initial experience with split-site persistency in a production service

    NASA Astrophysics Data System (ADS)

    Peters, A. J.; Mascetti, L.; Iven, J.; Espinal Curull, X.

    2014-06-01

    In order to accommodate growing demand for storage and computing capacity from the LHC experiments, in 2012 CERN tendered for a remote computer centre. The potential negative performance implications of the geographical distance (aka network latency) within the same "site" and storage service on physics computing have been investigated. Overall impact should be acceptable, but for some access patterns might be significant. Recent EOS changes should help to mitigate the effects, but experiments may need to adjust their job parameters.

  3. Controlled synthesis of biodegradable lactide polymers and copolymers using novel in situ generated or single-site stereoselective polymerization initiators.

    PubMed

    Zhong, Zhiyuan; Dijkstra, Pieter J; Feijen, Jan

    2004-01-01

    Polylactides and their copolymers are key biodegradable polymers used widely in biomedical, pharmaceutical and ecological applications. The development of synthetic pathways and catalyst/initiator systems to produce pre-designed polylactides, as well as the fundamental understanding of the polymerization reactions, has continuously been an important topic. Here, we will address the recent advances in the ring-opening polymerization of lactides, with an emphasis on the highly versatile in situ generated initiator systems and single-site stereoselective initiators. The in situ generated initiators including in situ formed yttrium, calcium and zinc alkoxides all have been shown to bring about a rapid and living polymerization of lactides under mild conditions, which facilitated the preparation of a variety of advanced lactide-based biomaterials. For example, well-defined di- and tri-block copolymers consisting of hydrophilic poly(ethylene glycol) blocks and hydrophobic polyester blocks, which form novel biodegradable polymersomes or biodegradable thermosensitive hydrogels, have been prepared. In the past few years, significant progress has also been made in the area of stereoselective polymerization of lactides. This new generation of initiators has enabled the production of polylactide materials with novel microstructures and/or properties, such as heterotactic (--RRSSRRSSRRSS--) polylactide, crystalline syndiotactic (--RSRSRSRSRSRS--) polylactide and isotactic stereoblock (--Rn Sn Rn Sn--) polylactide, exhibiting a high melting temperature. The recently developed polymerizations using in situ generated initiators and stereoselective polymerizations have no doubt opened a brand-new avenue for the design and exploration of polylactides and their copolymers.

  4. Some Properties of Site-Specific and General Recombination Inferred from Int-Initiated Exchanges by Bacteriophage Lambda

    PubMed Central

    Echols, Harrison; Green, Linda

    1979-01-01

    The site-specific recombination at the attachment site for prophage integration might proceed by two general mechanisms: (1) a concerted reaction without a free intermediate; (2) a sequential mechanism differing from typical general recombination only by an inability of the cross-strand intermediate structure to migrate into the region of nonhomology adjacent to the attachment site. The blocked-migration, sequential model predicts frequent genetic exchange in the int xis region near the attachment site if Int-mediated recombination occurs between λ phage with homologous attachment sites. We find such additional int xis exchanges, but only at very low frequency (1% of the Int-mediated recombination). We conclude that the resolution point only rarely moves away from the initial crossover point specified by Int and, therefore, that the Int reaction is mainly concerted. We interpret the rare additional int xis recombinants as indicative of occasional branch migration from an initial Int-mediated crossover. The frequency of the rare int xis recombinants is not simply related to distance from the attachment site to an int- or xis- mutation, suggesting that the heteroduplex distance is often at least a gene in length. The frequency of these additional exchanges is also not a strong function of distance between two mutations; from this we conclude that the resolution to the observed recombinant structure in the sequential cases occurs often by mismatch repair. We have found no marked effect of mutations in the bacterial recA, recB, recC, recF, or recL genes on the frequency of the int xis recombinants; this may indicate that none of these genes specifies a product uniquely required for resolution of a cross-strand intermediate. PMID:161242

  5. Initial stages of Ba adsorption on the Si(100)-(2×1) surface at room temperature

    NASA Astrophysics Data System (ADS)

    Yao, X.; Hu, Xiaoming; Sarid, D.; Yu, Z.; Wang, J.; Marshall, D. S.; Droopad, R.; Abrokwah, J. K.; Hallmark, J. A.; Ooms, W. J.

    1999-02-01

    Scanning tunneling microscopy has been used to study the initial stages of room-temperature adsorption of Ba atoms on a clean Si(100)-(2×1) surface. It is found that most Ba atoms are located at type C defects, which stabilizes the dimer buckling in their vicinity, while the rest are located at single dimer sites on top of the Si dimer rows. Analysis of the images reveals that the Ba-Si(100) interactions are mediated by a partial charge transfer from the adsorbate to the substrate.

  6. Diffusional kinetics of SiGe Dimers on Si(100) using atom-tracking scanning tunneling microscopy

    SciTech Connect

    QIN,X.R.; SWARTZENTRUBER,BRIAN S.; LAGALLY,M.G.

    2000-06-14

    Quantitative measurements of the diffusion of adsorbed mixed Ge-Si dimers on the Si(100) surface have been made as a function of temperature using atom-tracking scanning tunneling microscopy. These mixed dimers are distinguishable from pure Si-Si dimers by their characteristic kinetics--a 180-degree rotation between two highly buckled configurations. At temperatures at which the mixed dimers diffuse, atomic-exchange events occur, in which the Ge atom in the adsorbed dimer exchanges with a substrate Si atom. Re-exchange can also occur when the diffusing Si-Si dimer revisits the original site of exchange.

  7. Alteration of the major phosphorylation site of eukaryotic protein synthesis initiation factor 4E prevents its association with the 48 S initiation complex.

    PubMed

    Joshi-Barve, S; Rychlik, W; Rhoads, R E

    1990-02-15

    Site-directed mutagenesis was used to replace the serine residue at the primary phosphorylation site of human eukaryotic initiation factor (eIF) 4E with an alanine residue. The mutated cDNA was transcribed in vitro, and the transcript was used to direct protein synthesis in a reticulocyte lysate system. The variant protein (eIF-4EAla) was retained on a 7-methylguanosine 5'-triphosphate (m7GTP)-Sepharose affinity column and was specifically eluted by m7GTP. Examination of eIF-4EAla by isoelectric focusing revealed two species which had the same pI values as the phosphorylated and nonphosphorylated forms of unaltered eIF-4E (here designated eIF-4ESer). However, conversion of unphosphorylated eIF-4EAla to the putative phosphorylated eIF-4EAla in the reticulocyte lysate system was slower than the corresponding conversion of eIF-4ESer. The possibility that the more acidic form of eIF-4EAla was due to NH2-terminal acetylation was ruled out by an experiment in which the acetyl-CoA pool of the reticulocyte lysate system was depleted with oxaloacetate and citrate synthase. The more acidic form of eIF-4EAla was, however, eliminated by treatment with calf intestine alkaline phosphatase, suggesting that it results from a second-site phosphorylation. When translation reaction mixtures were resolved on sucrose density gradients, the 35S-labeled eIF-4ESer was found on the 48 S initiation complex in the presence of guanylyl imidodiphosphate, as reported earlier (Hiremath, L.S., Hiremath, S.T., Rychlik, W., Joshi, S., Domier, L.L., and Rhoads, R.E. (1989) J. Biol. Chem. 264, 1132-1138). eIF-4EAla, by contrast, was not found on the 48 S complex, suggesting that phosphorylation of eIF-4E is necessary for it to carry out its role of transferring mRNA to the 48 S complex. Supporting this interpretation was the finding that eIF-4ESer isolated from 48 S initiation complexes consisted predominantly of the phosphorylated form.

  8. Inequivalent models of irreversible dimer filling: ``Transition state'' dependence

    NASA Astrophysics Data System (ADS)

    Nord, R. S.; Evans, J. W.

    1990-12-01

    Irreversible adsorption of diatomics on crystalline surfaces is sometimes modeled as random dimer filling of adjacent pairs of sites on a lattice. We note that this process can be implemented in two distinct ways: (i) randomly pick adjacent pairs of sites, jj', and fill jj' only if both are empty (horizontal transition state); or (ii) randomly pick a single site, j, and if j and at least one neighbor are empty, then fill j and a randomly chosen empty neighbor (vertical transition state). Here it is instructive to consider processes which also include competitive random monomer filling of single sites. We find that although saturation (partial) coverages differ little between the models for pure dimer filling, there is a significant difference for comparable monomer and dimer filling rates. We present exact results for saturation coverage behavior for a linear lattice, and estimates for a square lattice. Ramifications for simple models of CO oxidation on surfaces are indicated.

  9. Initial source and site characterization studies for the U. C. San Diego campus

    SciTech Connect

    Day, S.; Erick, F.; Heuze, F.E.; Mellors, R.; Minster, B.; Park, S.; Wagoner, J.

    1999-07-01

    The basic approach of the Campus Laboratory Collaboration (CLC) project is to combine the substantial expertise that exists within the University of California (UC) system in geology, seismology, geotechnical engineering, and structural engineering to evaluate the effects of large earthquakes on UC facilities. These estimates draw upon recent advances in hazard assessment, seismic wave propagation modeling in rocks and soils, dynamic soil testing, and structural dynamics. The UC campuses currently chosen for applications of our integrated methodology are Riverside, San Diego, and Santa Barbara. The basic procedure is first to identify possible earthquake source regions and local campus site conditions that may affect estimates of strong ground motion. Combined geological , geophysical, and geotechnical studies are conducted to characterize each campus with specific focus on the location of particular target buildings of special interest to the campus administrators. The project will then drill and log deep boreholes next to the target structure, to provide direct in-situ measurements of subsurface material properties and to install uphole and downhole 3-component seismic sensors capable of recording both weak and strong motions. The boreholes provide access to deeper materials, below the soil layers, that have relatively high seismic shear-wave velocities. Analysis of conjugate downhole and uphole records provides a basis for optimizing the representation of the low-strain response of the sites. Earthquake rupture scenarios of identified causative faults are combined with the earthquake records and nonlinear soil models to provide site-specific estimates of strong motions at the selected target locations. The predicted ground motions are then used as input to the dynamic analysis of the buildings.

  10. Site characterization of the Romanian Seismic Network stations: a national initiative and its first preliminary results

    NASA Astrophysics Data System (ADS)

    Grecu, Bogdan; Zahria, Bogdan; Manea, Elena; Neagoe, Cristian; Borleanu, Felix; Diaconescu, Mihai; Constantinescu, Eduard; Bala, Andrei

    2017-04-01

    The seismic activity in Romania is dominated by the intermediate-depth earthquakes occurring in Vrancea region, although weak to moderate crustal earthquakes are produced regularly in different areas of the country. The National Institute for Earth Physics (NIEP) built in the last years an impressive infrastructure for monitoring this activity, known as the Romanian Seismic Network (RSN). At present, RSN consists of 122 seismic stations, of which 70 have broadband velocity sensors and 42 short period sensors. One hundred and eleven stations out of 122 have accelerometer sensors collocated with velocity sensors and only 10 stations have only accelerometers. All the stations record continuously the ground motion and the data are transmitted in real-time to the Romanian National Data Center (RoNDC), in Magurele. Last year, NIEP has started a national project that addresses the characterization of all real-time seismic stations that constitute the RSN. We present here the steps that were undertaken and the preliminary results obtained since the beginning the project. The first two activities consisted of collecting all the existent technical and geological data, with emphasize on the latter. Then, we performed station noise investigations and analyses in order to characterize the noise level and estimate the resonances of the sites. The computed H/V ratios showed clear resonant peaks at different frequencies which correlate relatively well with the thickness of the sedimentary package beneath the stations. The polarization analysis of the H/V ratios indicates for some stations a strong directivity of the resonance peak which suggests possible topographic effects at the stations. At the same time, special attention was given to the estimation of the site amplification from earthquake data. The spectral ratios obtained from the analysis of more than 50 earthquakes with magnitudes (Mw) larger than 4.1 are characterized by similar resonance peaks as those obtained from

  11. Atomic-scale dynamics of atoms and dimers on the Si(001) surface

    SciTech Connect

    Swartzentruber, B.S.

    1996-12-31

    The kinetics of adsorbed Si monomers and dimers, at submonolayer coverage, are measured using scanning tunneling microscopy (STM). Si monomers are observed in empty-state STM images acquired between room temperature and 115 C. The monomers are trapped at the ends of rebonded-SB type dimer rows. When monomers thermally escape from the traps, they rapidly diffuse along the substrate dimer row until they find another unoccupied trap or return to their original trap. The binding activation barrier at isolated traps is {approximately}1.0 eV. A slightly lower barrier exists for monomers to hop between the ends of neighboring dimer rows - a process facilitating diffusion along segments of SB type steps. In addition to monomers, the interactions of adsorbed Si dimers with steps and islands on Si(001) are quantified using atom-tracking STM. Diffusing dimers are reflected from steps, sides of islands, and certain surface defect structures. Site-specific free energies are extracted from measurements of lattice-site occupation probabilities of dimers trapped between these reflecting barriers. Relative to the free energy of isolated dimers on a terrace, dimers located at the first lattice site next to SA steps and the sides of islands are bound by {approximately}0.03-0.06 eV. The binding decreases to half that at the second lattice site, and is indistinguishable from the free-terrace value at a distance of three or more lattice sites.

  12. Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination.

    PubMed

    Eriksen, Mette; Sneppen, Kim; Pedersen, Steen; Mitarai, Namiko

    2017-01-01

    Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be "hit" by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination.

  13. Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination

    PubMed Central

    Eriksen, Mette; Sneppen, Kim; Pedersen, Steen; Mitarai, Namiko

    2017-01-01

    Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be “hit” by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination. PMID:28382022

  14. Two translational initiation sites in the infB gene are used to express initiation factor IF2 alpha and IF2 beta in Escherichia coli.

    PubMed Central

    Plumbridge, J A; Deville, F; Sacerdot, C; Petersen, H U; Cenatiempo, Y; Cozzone, A; Grunberg-Manago, M; Hershey, J W

    1985-01-01

    The gene infB codes for the two forms of translational initiation factor IF2: IF2 alpha (97 300 daltons) and IF2 beta (79 700 daltons). To determine whether the two forms differ at their N terminus, purified IF2 alpha and IF2 beta were subjected to 11 or more steps of Edman degradation. The N-terminal amino acid sequences are completely different, but match perfectly the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream. A fusion was constructed between the proximal half of the infB gene and the lacZ gene lacking the region coding for the first eight amino acids. The fused gene expresses two products of 170 000 and 150 000 daltons, corresponding to the fused proteins IF2 alpha-beta-galactosidase and IF2 beta-beta-galactosidase, which confirms in vivo that the IF2 forms differ at their N terminus. A deletion of the 5'-non-translated region of the fused gene, including the Shine/Dalgarno ribosomal binding site, results in the expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This strongly suggests that IF2 beta results from independent translation rather than from a precise proteolytic cleavage of IF2 alpha. Further evidence for initiation of protein synthesis at the putative IF2 alpha and IF2 beta start sites was sought by using an in vitro dipeptide synthesis assay. A DNA fragment containing the entire infB gene was cloned into three plasmid vectors and the resulting recombinant DNAs were used as templates in assays containing fMet-tRNA and various labelled aminoacyl-tRNAs.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 2. PMID:3894004

  15. Defining the site of light perception and initiation of phototropism in Arabidopsis.

    PubMed

    Preuten, Tobias; Hohm, Tim; Bergmann, Sven; Fankhauser, Christian

    2013-10-07

    Phototropism is an adaptive response allowing plants to optimize photosynthetic light capture. This is achieved by asymmetric growth between the shaded and lit sides of the stimulated organ. In grass seedlings, the site of phototropin-mediated light perception is distinct from the site of bending; however, in dicotyledonous plants (e.g., Arabidopsis), spatial aspects of perception remain debatable. We use morphological studies and genetics to show that phototropism can occur in the absence of the root, lower hypocotyl, hypocotyl apex, and cotyledons. Tissue-specific expression of the phototropin1 (phot1) photoreceptor demonstrates that light sensing occurs in the upper hypocotyl and that expression of phot1 in the hypocotyl elongation zone is sufficient to enable a normal phototropic response. Moreover, we show that efficient phototropism occurs when phot1 is expressed from endodermal, cortical, or epidermal cells and that its local activation rapidly leads to a global response throughout the seedling. We propose that spatial aspects in the steps leading from light perception to growth reorientation during phototropism differ between grasses and dicots. These results are important to properly interpret genetic experiments and establish a model connecting light perception to the growth response, including cellular and morphological aspects. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. The Field Lysimeter Test Facility (FLTF) at the Hanford Site: Installation and initial tests

    SciTech Connect

    Gee, G.W.; Kirkham, R.R.; Downs, J.L.; Campbell, M.D.

    1989-02-01

    The objectives of this program are to test barrier design concepts and to demonstrate a barrier design that meets established performance criteria for use in isolating wastes disposed of near-surface at the Hanford Site. Specifically, the program is designed to assess how well the barriers perform in controlling biointrusion, water infiltration, and erosion, as well as evaluating interactions between environmental variables and design factors of the barriers. To assess barrier performance and design with respect to infiltration control, field lysimeters and small- and large-scale field plots are planned to test the performance of specific barrier designs under actual and modified (enhanced precipitation) climatic conditions. The Field Lysimeter Test Facility (FLTF) is located in the 600 Area of the Hanford Site just east of the 200 West Area and adjacent to the Hanford Meteorological Station. The FLTF data will be used to assess the effectiveness of selected protective barrier configurations in controlling water infiltration. The facility consists of 14 drainage lysimeters (2 m dia x 3 m deep) and four precision weighing lysimeters (1.5 m x 1.5 m x 1.7 m deep). The lysimeters are buried at grade and aligned in a parallel configuration, with nine lysimeters on each side of an underground instrument chamber. The lysimeters were filled with materials to simulate a multilayer protective barrier system. Data gathered from the FLTF will be used to compare key barrier components and to calibrate and test models for predicting long-term barrier performance.

  17. Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II.

    PubMed

    Fishburn, James; Galburt, Eric; Hahn, Steven

    2016-06-17

    Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

  18. Minimization of a Protein–DNA Dimerizer

    PubMed Central

    Stafford, Ryan L.; Arndt, Hans-Dieter; Brezinski, Mary L.; Ansari, Aseem Z.; Dervan, Peter B.

    2011-01-01

    A protein–DNA dimerizer constructed from a DNA-binding polyamide and the peptide FYPWMKG facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. The Exd binding domain can be reduced to a dipeptide WM attached to the polyamide through an ε-aminohexanoic acid linker with retention of protein–DNA dimerizer activity. Screening a library of analogues indicated that the tryptophan indole moiety is more important than methionine’s side chain or the N-terminal acetamide. Remarkably, switching the stereochemistry of the tryptophan residue (l to d) stabilizes the dimerizer•Exd•DNA ternary complex at 37 °C. These observations provide design principles for artificial transcription factors that may function in concert with the cellular regulatory circuitry. PMID:17290996

  19. GliaSite Brachytherapy Boost as Part of Initial Treatment of Glioblastoma Multiforme: A Retrospective Multi-Institutional Pilot Study

    SciTech Connect

    Welsh, James; Sanan, Abhay; Gabayan, Arash J.; Green, Sylvan B.; Lustig, Robert; Burri, Stuart; Kwong, Edmund; Stea, Baldassarre . E-mail: bstea@email.ariozna.edu

    2007-05-01

    Purpose: To report on a retrospective analysis of the cumulative experience from eight institutions using the GliaSite Radiotherapy System as a brachytherapy boost in the initial management of glioblastoma multiforme. Methods and Materials: Eight institutions provided data on 20 patients with histologically proven glioblastoma multiforme with a median age of 59 years (range, 39-76) and median Karnofsky performance scale of 80 (range, 50-100). After maximal surgical debulking, patients were treated with GliaSite brachytherapy to a median dose of 50 Gy, followed by external beam radiotherapy to a median dose of 60 Gy (range, 46-60 Gy), for a cumulative dose escalation of 110 Gy (range, 84-130 Gy). Results: The average survival for this study population was 11.4 months (range, 4-29). When the patients' survival was compared with that of historical controls according to their Radiation Therapy Oncology Group recursive partitioning analysis class, the average survival was increased by 3 months (95% confidence interval, 0.23-4.9) corresponding to a 43% increase (p = 0.033). Three patients (14%) experienced Radiation Therapy Oncology Group Grade 3 central nervous system toxicity. Of the treatment failures, 50% were >2 cm from the edge of the balloon. Conclusion: The results of this analysis have demonstrated that dose escalation (>100 Gy) with GliaSite is well tolerated and associated with minimal toxicity. Local control improved with the use of GliaSite brachytherapy. The putative survival advantage seen in this study needs to be interpreted with caution; nevertheless, the data provide sufficient justification to investigate the potential role of radiation dose escalation in conjunction with GliaSite in the initial treatment of glioblastoma multiforme.

  20. Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

    NASA Astrophysics Data System (ADS)

    Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

    2016-07-01

    Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

  1. Final report on the amended safety assessment of diisopropyl dimer dilinoleate, dicetearyl dimer dilinoleate, diisostearyl dimer dilinoleate, dioctyl dimer dilinoleate, dioctyldodecyl dimer dilinoleate, and ditridecyl dimer dilinoleate.

    PubMed

    Fiume, Monice Zondlo

    2003-01-01

    Diisopropyl Dimer Dilinoleate, Dicetearyl Dimer Dilinoleate, Diisostearyl Dimer Dilinoleate, Dioctyl Dimer Dilinoleate, Dioctyldodecyl Dimer Dilinoleate, and Ditridecyl Dimer Dilinoleate are diesters of their respective alcohols and dilinoleic acid. They function as skin-conditioning agents in a variety of cosmetic products at concentrations around 10%, but may be used at concentrations up to 53% in lipsticks. These ingredients do not absorb radiation in the ultraviolet (UV) UVA or UVB range and the only impurities expected are <0.5% dilinoleic acid, <0.1% isopropyl alcohol or <1% isostearyl alcohol, and/or small amounts of dilinoleic acid and cetearyl alcohol or octyldodecanol, depending on which diester is used. The potential skin penetration of these ingredients was evaluated using an estimate of the octanol/water partition coefficient (logP of 17.7) based on the structure of Diisopropyl Dimer Dilinoleate. This is consistent with the insolubility of these ingredients in water. Safety test data on dilinoleic acid (no adverse effects) were considered relevant because dilinoleic acid is a component of these diesters and a likely breakdown product. The acute oral and dermal LD(50) values for rats of Diisopropyl, Diisostearyl, and Dioctyldodecyl Dimer Dilinoleate were >5.0 g/kg. In a subchronic feeding study, macrophage aggregation was seen in the mesenteric lymph node at the lowest dose level (0.1% in the diet). These ingredients did not produce skin or ocular irritation in animal tests, nor were they comedogenic. Ames testing, clastogenesis in human lymphocytes in culture, and L5178Y mouse lymphoma cell forward mutations were all negative, indicating no dilinoleic acid genotoxicity. No carcinogenicity or reproductive/developmental toxicity data were available; however, structural alerts that would suggest a mutagenic or carcinogenic risk are absent. Significant reproductive/developmental toxicity or other systemic toxicity is not expected with these ingredients

  2. Depressing effect of caffeine at crayfish neuromuscular synapses II. Initial search for possible sites of action.

    PubMed

    Celenza, Kathryn M; Shugert, Elizabeth; Vélez, Samuel J

    2007-05-01

    Caffeine's unexpected depression of synaptic transmission in the superficial flexor muscle system (SFM) of Procambarus clarkii was studied by looking at three known sites of action of this drug: via adenosine and ryanodine receptors and inhibition of phosphodiesterase.1. JPs did not change in size when exposed to physiological concentrations of adenosine, suggesting that the SFM system lacks presynaptic adenosine receptors.2. JPs slightly increased in size in the presence of a phosphodiesterase inhibitor, the opposite response to that obtained with caffeine, suggesting that caffeine is not acting via this pathway.3. A calcium ionophore immediately enhanced synaptic transmission in the SFM system but when given in combination with caffeine the enhancement is reduced and declines over time.4. Serotonin enhanced synaptic transmission in the SFM system, but when given in combination with caffeine this enhancement was not observed.5. These caffeine effects are interpreted in terms of alterations to the calcium homeostatic mechanisms of the terminals.

  3. Repair of DNA-containing pyrimidine dimers

    SciTech Connect

    Grossman, L.; Caron, P.R.; Mazur, S.J.; Oh, E.Y.

    1988-08-01

    Ultraviolet light-induced pyrimidine dimers in DNA are recognized and repaired by a number of unique cellular surveillance systems. The most direct biochemical mechanism responding to this kind of genotoxicity involves direct photoreversal by flavin enzymes that specifically monomerize pyrimidine:pyrimidine dimers monophotonically in the presence of visible light. Incision reactions are catalyzed by a combined pyrimidine dimer DNA-glycosylase:apyrimidinic endonuclease found in some highly UV-resistant organisms. At a higher level of complexity, Escherichia coli has a uvr DNA repair system comprising the UvrA, UvrB, and UvrC proteins responsible for incision. There are several preincision steps governed by this pathway, which includes an ATP-dependent UvrA dimerization reaction required for UvrAB nucleoprotein formation. This complex formation driven by ATP binding is associated with localized topological unwinding of DNA. This same protein complex can catalyze an ATPase-dependent 5'----3'-directed strand displacement of D-loop DNA or short single strands annealed to a single-stranded circular or linear DNA. This putative translocational process is arrested when damaged sites are encountered. The complex is now primed for dual incision catalyzed by UvrC. The remainder of the repair process involves UvrD (helicase II) and DNA polymerase I for a coordinately controlled excision-resynthesis step accompanied by UvrABC turnover. Furthermore, it is proposed that levels of repair proteins can be regulated by proteolysis. UvrB is converted to truncated UvrB* by a stress-induced protease that also acts at similar sites on the E. coli Ada protein. Although UvrB* can bind with UvrA to DNA, it cannot participate in helicase or incision reactions. It is also a DNA-dependent ATPase.21 references.

  4. Implementation of a referral to discharge glycemic control initiative for reduction of surgical site infections in gynecologic oncology patients.

    PubMed

    Hopkins, Laura; Brown-Broderick, Jennifer; Hearn, James; Malcolm, Janine; Chan, James; Hicks-Boucher, Wendy; De Sousa, Filomena; Walker, Mark C; Gagné, Sylvain

    2017-08-01

    To evaluate the frequency of surgical site infections before and after implementation of a comprehensive, multidisciplinary perioperative glycemic control initiative. As part of a CUSP (Comprehensive Unit-based Safety Program) initiative, between January 5 and December 18, 2015, we implemented comprehensive, multidisciplinary glycemic control initiative to reduce SSI rates in patients undergoing major pelvic surgery for a gynecologic malignancy ('Group II'). Key components of this quality of care initiative included pre-operative HbA1c measurement with special triage for patients meeting criteria for diabetes or pre-diabetes, standardization of available intraoperative insulin choices, rigorous pre-op/intra-op/post-op glucose monitoring with control targets set to maintain BG ≤10mmol/L (180mg/dL) and communication/notification with primary care providers. Effectiveness was evaluated against a similar control group of patients ('Group I') undergoing surgery in 2014 prior to implementation of this initiative. We studied a total of 462 patients. Subjects in the screened (Group II) and comparison (Group I) groups were of similar age (avg. 61.0, 60.0years; p=0.422) and BMI (avg. 31.1, 32.3kg/m(2); p=0.257). Descriptive statistics served to compare surgical site infection (SSI) rates and other characteristics across groups. Women undergoing surgery prior to implementation of this algorithm (n=165) had an infection rate of 14.6%. Group II (n=297) showed an over 2-fold reduction in SSI compared to Group I [5.7%; p=0.001, adjRR: 0.45, 95% CI: (0.25, 0.81)]. Additionally, approximately 19% of Group II patients were newly diagnosed with either prediabetes (HbA1C 6.0-6.4) or diabetes (HbA1C≥6.5) and were referred to family or internal medicine for appropriate management. Implementation of a comprehensive multidisciplinary glycemic control initiative can lead to a significant reduction in surgical site infections in addition to early identification of an important health

  5. Crosslinking Evidence for Motional Constraints within Chemoreceptor Trimers of Dimers

    PubMed Central

    Massazza, Diego A.; Parkinson, John S.; Studdert, Claudia A.

    2011-01-01

    Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In E. coli, chemoreceptors show higher order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer of dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo crosslinking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed crosslinks, whereas reporters lying at different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernable effect on the crosslinking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced crosslinking at most of the reporter sites, indicating that individual dimers may move closer together under this condition. PMID:21174433

  6. Slow assembly and disassembly of lambda Cro repressor dimers.

    PubMed

    Jia, Haifeng; Satumba, W John; Bidwell, Gene L; Mossing, Michael C

    2005-07-29

    Dimers of Cro are required to recognize operator DNA and repress transcription, but dimerization is weak compared to DNA binding. Fluorophore-conjugated, single-cysteine variants of Cro have been used to investigate the equilibria and kinetics of dimer assembly. Equilibrium distributions of mixed dimers, monitored by fluorescence resonance energy transfer (FRET), confirm that labeled variants have equilibrium dimer dissociation constants in the micromolar concentration range. Subunit exchange experiments yield first order rate constants for dimer dissociation that range from 0.02 s(-1) to 0.04 s(-1). Association rate constants calculated from the ratios of dissociation equilibrium and rate constants range from 0.7x10(4) M(-1) s(-1) to 3x10(4) M(-1) s(-1), depending on the site of the fluorescent label. At nanomolar concentrations of subunits, assembly can be driven by addition of DNA. The bimolecular association rate constants measured under these conditions are not dramatically enhanced, ranging from 7x10(4) M(-1) s(-1) to 9x10(4) M(-1) s(-1). The association rate is second order in protein but independent of DNA concentration between 10 nM and 200 nM. The association of subunits under native conditions is more than four orders of magnitude slower than the fast assembly phase measured previously in refolding experiments, and is unaffected by peptidyl-prolyl isomerases. Stabilization of the folded structure of the protein by residue substitution in Cro F58W or reduced temperature increases the ratio of dimers to monomers and decreases the rate of subunit exchange. These data suggest that native monomers have compact structures with substantial barriers to unfolding and that unfolded or partially folded monomers are the preferred substrates for dimer assembly. Cro binding in vivo may be under kinetic rather than thermodynamic control. The slow assembly of Cro dimers demonstrated here provides a new perspective on the lysis/lysogeny switch of bacteriophage lambda.

  7. Effects of Dimerization of Serratia marcescens Endonuclease on Water Dynamics.

    SciTech Connect

    Chen, Chuanying; Beck, Brian W.; Krause, Kurt; Weksberg, Tiffany E.; Pettitt, Bernard M.

    2007-02-15

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.

  8. Quantitation of cyclobutane pyrimidine dimer formation in double- and single-stranded DNA fragments of defined sequence

    SciTech Connect

    Gordon, L.K., Haseltine, W.A.

    1982-01-01

    The distribution of cyclobutane pyrimidine dimers in defined sequences of ultraviolet light-irradiated DNA was determined. The results demonstrate that the extent of dimer formation at a potential dimer site as a function of the dose reaches a steady-state level for all dimers at doses above 2000/Jm/sup 2/. The steady-state level is primarily dependent upon the composition of the dimer, varying from a maximum of about 10% dimer formation at sites of adjacent thymines to less than 1% for sites of adjacent cytosines. The extent of dimer formation is also affected by the two bases that immediately flank the potential dimer site as well as by longer-range sequence effects. The rates of dimer formation and the steady-state levels at most dimers are similar in single- and double-stranded DNA. The dose rate of irradiation does not affect the distribution of pyrimidine dimers over the range of 1.8-7.5 J/m/sup 2//sec. The implications of these observations for understanding mutation rates at different sites within a gene are discussed.

  9. Initial computational fluid dynamics modeling of the Giant Magellan Telescope site and enclosure

    NASA Astrophysics Data System (ADS)

    Danks, Ryan; Smeaton, William; Bigelow, Bruce; Burgett, William

    2016-08-01

    In the era of extremely large telescopes (ELTs), with telescope apertures growing in size and tighter image quality requirements, maintaining a controlled observation environment is critical. Image quality is directly influenced by thermal gradients, the level of turbulence in the incoming air flow and the wind forces acting on the telescope. Thus any ELT enclosure must be able to modulate the speed and direction of the incoming air and limit the inflow of disturbed ground-layer air. However, gaining an a priori understanding of the wind environment's impacts on a proposed telescope is complicated by the fact that telescopes are usually located in remote, mountainous areas, which often do not have high quality historic records of the wind conditions, and can be subjected to highly complex flow patterns that may not be well represented by the traditional analytic approaches used in typical building design. As part of the design process for the Giant Magellan Telescope at Cerro Las Campanas, Chile; the authors conducted a parametric design study using computational fluid dynamics which assessed how the telescope's position on the mesa, its ventilation configuration and the design of the enclosure and windscreens could be optimized to minimize the infiltration of ground-layer air. These simulations yielded an understanding of how the enclosure and the natural wind flows at the site could best work together to provide a consistent, well controlled observation environment. Future work will seek to quantify the aerothermal environment in terms of image quality.

  10. NASA's Beachside Corrosion Test Site and Current Environmentally Friendly Corrosion Control Initiatives

    NASA Technical Reports Server (NTRS)

    Russell, Richard W.; Calle, Luz Marina; Johnston, Frederick; Montgomery, Eliza L.; Curran, Jerome P.; Kolody, Mark R.

    2013-01-01

    NASA began corrosion studies at the Kennedy Space Center (KSC) in 1966 during the Gemini/Apollo Programs with the evaluation of long-term corrosion protective coatings for carbon steel. KSC's Beachside Corrosion Test Site (BCTS), which has been documented by the American Society of Materials (ASM) as one of the most corrosive, naturally occurring, environments in the world, was established at that time. With the introduction of the Space Shuttle in 1981, the already highly corrosive conditions at the launch pad were rendered even more severe by the acid ic exhaust from the solid rocket boosters. In the years that followed, numerous studies have identified materials, coatings, and maintenance procedures for launch hardware and equipment exposed to the highly corrosive environment at the launch pad. This paper presents a historical overview of over 45 years of corrosion and coating evaluation studies and a description of the BCTS's current capabilities. Additionally, current research and testing programs involving chromium free coatings, environmentally friendly corrosion preventative compounds, and alternates to nitric acid passivation will be discussed.

  11. Slimhole drilling and directional drilling for on-site inspections under a Comprehensive Test Ban: An initial assessment

    SciTech Connect

    Heuze, F. E.

    1995-07-01

    On Site-Inspection (OSI), under the Comprehensive Test Ban being negotiated in the Conference on Disarmament in Geneva, may include drilling at the site of a suspected clandestine underground nuclear explosion to recover radioactive samples. It is in the interest of the drilling party to operate as light and compact a system as possible because it is likely that the drilling equipment will first be airlifted to the country being inspected, and then will be carried by air or surface to the inspection site. It will be necessary for the inspection party to have the capability for more than vertical drilling since there may not be a drilling site available vertically above the suspected nuclear cavity location. This means having, the ability to perform directional drilling and to obtain accurate positioning of the drilling tool. Consequently, several directions may be explored from a single surface drilling pad. If the target depth is expected to be at or less than 600 m (2000 ft), slant drilling may be required to a length well in excess of 600 m. Clearly, the operation must be designed with health and safety features to prevent radioactive exposure if the drilling encounters a nuclear source region. The DOE/LLNL community has developed a strong expertise in this regard. In this initial assessment we focus on the portability and directionality of drilling systems.

  12. Replicative Intermediates of Human Papillomavirus Type 11 in Laryngeal Papillomas: Site of Replication Initiation and Direction of Replication

    NASA Astrophysics Data System (ADS)

    Auborn, K. J.; Little, R. D.; Platt, T. H. K.; Vaccariello, M. A.; Schildkraut, C. L.

    1994-07-01

    We have examined the structures of replication intermediates from the human papillomavirus type 11 genome in DNA extracted from papilloma lesions (laryngeal papillomas). The sites of replication initiation and termination utilized in vivo were mapped by using neutral/neutral and neutral/alkaline two-dimensional agarose gel electrophoresis methods. Initiation of replication was detected in or very close to the upstream regulatory region (URR; the noncoding, regulatory sequences upstream of the open reading frames in the papillomavirus genome). We also show that replication forks proceed bidirectionally from the origin and converge 180circ opposite the URR. These results demonstrate the feasibility of analysis of replication of viral genomes directly from infected tissue.

  13. Sequences at the 3' ends of yeast viral dsRNAs: proposed transcriptase and replicase initiation sites.

    PubMed Central

    Brennan, V E; Field, L; Cizdziel, P; Bruenn, J A

    1981-01-01

    ScV is a double-stranded RNA virus of yeast consisting of two separately encapsidated dsRNAs (L and M). ScV-1 and ScV-2 are two dsRNA viruses present in two different yeast killer strains, K1 and K2. Our 3' end sequence analysis shows that the two sets of viral dsRNAs from ScV-1 and ScV-2 are very similar. Consensus sequences for transcriptase and replicase initiation are proposed. A stem and loop structure with a 3' terminal AUGC sequence, like that of several plant virus plus strand RNAs, is present at the putative replicase initiation site of one of the yeast viral RNA plus strands. Images PMID:7029463

  14. In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site.

    PubMed

    Caldentey, J; Blanco, L; Bamford, D H; Salas, M

    1993-08-11

    Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using single-stranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3' end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3' end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3' end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin-containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process.

  15. Prediction of Initiation Site of Destruction of Flat Braided Carbon Fiber Composites Using HTS-SQUID Gradiometer

    NASA Astrophysics Data System (ADS)

    Shinyama, Y.; Hatsukade, Y.; Tanaka, S.; Takai, Y.; Aly-Hassan, M. S.; Nakai, A.; Hamada, H.

    Carbon fiber reinforced polymers (CFRPs) are composite materials with lightweight and high specific strength. As the braided CFRPs have continuous carbon-fiber bundles in their longitudinal direction, they are stronger than conventional CFRPs. In this study, we applied a current-injection-based NDE method using a HTS-SQUID gradiometer to the flat braided CFRPs with and without carbon nanotubes (CNTs), and estimated conditions of the carbon fibers while applying a step-by-step tensile load to the CFRPs. From the results, a possibility to predict an initiation site of the destruction in the braided CFRPs was demonstrated.

  16. Multiple phosphorylation sites of DNA polymerase alpha-primase cooperate to regulate the initiation of DNA replication in vitro.

    PubMed

    Schub, O; Rohaly, G; Smith, R W; Schneider, A; Dehde, S; Dornreiter, I; Nasheuer, H P

    2001-10-12

    DNA polymerase alpha-primase (pol-prim) is the only enzyme that can start DNA replication de novo. The 180-kDa (p180) and 68-kDa (p68) subunits of the human four-subunit enzyme are phosphorylated by Cyclin-dependent kinases (Cdks) in a cell cycle-dependent manner. Cyclin A-Cdk2 physically interacts with pol-prim and phosphorylates N-terminal amino acids of the p180 and the p68 subunits, leading to an inhibition of pol-prim in initiating cell-free SV40 DNA replication. Mutation of conserved putative Cdk phosphorylation sites in the N terminus of human p180 and p68 reduced their phosphorylation by Cyclin A-Cdk2 in vitro. In contrast to wild-type pol-prim these mutants were no longer inhibited by Cyclin A-Cdk2 in the initiation of viral DNA replication. Importantly, rather than inhibiting it, Cyclin A-Cdk2 stimulated the initiation activity of pol-prim containing a triple N-terminal alanine mutant of the p180 subunit. Together these results suggest that Cyclin A-Cdk2 executes both stimulatory and inhibitory effects on the activity of pol-prim in initiating DNA replication.

  17. Savannah River Site waste vitrification projects initiated throughout the United States: Disposal and recycle options

    SciTech Connect

    Jantzen, C.M.

    2000-04-10

    A vitrification process was developed and successfully implemented by the US Department of Energy's (DOE) Savannah River Site (SRS) and at the West Valley Nuclear Services (WVNS) to convert high-level liquid nuclear wastes (HLLW) to a solid borosilicate glass for safe long term geologic disposal. Over the last decade, SRS has successfully completed two additional vitrification projects to safely dispose of mixed low level wastes (MLLW) (radioactive and hazardous) at the SRS and at the Oak Ridge Reservation (ORR). The SRS, in conjunction with other laboratories, has also demonstrated that vitrification can be used to dispose of a wide variety of MLLW and low-level wastes (LLW) at the SRS, at ORR, at the Los Alamos National Laboratory (LANL), at Rocky Flats (RF), at the Fernald Environmental Management Project (FEMP), and at the Hanford Waste Vitrification Project (HWVP). The SRS, in conjunction with the Electric Power Research Institute and the National Atomic Energy Commission of Argentina (CNEA), have demonstrated that vitrification can also be used to safely dispose of ion-exchange (IEX) resins and sludges from commercial nuclear reactors. In addition, the SRS has successfully demonstrated that numerous wastes declared hazardous by the US Environmental Protection Agency (EPA) can be vitrified, e.g. mining industry wastes, contaminated harbor sludges, asbestos containing material (ACM), Pb-paint on army tanks and bridges. Once these EPA hazardous wastes are vitrified, the waste glass is rendered non-hazardous allowing these materials to be recycled as glassphalt (glass impregnated asphalt for roads and runways), roofing shingles, glasscrete (glass used as aggregate in concrete), or other uses. Glass is also being used as a medium to transport SRS americium (Am) and curium (Cm) to the Oak Ridge Reservation (ORR) for recycle in the ORR medical source program and use in smoke detectors at an estimated value of $1.5 billion to the general public.

  18. Stochastic analysis of dimerization systems.

    PubMed

    Barzel, Baruch; Biham, Ofer

    2009-09-01

    The process of dimerization, in which two monomers bind to each other and form a dimer, is common in nature. This process can be modeled using rate equations, from which the average copy numbers of the reacting monomers and of the product dimers can then be obtained. However, the rate equations apply only when these copy numbers are large. In the limit of small copy numbers the system becomes dominated by fluctuations, which are not accounted for by the rate equations. In this limit one must use stochastic methods such as direct integration of the master equation or Monte Carlo simulations. These methods are computationally intensive and rarely succumb to analytical solutions. Here we use the recently introduced moment equations which provide a highly simplified stochastic treatment of the dimerization process. Using this approach, we obtain an analytical solution for the copy numbers and reaction rates both under steady-state conditions and in the time-dependent case. We analyze three different dimerization processes: dimerization without dissociation, dimerization with dissociation, and heterodimer formation. To validate the results we compare them with the results obtained from the master equation in the stochastic limit and with those obtained from the rate equations in the deterministic limit. Potential applications of the results in different physical contexts are discussed.

  19. Site of impulse initiation in tendon organs of cat soleus muscle.

    PubMed

    Gregory, J E; Morgan, D L; Proske, U

    1985-12-01

    A continuing controversy surrounds the question of whether Golgi tendon organs are examples of receptors in which impulses may be generated at more than one site. This paper reports a systematic examination of a number of models incorporating single or multiple impulse generators and of the compatibility of their predictions with experimental observations. Two phenomena, in particular, that must be accounted for are nonlinear summation and cross-adaptation. When two motor units each with a direct effect on the tendon organ are stimulated together, the rate of discharge is greater than either individual rate but is less than their sum. In cross-adaptation a conditioning response elicited by one motor unit contraction produces adaptation of the discharge associated with stimulation of a second motor unit. A model with a central impulse generator can be modified to account for nonlinear summation by postulating a nonlinear transformation in the generator current-to-impulse rate conversion. Experiments measuring summation of responses to stimulation of three inputs produced results that did not support this model. Another variation of the model, which had a nonlinearity in the tension-to-current step and cross-connections (mechanical or neural) between tendon strands stressed by contracting muscle fibers, was able to account for the observations. A second model that provided the right predictions was a multiple impulse generator with cross-connections. Which of the two models best fits the experimental observations can be decided by comparing the calculated summation coefficients and cross-adaptation coefficients. A central impulse generator predicts a negative correlation, the multiple impulse generator a positive correlation. All of the observations were made using tendon organs of cat soleus muscle. Responses were recorded to stimulation of filaments of ventral root. In a comparison between 20 pairs of responses from six tendon organs the correlation between

  20. Breast conserving surgery and accelerated partial breast irradiation using the MammoSite system: initial clinical experience.

    PubMed

    DiFronzo, L Andrew; Tsai, Peter I; Hwang, Julie M; Lee, John J; Ryoo, Monica C; Rahimian, Javad; Tome, Michael; Takasugi, Jan K; Haigh, Philip I

    2005-08-01

    Balloon catheter-based accelerated partial breast irradiation (APBI) may result in desirable short-term outcomes in patients undergoing breast conserving surgery. Prospective consecutive case series. Tertiary multidisciplinary referral center. Forty selected patients with invasive breast carcinoma undergoing breast conserving surgery and MammoSite device placement. Breast conserving surgery, sentinel and/or axillary node dissection, placement of the new balloon catheter applicator (MammoSite device), and APBI. Infection, early and late seroma, device explantation, time to initiating APBI, acute toxic effects on the skin, and cosmesis using the Harvard Scale. Thirty-nine patients underwent MammoSite device placement at the time of lumpectomy; 1 patient underwent percutaneous device placement after lumpectomy. Nineteen patients (49%) had drainage catheters placed in the breast cavity at the time of lumpectomy. Wound infection developed in 3 patients (8%). Five devices (12%) were explanted because of unfavorable final pathological findings or infection. The mean time to the start of APBI in patients who did not undergo simultaneous drain placement was 7.2 days (range, 5-12 days), compared with 5.1 days (range, 3-8 days) in patients who did (P = .008). With a mean follow-up of 13.3 months (range, 2-28 months), patients completing APBI had limited toxic effects on the skin, with excellent or good cosmetic results in 39 patients (97%). Use of the MammoSite system in APBI has favorable short-term outcomes. Infection and radiation treatment delay are common and may warrant use of perioperative antibiotics and drain placement, respectively. A small number of patients who have device placement at the time of lumpectomy will require explantation because of unfavorable final pathological findings. Short-term outcomes of MammoSite brachytherapy support further studies comparing APBI with standard whole breast irradiation in patients undergoing breast conserving surgery.

  1. Initial recommendations for restricting gamma-ray spectrometry measurements of radionuclides for on-site inspections

    SciTech Connect

    Buckley, W F; Kreek, S A; Wild, J F

    1998-11-06

    The US paper "Radionuclide Sampling, Sample Handling and Analytical Laboratory Equipment for Comprehensive Test Ban Treaty On-Site Inspections," CTBT/PC/V/OSI/WSII/PR/29 identified the radionuclides of interest to an OS1 as 144Ce, 147Nd, 141Ce, 149Ba140La), 95 Zr(95Nb), 131mXe, 133mXe, 133gXe, 135gXe, and 37Ar. All of these nuclides (except 37Ar) can be measured via some form of conventional or coincidence-based gamma-ray spectrometry. The non-gaseous radionuclides [144Ce, 147Nd, 141Ce, 140Ba(140La), and 95Zr(95Nb)] can be measured via conventional high-resolution gamma-ray spectrometry using a shielded, high-purity germanium (HPGe) detector. The gaseous radionuclides 131mXe, 133mXe, 133gXe, and 135gXe are best measured (after separation from their homologous elements) via a gamma & beta/electron coincidence technique such as that described in CTBT/WGB/TL-11/5 which could utilize either a HPGe or low-resolution (NaI(TI)) gamma-ray spectrometer to detect the gamma-ray/x-ray and a plastic scintillator to detect the beta particle/electron from the decay of the various Xe isotopes. The US paper CTBT/PC/V/IOSI/WSII/PR/29 (and other papers) identified a need to limit the information that can be extracted from high-resolution gamma-ray spectra to ensure that only information relevant to an OSI is accessible. The term "blinding" has been used to describe the need to limit the information available to the Inspection Team from the high-resolution gamma-ray measurement. A better term is "measurement restriction"; the need for restricting the information is particularly relevant to conventional high-resolution gamma-ray spectrometry measurements, but not to the gamma & beta/electron coincidence-type measurements

  2. Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics.

    PubMed

    Hortal, Sara; Pera, Joan; Parladé, Javier

    2009-03-01

    Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan(R) real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical-chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.

  3. Initial Observations and Activities of Curiosity's Mars Hand Lens Imager (MAHLI) at the Gale Field Site

    NASA Astrophysics Data System (ADS)

    Aileen Yingst, R.; Edgett, Kenneth; MSL Science Team

    2013-04-01

    The Mars Hand Lens Imager (MAHLI) is a 2-megapixel focusable macro lens color camera on the turret on the Mars Science Laboratory rover, Curiosity's, robotic arm. The investigation centers on stratigraphy, grain-scale texture, structure, mineralogy, and morphology. MAHLI acquires focused images at working distances of 2.1 cm to infinity; at 2.1 cm the scale is 14 µm/pixel; at 6.9 cm it is 31 µm/pixel, like the Spirit and Opportunity Microscopic Imagers (MI). Most MAHLI use during the first 100 Martian days (sols) was focused on instrument, rover, and robotic arm engineering check-outs and risk reduction, including (1) interrogation of an eolian sand shadow for suitability for scooping, decontamination of the sample collection and processing system (CHIMRA, Collection and Handling for In-Situ Martian Rock Analysis), and first solid sample delivery to the Chemistry and Mineralogy (CheMin) and Sample Analysis at Mars (SAM) instruments; (2) documentation of the nature of this sand; (3) verification that samples were delivered to SAM and passed through a 150 µm mesh and a 2 mm funnel throat in the CheMin inlet; (4) development of methods for future precision robotic arm positioning of MAHLI and the Alpha Particle X-Ray Spectrometer (APXS); and (5) use of MAHLI autofocus for range-finding to determine locations to position the scoop before each scooping event. Most Sol 0-100 MAHLI images were obtained at scales of 31-110 µm/pixel; some geologic targets were imaged at 21-31 µm/pixel. No opportunities to position the camera close enough to obtain 14-20 µm/pixel images were available during this initial period. Only two rocks, named Jake Matijevic and Bathurst Inlet, were imaged at a resolution higher than MI. Both were dark gray and mantled with dust and fine/very fine sand. In both cases, the highest resolution images of these rocks show no obvious, indisputable grains, suggesting that grain sizes (as expressed at the rock surfaces) are < 80 µm. However, because of

  4. Paleomagnetic results from IODP Expedition 344 Site U1381 and implications for the initial subduction of the Cocos Ridge

    NASA Astrophysics Data System (ADS)

    Li, Yong-Xiang; Zhao, Xixi; Jovane, Luigi; Petronotis, Katerina; Gong, Zheng; Xie, Siyi

    2016-04-01

    Understanding the processes that govern the strength, nature, and distribution of slip along subduction zones is a fundamental and societally relevant goal of modern earth science. The Costa Rica Seismogenesis Project (CRISP) is specially designed to understand the processes that control nucleation and seismic rupture of large earthquakes at erosional subduction zones. Drilling directly on the Cocos Ridge (CR) during International Ocean Drilling Program (IODP) Expedition 344 discovered a sedimentary hiatus in Site U1381 cores. In this study, we conducted a magnetostratigraphic and rock magnetic study on the Cenozoic sedimentary sequences of site U1381. Anisotropy of magnetic susceptibility data from sediments above and below the hiatus show oblate fabrcis, but the Kmin axes of the AMS data from sediments below the hiatus are more dispersed than those from sediments above the hiatus, implying that formation of hiatus may have affected AMS. Paleomagnetic results of the U1381 core, together with available Ar-Ar dates of ash layers from sediments below the hiatus, allow us to establish a geomagnetic polarity timescale that brackets the hiatus between ca. 9.61 and 1.52 Ma. Analyses of sedimentary records from ODP/IODP cores in the vicinity reveal that the hiatus appears to be regional, spanning the northeastern end of the CR. Also, the hiatus appears to occur only at certain locations. Its regional occurrence at unique locations implies a link to the initial shallow subduction of the Cocos Ridge. The hiatus was probably produced by either bottom current erosion or the CR buckling upon its initial collision with the Middle American trench (MAT). Thus, the initial subduction of the CR must have taken place on or before 1.52 Ma.

  5. Formation and repair of psoralen-DNA adducts and pyrimidine dimers in human DNA and chromatin.

    PubMed Central

    Cleaver, J E; Killpack, S; Gruenert, D C

    1985-01-01

    DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site. PMID:3002774

  6. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition.

    PubMed

    Dey, Madhusudan; Cao, Chune; Dar, Arvin C; Tamura, Tomohiko; Ozato, Keiko; Sicheri, Frank; Dever, Thomas E

    2005-09-23

    The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.

  7. Equivalence between XY and dimerized models

    SciTech Connect

    Campos Venuti, Lorenzo; Roncaglia, Marco

    2010-06-15

    The spin-1/2 chain with XY anisotropic coupling in the plane and the XX isotropic dimerized chain are shown to be equivalent in the bulk. For finite systems, we prove that the equivalence is exact in given parity sectors, after taking care of the precise boundary conditions. The proof is given constructively by finding unitary transformations that map the models onto each other. Moreover, we considerably generalized our mapping and showed that even in the case of fully site-dependent couplings the XY chain can be mapped onto an XX model. This result has potential application in the study of disordered systems.

  8. Serum D-Dimer Test Is Promising for the Diagnosis of Periprosthetic Joint Infection and Timing of Reimplantation.

    PubMed

    Shahi, Alisina; Kheir, Michael M; Tarabichi, Majd; Hosseinzadeh, Hamid R S; Tan, Timothy L; Parvizi, Javad

    2017-09-06

    Despite the availability of a battery of tests, the diagnosis of periprosthetic joint infection (PJI) continues to be challenging. Serum D-dimer assessment is a widely available test that detects fibrinolytic activities that occur during infection. We hypothesized that patients with PJI may have a high level of circulating D-dimer and that the presence of a high level of serum D-dimer may be a sign of persistent infection in patients awaiting reimplantation. This prospective study was initiated to enroll patients undergoing primary and revision arthroplasty. Our cohort consisted of 245 patients undergoing primary arthroplasty (n = 23), revision for aseptic failure (n = 86), revision for PJI (n = 57), or reimplantation (n = 29) or who had infection in a site other than a joint (n = 50). PJI was defined using the Musculoskeletal Infection Society criteria. In all patients, serum D-dimer level, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level were measured preoperatively. The median D-dimer level was significantly higher (p < 0.0001) for the patients with PJI (1,110 ng/mL [range, 243 to 8,487 ng/mL]) than for the patients with aseptic failure (299 ng/mL [range, 106 to 2,571 ng/mL). Using the Youden index, 850 ng/mL was determined as the optimal threshold value for serum D-dimer for the diagnosis of PJI. Serum D-dimer outperformed both ESR and serum CRP, with a sensitivity of 89% and a specificity of 93%. ESR and CRP had a sensitivity of 73% and 79% and a specificity of 78% and 80%, respectively. The sensitivity and specificity of ESR and CRP combined was 84% (95% confidence interval [CI], 76% to 90%) and 47% (95% CI, 36% to 58%), respectively. It appears that serum D-dimer is a promising marker for the diagnosis of PJI. This test may also have a great utility for determining the optimal timing of reimplantation. Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

  9. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed Central

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-01-01

    In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a

  10. Completion Report for Multi-Site Incentive MRT 2779 Implement ASC Tripod Initiative by 30SEP08

    SciTech Connect

    East, D; Cerutti, J; Noe, J; Cupps, K; Loncaric, J; Sturtevant, J

    2008-09-22

    This report provides documentation and evidence for the completion of the deployment of the Tripod common operating system (TripodOS, also known as and generally referred to below as TOSS). Background documents for TOSS are provided in Appendices A and B, including the initial TOSS proposal accepted by ASC HQ and Executives in July 2007 and a Governance Model defined by a Tri-Lab working group in September 2007. Appendix C contains a document that clarifies the intent and requirements for the completion criteria associated with MRT 2779. The deployment of TOSS is a Multi-Site Incentive from the ASC FY08-09 Implementation Plan due at the end of Quarter 4 in FY08.

  11. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding.

    PubMed

    Liko, Idlir; Degiacomi, Matteo T; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V

    2016-07-19

    Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance.

  12. Hydrogenated fullerenes dimer, peanut and capsule: An atomic comparison

    NASA Astrophysics Data System (ADS)

    EL-Barbary, A. A.

    2016-04-01

    Hydrogenated fullerenes are detected in the Universe in space but their identification is still unsolved task. Therefore, this paper provides useful information about hydrogenated fullerenes (dimer, peanut and capsule) using DFT method at the B3LYP/6-31G(d) level of theory. The stability, geometric structures, hydrogen adsorption energies and NMR chemical shifts are calculated. The results show that the energy of most stable isomer of C118 dimer is lower than the energies sum of C60 and C58 cages by 1.77 eV and the energy per carbon atom of C144 capsule is more stable than C60 cage by 126.98 meV. Also, endohedral Ti-doped C118 dimer and C128 peanut are found to be most stable structures than exohedral Ti-doped C118 dimer and C128 peanut by 2.19 eV/Ti and 3.52 eV/Ti, respectively. The hydrogenation process is found to be enhanced (especially at the caps) for endohedral Ti-doped C118 dimer and C128 peanut through electronic surface modifications. The most active hydrogenation sites are selected and it is found that the most stable hydrogenation sites are Houts1 and Houts3 for fullerenes and endohedral Ti-doped fullerenes, respectively.

  13. Divergent mtDNA lineages of goats in an Early Neolithic site, far from the initial domestication areas

    PubMed Central

    Fernández, Helena; Hughes, Sandrine; Vigne, Jean-Denis; Helmer, Daniel; Hodgins, Greg; Miquel, Christian; Hänni, Catherine; Luikart, Gordon; Taberlet, Pierre

    2006-01-01

    Goats were among the first farm animals domesticated, ≈10,500 years ago, contributing to the rise of the “Neolithic revolution.” Previous genetic studies have revealed that contemporary domestic goats (Capra hircus) show far weaker intercontinental population structuring than other livestock species, suggesting that goats have been transported more extensively. However, the timing of these extensive movements in goats remains unknown. To address this question, we analyzed mtDNA sequences from 19 ancient goat bones (7,300–6,900 years old) from one of the earliest Neolithic sites in southwestern Europe. Phylogenetic analysis revealed that two highly divergent goat lineages coexisted in each of the two Early Neolithic layers of this site. This finding indicates that high mtDNA diversity was already present >7,000 years ago in European goats, far from their areas of initial domestication in the Near East. These results argue for substantial gene flow among goat populations dating back to the early neolithisation of Europe and for a dual domestication scenario in the Near East, with two independent but essentially contemporary origins (of both A and C domestic lineages) and several more remote and/or later origins. PMID:17030824

  14. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    SciTech Connect

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-09-27

    A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-turn motif that also occurs in the 'off' state of the human ribosomal decoding site RNA.

  15. An environmental justice risk communication initiative at the U.S. Department of Energy`s Savannah River Site

    SciTech Connect

    Temple, J.Z.; Musham, C.; Bath, S.

    1997-08-01

    Four low-income and minority communities located in close proximity to the Savannah River Site (SRS), a DOE nuclear facility were involved in an environmental justice risk communication initiative under the auspices of a DOE Environmental Justice Strategy addressing Executive Order 12898. The initial phase of this project identified community perceptions of health concerns, SRS image and communication, and environmental concerns. Findings served as the foundation for the design, development, and delivery of four community-specific risk communication programs. In response to identified community health concerns, meetings were conducted to share public and worker health studies associated with SRS. Special emphasis was focused on a public health cancer study conducted in the SRS region of concern. SRS 1995 environmental monitoring data, with emphasis on radiation and its impact on air and water, was the focus of the final series of community meetings. Selection and development of a risk communication team comprised of SRS scientists and engineers was included. Project strategies involved active utilization of an advisory committee and a technical committee throughout the process. Interface with appropriate boards and committees involved in outreach activities at the nuclear complex also occurred.

  16. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  17. Identification of ubiquitin-modified lysine residues and novel phosphorylation sites on eukaryotic initiation factor 2B epsilon

    PubMed Central

    Tuckow, Alexander P.; Kazi, Abid A.; Kimball, Scot R.; Jefferson, Leonard S.

    2013-01-01

    Eukaryotic initiation factor 2Bε (eIF2Bε) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Bε followed by ubiquitin. Tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Bε. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC-MS/MS analysis, and an interaction between endogenous eIF2Bε with NEDD4 was confirmed by co-immunoprecipitation. PMID:23707720

  18. Mechanism and evolution of protein dimerization.

    PubMed Central

    Xu, D.; Tsai, C. J.; Nussinov, R.

    1998-01-01

    We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers

  19. Haldane relation for interacting dimers

    NASA Astrophysics Data System (ADS)

    Giuliani, Alessandro; Mastropietro, Vieri; Lucio Toninelli, Fabio

    2017-03-01

    We consider a model of weakly interacting close-packed dimers on the two-dimensional square lattice. In a previous paper, we computed both the multi-point dimer correlations, which display non-trivial critical exponents, continuously varying with the interaction strength; and the height fluctuations, which, after proper coarse graining and rescaling, converge to a massless Gaussian field with a suitable interaction-dependent pre-factor (‘amplitude’). In this paper, we prove the identity between the critical exponent of the two-point dimer correlation and the amplitude of this massless Gaussian field. This identity is the restatement, in the context of interacting dimers, of one of the Haldane universality relations, part of his Luttinger-liquid conjecture, originally formulated in the context of one-dimensional interacting Fermi systems. Its validity is a strong confirmation of the effective massless Gaussian field description of the interacting dimer model, which was proposed on the basis of formal bosonization arguments. We also conjecture that a certain discrete curve defined at the lattice level via the Temperley bijection converges in the scaling limit to an SLE κ process, with κ depending non-trivially on the interaction and related in a simple way to the amplitude of the limiting Gaussian field.

  20. Adatom-dimer interaction on the Si(001)-2 × 1 surface

    NASA Astrophysics Data System (ADS)

    Toh, C. P.; Ong, C. K.

    1994-02-01

    We use a modified form of the Stillinger-Weber potential to obtain the binding sites and diffusion barriers of a Si adatom in the vicinity of single F and B type dimers on the Si(001)-2 × 1 surface. We find that both kinds of dimer provide good sinks for adatoms and are therefore ideal nucleation sites, provided the temperature is not too high as to induce dimer breaking. Our results also show that adatoms can be trapped in non-lattice sites surrounding the F type dimer, leading to a disordering of the growing epitaxial film. Monte Carlo simulated annealing indicates that adatoms at these "defect" sites are vertically displaced with respect to those adsorbed on the epitaxial sites, giving rise to step structures that closely resemble those proposed by Falta and Henzler [Surf. Sci 269/270 (1992) 14] to account for their SPA-LEED results.

  1. Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles.

    PubMed

    Gazaryan, I G; Klyachko, N L; Dulkis, Y K; Ouporov, I V; Levashov, A V

    1997-12-01

    Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.

  2. Formation and properties of dimeric recombinant horseradish peroxidase in a system of reversed micelles.

    PubMed Central

    Gazaryan, I G; Klyachko, N L; Dulkis, Y K; Ouporov, I V; Levashov, A V

    1997-01-01

    Wild-type recombinant horseradish peroxidase purified and refolded from Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed micelles in octane. In contrast with native horseradish peroxidase the wild-type recombinant enzyme forms dimeric structures as judged by sedimentation analysis. Peroxidase substrates affect the equilibrium between monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the surfactant exhibits two maxima with pyrogallol, o-phenylene- diamine, guaiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences in activities of monomeric and dimeric forms of the recombinant horseradish peroxidase provide evidence for active-site screening in dimeric forms. This has been used to model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obtained, three of eight glycosylation sites were screened. This might explain the absence of dimeric structures in native enzyme peroxidase. The system of reversed micelles provides, for the first time, evidence for the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme. Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures. PMID:9371726

  3. Adventures in Holographic Dimer Models

    SciTech Connect

    Kachru, Shamit; Karch, Andreas; Yaida, Sho; /Stanford U., Phys. Dept.

    2011-08-12

    We abstract the essential features of holographic dimer models, and develop several new applications of these models. Firstly, semi-holographically coupling free band fermions to holographic dimers, we uncover novel phase transitions between conventional Fermi liquids and non-Fermi liquids, accompanied by a change in the structure of the Fermi surface. Secondly, we make dimer vibrations propagate through the whole crystal by way of double trace deformations, obtaining nontrivial band structure. In a simple toy model, the topology of the band structure experiences an interesting reorganization as we vary the strength of the double trace deformations. Finally, we develop tools that would allow one to build, in a bottom-up fashion, a holographic avatar of the Hubbard model.

  4. Adventures in holographic dimer models

    NASA Astrophysics Data System (ADS)

    Kachru, Shamit; Karch, Andreas; Yaida, Sho

    2011-03-01

    We abstract the essential features of holographic dimer models, and develop several new applications of these models. Firstly, semi-holographically coupling free band fermions to holographic dimers, we uncover novel phase transitions between conventional Fermi liquids and non-Fermi liquids, accompanied by a change in the structure of the Fermi surface. Secondly, we make dimer vibrations propagate through the whole crystal by way of double trace deformations, obtaining nontrivial band structure. In a simple toy model, the topology of the band structure experiences an interesting reorganization as we vary the strength of the double trace deformations. Finally, we develop tools that would allow one to build, in a bottom-up fashion, a holographic avatar of the Hubbard model.

  5. Dimer-flipping-assisted diffusion on a Si(001) surface

    NASA Astrophysics Data System (ADS)

    Zi, J.; Min, B. J.; Lu, Y.; Wang, C. Z.; Ho, K. M.

    2000-12-01

    The binding sites and diffusion pathways of Si adatoms on a c(4×2) reconstructed Si(001) surface are investigated by a tight-binding method with an environment-dependent silicon potential in conjunction with ab initio calculations using the Car-Parrinello method. A new diffusion pathway along the trough edge driven by dimer flipping is found with a barrier of 0.74 eV, comparable to that of 0.68 eV along the top of the dimer rows.

  6. Dimerization of polycyclic aromatic hydrocarbons in soot nucleation.

    PubMed

    Zhang, Hong-Bo; You, Xiaoqing; Wang, Hongmiao; Law, Chung K

    2014-02-27

    A possible pathway of soot nucleation, in which localized π electrons play an important role in binding the polycyclic aromatic hydrocarbon (PAH) molecules having multiradical characteristics to form stable polymer molecules through covalent bonds, is studied using density functional and semiempirical methods. Results show that the number of covalent bonds formed in the dimerization of two identical PAHs is determined by the radical character, and the sites to form bonds are related to the aromaticity of individual six-membered ring structure. It is further shown that the binding energy of dimerization increases linearly with the diradical character in the range relevant to soot nucleation.

  7. Initial results from seismic monitoring at the Aquistore CO2 storage site, Saskatchewan, Canada

    SciTech Connect

    White, D. J.; Roach, L. A.N.; Roberts, B.; Daley, T. M.

    2014-12-31

    of 2013. Comparison of the data from these surveys relative to the baseline 3D survey data from 2012 shows excellent repeatability (NRMS less than 10%) which will provide enhanced monitoring sensitivity to smaller amounts of CO2. The permanent array also provides continuous passive monitoring for injection-related microseismicity. Passive monitoring has been ongoing since the summer of 2012 in order to establish levels of background seismicity before CO2 injection starts in 2014. Microseismic monitoring was augmented in 2013 by the installation of 3 broadband seismograph stations surrounding the Aquistore site. These surface installations should provide a detection capability of seismic events with magnitudes as low as ~0. Downhole seismic methods are also being utilized for CO2 monitoring at the Aquistore site. Baseline crosswell tomographic images depict details (meters-scale) of the reservoir in the 150-m interval between the observation and injection wells. This level of resolution is designed to track the CO2 migration between the wells during the initial injection period. A baseline 3D vertical seismic profile (VSP) was acquired in the fall of 2013 to provide seismic images with resolution on a scale between that provided by the surface seismic array and the downhole tomography. The 3D VSP was recorded simultaneously using both a conventional array of downhole geophones (60-levels) and an optical fibre system. The latter utilized an optical fiber cable deployed on the outside of the monitor well casing and cemented in place. A direct comparison of these two methodologies will determine the suitability of using the fiber cable for ongoing time-lapse VSP monitoring.

  8. Initial geochemistry data of the Lake Ohrid (Macedonia, Albania) "DEEP" site sediment record: The ICDP SCOPSCO drilling project

    NASA Astrophysics Data System (ADS)

    Francke, Alexander; Wagner, Bernd; Krastel, Sebastian; Lindhorst, Katja; Mantke, Nicole; Klinghardt, Dorothea

    2014-05-01

    Lake Ohrid, located at the border of Macedonia and Albania is about 30 km long, 15 km wide and up to 290 m deep. Formed within a tectonic graben, Lake Ohrid is considered to be the oldest lake in Europe. The ICDP SCOPSCO (Scientific Collaboration of Past Speciation Conditions in Lake Ohrid) deep drilling campaign at Lake Ohrid in spring 2013 aimed (a) to obtain more precise information about the age and origin of the lake, (b) to unravel the seismotectonic history of the lake area including effects of major earthquakes and associated mass wasting events, (c) to obtain a continuous record containing information on volcanic activities and climate changes in the central northern Mediterranean region, and (d) to better understand the impact of major geological/environmental events on general evolutionary patterns and shaping an extraordinary degree of endemic biodiversity as a matter of global significance. Drilling was carried out by DOSECC (Salt Lake City, USA) using the DLDS (Deep Lake Drilling System) with a hydraulic piston corer for surface sediments and rotation drilling for harder, deeper sediments. Overall, about 2,100 m of sediment were recovered from 4 drill sites. At the "DEEP" site in the center of the lake, seismic data indicated a maximum sediment fill of ca. 700 m, of which the uppermost 568 m sediment were recovered. Initial data from core catcher samples and on-site susceptibility measurements indicate that the sediment sequence covers more than 1.2 million years and provides a continuous archive of environmental and climatological variability in the area. Currently, core opening, core description, XRF and MSCL -scanning, core correlation, and sub-sampling of the sediment cores from the "DEEP" site is conducted at the University of Cologne. High-resolution geochemical data obtained from XRF-scanning imply that the sediments from the "DEEP" site are highly sensitive to climate and environmental variations in the Balkan area over the last few glacial

  9. Prediction of translation initiation sites in human mRNA sequences with AUG start codon in weak Kozak context: A neural network approach.

    PubMed

    Tikole, Suhas; Sankararamakrishnan, Ramasubbu

    2008-05-16

    Translation of eukaryotic mRNAs is often regulated by nucleotides around the start codon. A purine at position -3 and a guanine at position +4 contribute significantly to enhance the translation efficiency. Algorithms to predict the translation initiation site often fail to predict the start site if the sequence context is not present. We have developed a neural network method to predict the initiation site of mRNA sequences that lack the preferred nucleotides at the positions -3 and +4 surrounding the translation initiation site. Neural networks of various architectures comprising different number of hidden layers were designed and tested for various sizes of windows of nucleotides surrounding translation initiation sites. We found that the neural network with two hidden layers showed a sensitivity of 83% and specificity of 73% indicating a vastly improved performance in successfully predicting the translation initiation site of mRNA sequences with weak Kozak context. WeakAUG server is freely available at http://bioinfo.iitk.ac.in/AUGPred/.

  10. Is Dimerization Required for the Catalytic Activity of Bacterial Biotin Carboxylase?

    SciTech Connect

    Shen,Y.; Chou, C.; Chang, G.; Tong, L.

    2006-01-01

    Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 Angstroms from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.

  11. Products and mechanism of acene dimerization. A computational study.

    PubMed

    Zade, Sanjio S; Zamoshchik, Natalia; Reddy, A Ravikumar; Fridman-Marueli, Galit; Sheberla, Dennis; Bendikov, Michael

    2011-07-20

    The high reactivity of acenes can reduce their potential applications in the field of molecular electronics. Although pentacene is an important material for use in organic field-effect transistors because of its high charge mobility, its reactivity is a major disadvantage hindering the development of pentacene applications. In this study, several reaction pathways for the thermal dimerization of acenes were considered computationally. The formation of acene dimers via a central benzene ring and the formation of acene-based polymers were found to be the preferred pathways, depending on the length of the monomer. Interestingly, starting from hexacene, acene dimers are thermodynamically disfavored products, and the reaction pathway is predicted to proceed instead via a double cycloaddition reaction (polymerization) to yield acene-based polymers. A concerted asynchronous reaction mechanism was found for benzene and naphthalene dimerization, while a stepwise biradical mechanism was predicted for the dimerization of anthracene, pentacene, and heptacene. The biradical mechanism for dimerization of anthracene and pentacene proceeds via syn or anti transition states and biradical minima through stepwise biradical pathways, while dimerization of heptacene proceeds via asynchronous ring closure of the complex formed by two heptacene molecules. The activation barriers for thermal dimerization decrease rapidly with increasing acene chain length and are calculated (at M06-2X/6-31G(d)+ZPVE) to be 77.9, 57.1, 33.3, -0.3, and -12.1 kcal/mol vs two isolated acene molecules for benzene, naphthalene, anthracene, pentacene, and heptacene, respectively. If activation energy is calculated vs the initially formed complex of two acene molecules, then the calculated barriers are 80.5, 63.2, 43.7, 16.7, and 12.3 kcal/mol. Dimerization is exothermic from anthracene onward, but it is endothermic at the terminal rings, even for heptacene. Phenyl substitution at the most reactive meso

  12. Enhanceosome transcription factors preferentially dimerize with high mobility group proteins.

    PubMed

    Jankowski, Aleksander; Obara, Paulina; Mathur, Utsav; Tiuryn, Jerzy

    2016-02-04

    The enhanceosome is an enhancer located upstream of the human interferon β gene, bound by transcription factor (TF) complex of extremely rigid structure. Within these rigid constraints, even a slight change of distances between transcription factor binding sites (TFBS) results in loss of functionality of the enhanceosome. We hypothesized that smaller subunits of the enhanceosome may entail TF complex formation in other regulatory regions. In order to verify this hypothesis we systematically searched for dimerization preferences of the TFs that have TFBS in the enhanceosome. For this we utilized our recently developed tool, TACO. We performed this computational experiment in a cell-type-specific manner by utilizing cell-type-specific DNase-seq data for 105 human cell types. We also used 20 TRANSFAC motifs comprising not only the usual TFs constituting the enhanceosome but also the architectural proteins of High Mobility Group I(Y) (HMG I). A similar experiment used 42 DNase-seq data sets for mouse cell types. We found 137 statistically significant dimer predictions in the human genome, and 37 predictions in the mouse genome, that matched the positioning on the enhanceosome with ±2 bp tolerance. To characterize these predicted TF dimers, we performed functional analysis (Gene Ontology enrichment) for sets of genes which were in the neighbourhood of predicted dimer instances. A notable feature of these instances is that (1) most of them are located in introns of genes, (2) they are enriched in regulatory states, and (3) those instances that are located near transcription start sites are enriched for inclusion in computationally predicted enhancers. We also investigated similarity of dimer predictions between human and mouse. It follows from our experiments that, except for homodimer formed by IRF proteins, the rest of the dimers were formed exclusively between one of the transcriptional activators (ATF-2/c-Jun and IRF) and a HMG I protein. NF- κB did not

  13. New View on the Initial Development Site and Radiographic Classification System of Osteoarthritis of the Knee Based on Radiographic Analysis

    PubMed Central

    Moon, Ki-Ho

    2012-01-01

    Introduction: Radiographic pathology of severe osteoarthritis of the knee (OAK) such as severe osteophyte at tibial spine (TS), compartment narrowing, marginal osteophyte, and subchondral sclerosis is well known. Kellgren-Lawrence grading system, which is widely used to diagnose OAK, describes narrowing-marginal osteophyte in 4-grades but uses osteophyte at TS only as evidence of OAK without detailed-grading. However, kinematically the knee employs medial TS as an axis while medial and lateral compartments carry the load, suggesting that early OAK would occur sooner at TS than at compartment. Then, Kellgren-Lawrence system may be inadequate to diagnose early-stage OAK manifested as a subtle osteophyte at TS without narrowing-marginal osteophyte. This undiagnosed-OAK will deteriorate becoming a contributing factor in an increasing incidence of OAK. Methods: This study developed a radiographic OAK-marker based on both osteophyte at TS and compartment narrowing-marginal osteophyte and graded as normal, mild, moderate, and severe. With this marker, both knee radiographs of 1,728 patients with knee pain were analyzed. Results: Among 611 early-stage mild OAK, 562 or 92% started at TS and 49 or 8% at compartment. It suggests the initial development site of OAK, helping develop new site-specific radiographic classification system of OAK accurately to diagnose all severity of OAK at early, intermediate, or late-stage. It showed that Kellgren-Lawrence system missed 92.0% of early-stage mild OAK from diagnosis. Conclusions: A subtle osteophyte at TS is the earliest radiographic sign of OAK. A new radiographic classification system of OAK was suggested for accurate diagnosis of all OAK in severity and at stage. PMID:23675278

  14. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

    PubMed Central

    Fabbretti, Attilio; Schedlbauer, Andreas; Brandi, Letizia; Kaminishi, Tatsuya; Giuliodori, Anna Maria; Garofalo, Raffaella; Ochoa-Lizarralde, Borja; Takemoto, Chie; Yokoyama, Shigeyuki; Connell, Sean R.; Gualerzi, Claudio O.

    2016-01-01

    In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon–anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state. PMID:27071098

  15. The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.

    PubMed

    López-Carrasco, Amparo; Gago-Zachert, Selma; Mileti, Giuseppe; Minoia, Sofia; Flores, Ricardo; Delgado, Sonia

    2016-01-01

    Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5' ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (-) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.

  16. The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations

    PubMed Central

    López-Carrasco, Amparo; Gago-Zachert, Selma; Mileti, Giuseppe; Minoia, Sofia; Flores, Ricardo; Delgado, Sonia

    2016-01-01

    ABSTRACT Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (−) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae. PMID:26618399

  17. A tertiary structure model of the internal ribosome entry site (IRES) for methionine-independent initiation of translation.

    PubMed Central

    Kanamori, Y; Nakashima, N

    2001-01-01

    Cricket paralysis-like viruses have a dicistronic positive-strand RNA genome. These viruses produce capsid proteins through internal ribosome entry site (IRES)-mediated translation. The IRES element of one of these viruses, Plautia stall intestine virus (PSIV), forms a pseudoknot immediately upstream from the capsid coding sequence, and initiates translation from other than methionine. Previously, we estimated that the IRES element of PSIV consists of seven stem-loops using the program MFOLD; however, experimental evidence of the predicted structures was not shown, except for stem-loop VI, which was responsible for formation of the pseudoknot. To determine the whole structure of the PSIV-IRES element, we introduced compensatory mutations into the upstream MFOLD-predicted helical segments. Mutation analysis showed that stem-loop V exists as predicted, but stem-loop IV is shorter than predicted. The structure of stem-loop III is different from predicted, and stem-loops I and II are not necessary for IRES activity. In addition, we identified two new pseudoknots in the IRES element of PSIV. The complementary sequence segments that are responsible for formation of the two pseudoknots are also observed in cricket paralysis virus (CrPV) and CrPV-like viruses such as Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), himetobi P virus (HiPV), Triatoma virus (TrV), and black queen-cell virus (BQCV), although each sequence is distinct in each virus. Considering the three pseudoknots, we constructed a tertiary structure model of the PSIV-IRES element. This structural model is applicable to other CrPV-like viruses, indicating that other CrPV-like viruses can also initiate translation from other than methionine. PMID:11233983

  18. Initial single-site experience with the Ovation abdominal stent-graft system in patients with challenging aortoiliac anatomy.

    PubMed

    Irace, Luigi; Venosi, Stefano; Gattuso, Roberto; Laurito, Antonella; Pompa, Valentina; Pasinati, Giacomo; Bresadola, Luciano

    2016-12-01

    Aim of the study was to evaluate the initial results of endovascular aneurysm repair with the Ovation abdominal stent-graft system in patients with challenging aortoiliac anatomy. The Ovation stent-graft is an ultra-low profile, modular endovascular graft characterized by a 14F OD delivery system, active suprarenal fixation, and polymer-filled proximal rings that allow sealing in short (≥7 mm) proximal necks. Between November 2010 and January 2012, 14 patients with abdominal aortic aneurysms (AAA) (male: 100%, mean age: 76 years, mean AAA diameter: 54 mm) and challenging aortoiliac anatomy were treated with the Ovation endograft at a single centre. Mean patient follow-up was 5 months. All cases were performed under local or epidural anesthesia. No operative deaths or major complications were noted. A type I endoleak was detected on final angiogram in one case, which was successfully treated with additional ballooning and Palmaz stenting of the hooking landing zone. One patient died at 2 months due to myocardial infarction unrelated to the device or procedure. AAA-related mortality was 0%. No AAA rupture, AAA enlargement, type I or III endoleak, stent migration, access site complication, or conversion to open surgery was reported during follow-up. A type II endoleak was successfully treated with a right lumbar artery embolization at 4 months. Occlusion of an iliac axis was successfully managed with local fibrinolysis and implantation of a covered stent in the external iliac artery. Initial outcomes of this single-center experience suggest that the Ovation abdominal stent-graft system is a promising treatment in AAA patients with challenging aortoiliac anatomy.

  19. Dimerization in Highly Concentrated Solutions of Phosphoimidazolide Activated Mononucleotides

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia

    1997-01-01

    Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2- methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2(+) in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MelmpU and 2-MelmpC produce about 65% of oligomers including 4% of the 3',5'-Iinked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of intemucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MelmpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

  20. Dimerization in Highly Concentrated Solutions of Phosphoimidazolide Activated Monomucleotides

    NASA Astrophysics Data System (ADS)

    Kanavarioti, Anastassia

    1997-08-01

    Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3',5'-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

  1. Blockage of RNA polymerase II at a cyclobutane pyrimidine dimer and 6-4 photoproduct.

    PubMed

    Mei Kwei, Joan Seah; Kuraoka, Isao; Horibata, Katsuyoshi; Ubukata, Manabu; Kobatake, Eiry; Iwai, Shigenori; Handa, Hiroshi; Tanaka, Kiyoji

    2004-08-06

    The blockage of transcription elongation by RNA polymerase II (pol II) at a DNA damage site on the transcribed strand triggers a transcription-coupled DNA repair (TCR), which rapidly removes DNA damage on the transcribed strand of the expressed gene and allows the resumption of transcription. To analyze the effect of UV-induced DNA damage on transcription elongation, an in vitro transcription elongation system using pol II and oligo(dC)-tailed templates containing a cyclobutane pyrimidine dimer (CPD) or 6-4 photoproduct (6-4PP) at a specific site was employed. The results showed that pol II incorporated nucleotides opposite the CPD and 6-4PP and then stalled. Pol II formed a stable ternary complex consisting of pol II, the DNA damage template, and the nascent transcript. Furthermore, atomic force microscopy imaging revealed that pol II stalled at the damaged region. These findings may provide the basis for analysis of the initiation step of TCR.

  2. Properties of the two-dimensional heterogeneous Lennard-Jones dimers: An integral equation study

    NASA Astrophysics Data System (ADS)

    Urbic, Tomaz

    2016-11-01

    Structural and thermodynamic properties of a planar heterogeneous soft dumbbell fluid are examined using Monte Carlo simulations and integral equation theory. Lennard-Jones particles of different sizes are the building blocks of the dimers. The site-site integral equation theory in two dimensions is used to calculate the site-site radial distribution functions and the thermodynamic properties. Obtained results are compared to Monte Carlo simulation data. The critical parameters for selected types of dimers were also estimated and the influence of the Lennard-Jones parameters was studied. We have also tested the correctness of the site-site integral equation theory using different closures.

  3. Properties of the two-dimensional heterogeneous Lennard-Jones dimers: An integral equation study.

    PubMed

    Urbic, Tomaz

    2016-11-21

    Structural and thermodynamic properties of a planar heterogeneous soft dumbbell fluid are examined using Monte Carlo simulations and integral equation theory. Lennard-Jones particles of different sizes are the building blocks of the dimers. The site-site integral equation theory in two dimensions is used to calculate the site-site radial distribution functions and the thermodynamic properties. Obtained results are compared to Monte Carlo simulation data. The critical parameters for selected types of dimers were also estimated and the influence of the Lennard-Jones parameters was studied. We have also tested the correctness of the site-site integral equation theory using different closures.

  4. Regulation of L-Selectin–mediated Rolling through Receptor Dimerization

    PubMed Central

    Li, Xuan; Steeber, Douglas A.; Tang, Mimi L.K.; Farrar, Michael A.; Perlmutter, Roger M.; Tedder, Thomas F.

    1998-01-01

    L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin–GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by ∼700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors. PMID:9763619

  5. Palladium dimers adsorbed on graphene: A DFT study

    SciTech Connect

    Kaur, Gagandeep; Gupta, Shuchi; Dharamvir, Keya

    2015-05-15

    The 2D structure of graphene shows a great promise for enhanced catalytic activity when adsorbed with palladium. We performed a systematic density functional theory (DFT) study of the adsorption of palladium dimer (Pd{sub 2}) on graphene using SIESTA package, in the generalized gradient approximation (GGA). The adsorption energy, geometry, and charge transfer of Pd{sub 2}-graphene system are calculated. Both horizontal and vertical orientations of Pd{sub 2} on graphene are studied. Our calculations revealed that the minimum energy configuration for Pd dimer is parallel to the graphene sheet with its two atoms occupying centre of adjacent hexagonal rings of graphene sheet. Magnetic moment is induced for Pd dimer adsorbed on graphene in vertical orientation while horizontal orientation of Pd dimer on graphene do not exhibit magnetism. Insignificant energy differences among adsorption sites means that dimer mobility on the graphene sheet is high. There is imperceptible distortion of graphene sheet perpendicular to its plane. However, some lateral displacements are seen.

  6. Core Promoter Plasticity Between Maize Tissues and Genotypes Contrasts with Predominance of Sharp Transcription Initiation Sites[OPEN

    PubMed Central

    Li, Wei; Vidal, Mabel; Gray, John; Doseff, Andrea I.; Grotewold, Erich

    2015-01-01

    Core promoters are crucial for gene regulation, providing blueprints for the assembly of transcriptional machinery at transcription start sites (TSSs). Empirically, TSSs define the coordinates of core promoters and other regulatory sequences. Thus, experimental TSS identification provides an essential step in the characterization of promoters and their features. Here, we describe the application of CAGE (cap analysis of gene expression) to identify genome-wide TSSs used in root and shoot tissues of two maize (Zea mays) inbred lines (B73 and Mo17). Our studies indicate that most TSS clusters are sharp in maize, similar to mice, but distinct from Arabidopsis thaliana, Drosophila melanogaster, or zebra fish, in which a majority of genes have broad-shaped TSS clusters. We established that ∼38% of maize promoters are characterized by a broader TATA-motif consensus, and this motif is significantly enriched in genes with sharp TSSs. A noteworthy plasticity in TSS usage between tissues and inbreds was uncovered, with ∼1500 genes showing significantly different dominant TSSs, sometimes affecting protein sequence by providing alternate translation initiation codons. We experimentally characterized instances in which this differential TSS utilization results in protein isoforms with additional domains or targeted to distinct subcellular compartments. These results provide important insights into TSS selection and gene expression in an agronomically important crop. PMID:26628745

  7. Transcription initiation sites within an IS2 insertion in a Gal-constitutive mutant of Escherichia coli.

    PubMed Central

    Hinton, D M; Musso, R E

    1982-01-01

    Insertion of the insertion sequence Is2(I) directly before the galE gene of the galactose operon results in a Gal minus phenotype (1, 2). The Gal-constitutive allele galc200 (and its deletion derivative galc200 delta 31) arise from such a Gal minus mutant by the insertion of LS2(II) DNA within the LS2(I) sequence (3). We have transcribed in vitro a DNA template representing the IS2-galE region of galc200 delta 31. Gal-directed transcription initiates at two sites within the IS2(I) sequence, 51 and 52 bp from the IS2-galE junction. The promoter for these transcripts, Pgal200 delta 31, is composed of a novel joint between a -10 region from the IS2(I) DNA and a -35 region contributed by the IS2(II) insertion. No promoters intrinsic to the 121 bp of the IS2(II) sequence also present on the template were detected. The relevance of Pgal200 delta 31 to the Galc phenotype of galc200 and to general mechanisms for the constitutive expression of genes adjacent to IS2 is discussed. Images PMID:6291000

  8. An initiation site of DNA replication with transcriptional enhancer activity present upstream of the c-myc gene.

    PubMed Central

    Iguchi-Ariga, S M; Okazaki, T; Itani, T; Ogata, M; Sato, Y; Ariga, H

    1988-01-01

    We have previously reported that c-myc protein may promote cellular DNA replication by binding to initiation sites of replication. Here we report that a putative origin of human cellular DNA replication (ori) is present at approximately 2 kb upstream of the coding region of the c-myc gene itself. The c-myc protein, or protein(s) complexed with c-myc protein, bind to the upstream region (approximately 200 bp in length) which has transcriptional enhancer activity as well as autonomously replicating activity in human cells, suggesting that the c-myc protein may be an enhancer binding protein as well as a DNA replication protein. Results with deletion mutants suggest that the sequence essential to the origin of DNA replication may be adjacent to, but cannot be clearly separated from, the sequence responsible for enhancer activity. Furthermore, when cloned DNA containing putative c-myc protein binding sequences was transfected as competitor into HL-60 cells, expression of c-myc was inhibited, suggesting that c-myc protein itself may be necessary for c-myc expression. Images PMID:3053161

  9. Intracranial meningeal hemangiopericytoma: Recurrences at the initial and distant intracranial sites and extraneural metastases to multiple organs.

    PubMed

    Wei, Guangquan; Kang, Xiaowei; Liu, Xianping; Tang, Xing; Li, Qinlong; Han, Juntao; Yin, Hong

    2015-07-01

    Regardless of the controversial pathogenesis, intracranial meningeal hemangiopericytoma (M-HPC) is a rare, highly cellular and vascularized mesenchymal tumor that is characterized by a high tendency for recurrence and extraneural metastasis, despite radical excision and postoperative radiotherapy. M-HPC shares similar clinical manifestations and radiological findings with meningioma, which causes difficulty in differentiation of this entity from those prognostically favorable mimics prior to surgery. Treatment of M-HPC, particularly in metastatic settings, remains a challenge. A case is described of primary M-HPC with recurrence at the initial and distant intracranial sites and extraneural multiple-organ metastases in a 36-year-old female. The metastasis of M-HPC was extremely extensive, and to the best of our knowledge this is the first case of M-HPC with delayed metastasis to the bilateral kidneys. The data suggests that preoperative computed tomography and magnetic resonance imaging could provide certain diagnostic clues and useful information for more optimal treatment planning. The results may imply that novel drugs, such as temozolomide and bevacizumab, as a component of multimodality therapy of M-HPC may deserve further investigation.

  10. Kinetics of DNA Tile Dimerization

    PubMed Central

    2015-01-01

    Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile–tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency. PMID:24794259

  11. Kinetics of DNA tile dimerization.

    PubMed

    Jiang, Shuoxing; Yan, Hao; Liu, Yan

    2014-06-24

    Investigating how individual molecular components interact with one another within DNA nanoarchitectures, both in terms of their spatial and temporal interactions, is fundamentally important for a better understanding of their physical behaviors. This will provide researchers with valuable insight for designing more complex higher-order structures that can be assembled more efficiently. In this report, we examined several spatial factors that affect the kinetics of bivalent, double-helical (DH) tile dimerization, including the orientation and number of sticky ends (SEs), the flexibility of the double helical domains, and the size of the tiles. The rate constants we obtained confirm our hypothesis that increased nucleation opportunities and well-aligned SEs accelerate tile-tile dimerization. Increased flexibility in the tiles causes slower dimerization rates, an effect that can be reversed by introducing restrictions to the tile flexibility. The higher dimerization rates of more rigid tiles results from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. We believe that the results presented here will assist in improved implementation of DNA tile based algorithmic self-assembly, DNA based molecular robotics, and other specific nucleic acid systems, and will provide guidance to design and assembly processes to improve overall yield and efficiency.

  12. Decreasing D-dimer after recent negative computed tomographic pulmonary angiogram does not rule out pulmonary embolism.

    PubMed

    Lo, Bruce M

    2013-06-01

    An algorithmic approach to testing utilizing risk stratification and quantitative D-dimer has been considered an acceptable approach to ruling out pulmonary embolism (PE). When D-dimer is elevated, further testing for PE is indicated. However, no evidence exists to guide practitioners when patients return after a recent negative workup for PE who previously had an elevated D-dimer. This case describes a patient who initially had an elevated D-dimer with negative workup for PE who, on repeat visit, had a decreasing D-dimer but was diagnosed with a PE. When evaluating patients after a negative workup for PE after an elevated D-dimer, a decrease in D-dimer cannot be used to rule out PE.

  13. Plasma D-dimer may predict poor functional outcomes through systemic complications after aneurysmal subarachnoid hemorrhage.

    PubMed

    Fukuda, Hitoshi; Lo, Benjamin; Yamamoto, Yu; Handa, Akira; Yamamoto, Yoshiharu; Kurosaki, Yoshitaka; Yamagata, Sen

    2016-08-12

    OBJECTIVE Plasma D-dimer levels elevate during acute stages of aneurysmal subarachnoid hemorrhage (SAH) and are associated with poor functional outcomes. However, the mechanism in which D-dimer elevation on admission affects functional outcomes remains unknown. The aim of this study is to clarify whether D-dimer levels on admission are correlated with systemic complications after aneurysmal SAH, and to investigate their additive predictive value on conventional risk factors for poor functional outcomes. METHODS A total of 187 patients with aneurysmal SAH were retrospectively analyzed from a single-center, observational cohort database. Correlations of plasma D-dimer levels on admission with patient characteristics, initial presentation, neurological complications, and systemic complications were identified. The authors also evaluated the additive value of D-dimer elevation on admission for poor functional outcomes by comparing predictive models with and without D-dimer. RESULTS D-dimer elevation on admission was associated with increasing age, female sex, and severity of SAH. Patients with higher D-dimer levels had increased likelihood of nosocomial infections (OR 1.22 [95% CI 1.07-1.39], p = 0.004), serum sodium disorders (OR 1.11 [95% CI 1.01-1.23], p = 0.033), and cardiopulmonary complications (OR 1.20 [95% CI 1.04-1.37], p = 0.01) on multivariable analysis. D-dimer elevation was an independent risk factor of poor functional outcome (modified Rankin Scale Score 3-6, OR 1.50 [95% CI 1.15-1.95], p = 0.003). A novel prediction model with D-dimer had significantly better discrimination ability for poor outcomes than conventional models without D-dimer. CONCLUSIONS Elevated D-dimer levels on admission were independently correlated with systemic complication, and had an additive value for outcome prediction on conventional risk factors after aneurysmal SAH.

  14. Optimized spin crossings and transition states for short-range electron transfer in transition metal dimers.

    PubMed

    Lundberg, Marcus; Siegbahn, Per E M

    2005-05-26

    Electron-transfer reactions in eight mixed-valence manganese dimers are studied using B3LYP. One of the dimers is a model of the active site of manganese catalase, while another represents a basic building block of the oxygen-evolving complex in photosystem II. The adiabatic reactions are characterized by fully optimized transition states where the single imaginary frequency represents the electron-transfer coordinate. When there is antiferromagnetic coupling between different high-spin centers, electron transfer must be accompanied by a spin transition. Spin transitions are characterized by minimum-energy crossing points between spin surfaces. Three reaction mechanisms have been investigated. First, a single-step reaction where spin flip is concerted with electron transfer. Second, an initial transition to a center with intermediate spin that can be followed by electron transfer. Third, an initial transition to a ferromagnetic state from which the electron can be transferred adiabatically. The complexes prefer the third route with rate-determining barriers ranging from 5.7 kcal/mol to 17.2 kcal/mol for different complexes. The origins of these differences are discussed in terms of oxidation states and ligand environments. Many DFT functionals overestimate charge-transfer interactions, but for the present complexes, the error should be limited because of short Mn-Mn distances.

  15. Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

    PubMed

    Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

    2016-07-01

    Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition.

  16. A Model for Dimerization of the SOX Group E Transcription Factor Family.

    PubMed

    Ramsook, Sarah N; Ni, Joyce; Shahangian, Shokofeh; Vakiloroayaei, Ana; Khan, Naveen; Kwan, Jamie J; Donaldson, Logan W

    2016-01-01

    Group E members of the SOX transcription factor family include SOX8, SOX9, and SOX10. Preceding the high mobility group (HMG) domain in each of these proteins is a thirty-eight amino acid region that supports the formation of dimers on promoters containing tandemly inverted sites. The purpose of this study was to obtain new structural insights into how the dimerization region functions with the HMG domain. From a mutagenic scan of the dimerization region, the most essential amino acids of the dimerization region were clustered on the hydrophobic face of a single, predicted amphipathic helix. Consistent with our hypothesis that the dimerization region directly contacts the HMG domain, a peptide corresponding to the dimerization region bound a preassembled HMG-DNA complex. Sequence conservation among Group E members served as a basis to identify two surface exposed amino acids in the HMG domain of SOX9 that were necessary for dimerization. These data were combined to make a molecular model that places the dimerization region of one SOX9 protein onto the HMG domain of another SOX9 protein situated at the opposing site of a tandem promoter. The model provides a detailed foundation for assessing the impact of mutations on SOX Group E transcription factors.

  17. Water dimer equilibrium constant of saturated vapor

    NASA Astrophysics Data System (ADS)

    Malomuzh, N. P.; Mahlaichuk, V. N.; Khrapatyi, S. V.

    2014-08-01

    The value and temperature dependence of the dimerization constant for saturated water vapor are determined. A general expression that links the second virial coefficient and the dimerization constant is obtained. It is shown that the attraction between water monomers and dimers is fundamental, especially at T > 350 K. The range of application for the obtained results is determined.

  18. Properties of the Lennard-Jones dimeric fluid in two dimensions: an integral equation study.

    PubMed

    Urbic, Tomaz; Dias, Cristiano L

    2014-03-07

    The thermodynamic and structural properties of the planar soft-sites dumbbell fluid are examined by Monte Carlo simulations and integral equation theory. The dimers are built of two Lennard-Jones segments. Site-site integral equation theory in two dimensions is used to calculate the site-site radial distribution functions for a range of elongations and densities and the results are compared with Monte Carlo simulations. The critical parameters for selected types of dimers were also estimated. We analyze the influence of the bond length on critical point as well as tested correctness of site-site integral equation theory with different closures. The integral equations can be used to predict the phase diagram of dimers whose molecular parameters are known.

  19. Properties of the Lennard-Jones dimeric fluid in two dimensions: An integral equation study

    SciTech Connect

    Urbic, Tomaz; Dias, Cristiano L.

    2014-03-07

    The thermodynamic and structural properties of the planar soft-sites dumbbell fluid are examined by Monte Carlo simulations and integral equation theory. The dimers are built of two Lennard-Jones segments. Site-site integral equation theory in two dimensions is used to calculate the site-site radial distribution functions for a range of elongations and densities and the results are compared with Monte Carlo simulations. The critical parameters for selected types of dimers were also estimated. We analyze the influence of the bond length on critical point as well as tested correctness of site-site integral equation theory with different closures. The integral equations can be used to predict the phase diagram of dimers whose molecular parameters are known.

  20. Proton collisions with the water dimer at keV energies

    NASA Astrophysics Data System (ADS)

    Quinet, O.; Deumens, E.; Öhrn, Y.

    Proton collisions with the water dimer are studied using a nonadiabatic, direct, time-dependent approach called electron nuclear dynamics (END). Fragmentation of the water dimer in collisions with protons at energies of 5.0, 1.0 keV and 200 eV is the primary aim of this initial study of water clusters using END. We report on the initial fragmentation dynamic, that is, for times less than 200 fs.

  1. Risk factors for superficial vs deep/organ-space surgical site infections: implications for quality improvement initiatives.

    PubMed

    Lawson, Elise H; Hall, Bruce Lee; Ko, Clifford Y

    2013-09-01

    Surgical site infections (SSIs) are the focus of numerous quality improvement initiatives because they are a common and costly cause of potentially preventable patient morbidity. Superficial and deep/organ-space SSIs differ in terms of anatomical location and clinical severity. To identify risk factors that are uniquely predictive of superficial vs deep/organ-space SSIs occurring after colectomy procedures. Retrospective cohort study. American College of Surgeons National Surgical Quality Improvement Program. Patients undergoing colectomy procedures in 2011 were identified by Current Procedural Terminology codes. Colectomy procedures. We compared rates of superficial SSI and deep/organ-space SSI associated with perioperative variables of interest: demographics; preoperative clinical severity, risk factors, and comorbidities and variables related to the hospitalization or procedure. Hierarchical multivariable logistic regression models were developed to identify risk-adjusted predictors of each SSI type. Among 27 011 patients identified from 305 hospitals, 6.2% developed a superficial SSI and 4.7% developed a deep/organ-space SSI. Risk factors common to the occurrence of both SSI types were identified: open surgery (vs laparoscopic) and current smoker. Risk factors with differential effects on each SSI type included specific postoperative diagnoses, disseminated cancer, and irradiation therapy, which were all associated with increased odds of deep/organ-space SSI only. The graded relationship between increasing body mass index and SSI occurrence appeared to be stronger for superficial SSI. Risk factors for superficial SSI and deep/organ-space SSI vary in terms of magnitude and significance, suggesting that these SSI types are somewhat different disease processes. Groups interested in preventing SSIs might improve success by considering these SSI types independently for root-cause analyses and development of best practices and interventions.

  2. Percolation of heteronuclear dimers irreversibly deposited on square lattices

    NASA Astrophysics Data System (ADS)

    Gimenez, M. C.; Ramirez-Pastor, A. J.

    2016-09-01

    The percolation problem of irreversibly deposited heteronuclear dimers on square lattices is studied. A dimer is composed of two segments, and it occupies two adjacent adsorption sites. Each segment can be either a conductive segment (segment type A ) or a nonconductive segment (segment type B ). Three types of dimers are considered: A A , B B , and A B . The connectivity analysis is carried out by accounting only for the conductive segments (segments type A ). The model offers a simplified representation of the problem of percolation of defective (nonideal) particles, where the presence of defects in the system is simulated by introducing a mixture of conductive and nonconductive segments. Different cases were investigated, according to the sequence of deposition of the particles, the types of dimers involved in the process, and the degree of alignment of the deposited objects. By means of numerical simulations and finite-size scaling analysis, the complete phase diagram separating a percolating from a nonpercolating region was determined for each case. Finally, the consistency of our results was examined by comparing with previous data in the literature for linear k -mers (particles occupying k adjacent sites) with defects.

  3. Percolation of heteronuclear dimers irreversibly deposited on square lattices.

    PubMed

    Gimenez, M C; Ramirez-Pastor, A J

    2016-09-01

    The percolation problem of irreversibly deposited heteronuclear dimers on square lattices is studied. A dimer is composed of two segments, and it occupies two adjacent adsorption sites. Each segment can be either a conductive segment (segment type A) or a nonconductive segment (segment type B). Three types of dimers are considered: AA, BB, and AB. The connectivity analysis is carried out by accounting only for the conductive segments (segments type A). The model offers a simplified representation of the problem of percolation of defective (nonideal) particles, where the presence of defects in the system is simulated by introducing a mixture of conductive and nonconductive segments. Different cases were investigated, according to the sequence of deposition of the particles, the types of dimers involved in the process, and the degree of alignment of the deposited objects. By means of numerical simulations and finite-size scaling analysis, the complete phase diagram separating a percolating from a nonpercolating region was determined for each case. Finally, the consistency of our results was examined by comparing with previous data in the literature for linear k-mers (particles occupying k adjacent sites) with defects.

  4. Site-nurse initiated Adherence and Symptom Support Telephone Calls for HIV-positive individuals starting antiretroviral therapy, ACTG 5031, a substudy of ACTG 384.

    PubMed Central

    Robbins, Gregory K.; Testa, Marcia A.; Su, Max; Safren, Steven A.; Morse, Gene; Lammert, Sara; Shafer, Robert W.; Reynolds, Nancy R.; Chesney, Margaret A.

    2013-01-01

    Background: Effective and easy to implement interventions to improve adherence to antiretroviral therapy are needed. Objective: To compare a site-nurse initiated adherence and symptom support telephone calls for HIV-positive individuals starting antiretroviral therapy compare to the study site’s standard of care. Methods: A randomized controlled trial of site-nurse initiated adherence and symptom support telephone calls for HIV-positive individuals starting antiretrovirals. Subjects were randomized to receive site-nurse initiated telephone calls (intervention) or no additional calls above the site’s standard of care (control). Subjects received calls 1-3 days after initiating antiretrovirals, weeks 1, 2, 3, 6, 10, 14, 18, 22, 26, and every 8 weeks thereafter. Self-reported adherence was captured during study visits. Results: A total of 333 subjects starting antiretrovirals as part of ACTG 384 were co-enrolled into ACTG 5031. Subjects were followed for up to 160 weeks and were contacted for 74% of scheduled calls. There was no significant difference in proportion of patients with >95% mean Total Adherence, 87.9% and 91.2% (p=0.34) and mean self-reported Total Adherence, 97.9% and 98.4% in the intervention and control, respectively, or in symptom distress and clinical endpoints. Conclusions: In the context of a clinical trial, where self-reported adherence was exceptionally high, the site-nurse initiated telephone calls did not further improve self-reported adherence, symptom distress or clinical outcomes. PMID:24144900

  5. Feasibility Study of Biopower in East Helena, Montana. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Moriarty, K.

    2013-02-01

    The U.S. Environmental Protection Agency (EPA) developed the RE-Powering America's Land initiative to reuse contaminated sites for renewable energy generation when aligned with the community's vision for the site. The former American Smelting and Refining Company (Asarco) smelter in East Helena, Montana, was selected for a feasibility study under the initiative. Biomass was chosen as the renewable energy resource based on the wood products industry in the area. Biopower was selected as the technology based on Montana's renewable portfolio standard (RPS) requiring utilities to purchase renewable power.

  6. Influence of Linker Length and Composition on Enzymatic Activity and Ribosomal Binding of Neomycin Dimers

    PubMed Central

    Watkins, Derrick; Kumar, Sunil; Green, Keith D.

    2015-01-01

    The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA. PMID:25896697

  7. Reactions of ultracold alkali-metal dimers

    SciTech Connect

    Zuchowski, Piotr S.; Hutson, Jeremy M.

    2010-06-15

    We investigate the energetics of reactions involving pairs of alkali-metal dimers. Atom exchange reactions to form homonuclear dimers are energetically allowed for some but not all of the heteronuclear dimers. We carry out high-level electronic structure calculations on the potential energy surfaces of all the heteronuclear alkali-metal trimers and show that trimer formation reactions are always energetically forbidden for low-lying singlet states of the dimers. The results have important implications for the stability of quantum gases of alkali-metal dimers.

  8. Fiber optic D dimer biosensor

    DOEpatents

    Glass, R.S.; Grant, S.A.

    1999-08-17

    A fiber optic sensor for D dimer (a fibrinolytic product) can be used in vivo (e.g., in catheter-based procedures) for the diagnosis and treatment of stroke-related conditions in humans. Stroke is the third leading cause of death in the United States. It has been estimated that strokes and stroke-related disorders cost Americans between $15-30 billion annually. Relatively recently, new medical procedures have been developed for the treatment of stroke. These endovascular procedures rely upon the use of microcatheters. These procedures could be facilitated with this sensor for D dimer integrated with a microcatheter for the diagnosis of clot type, and as an indicator of the effectiveness, or end-point of thrombolytic therapy. 4 figs.

  9. Fiber optic D dimer biosensor

    DOEpatents

    Glass, Robert S.; Grant, Sheila A.

    1999-01-01

    A fiber optic sensor for D dimer (a fibrinolytic product) can be used in vivo (e.g., in catheter-based procedures) for the diagnosis and treatment of stroke-related conditions in humans. Stroke is the third leading cause of death in the United States. It has been estimated that strokes and stroke-related disorders cost Americans between $15-30 billion annually. Relatively recently, new medical procedures have been developed for the treatment of stroke. These endovascular procedures rely upon the use of microcatheters. These procedures could be facilitated with this sensor for D dimer integrated with a microcatheter for the diagnosis of clot type, and as an indicator of the effectiveness, or end-point of thrombolytic therapy.

  10. Mechanism of FGF receptor dimerization and activation

    NASA Astrophysics Data System (ADS)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  11. mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

    PubMed Central

    Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

    2004-01-01

    Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

  12. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    PubMed

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  13. Mars Science Laboratory Curiosity rover initial Mastcam geomorphologic and multispectral characterization of the Gale crater field site

    NASA Astrophysics Data System (ADS)

    Bell, J. F.; Malin, M.; Maki, J.; Dietrich, W. E.; Edgett, K. S.; Edwards, L.; Garvin, J. B.; Hallet, B.; Herkenhoff, K. E.; Heydari, E.; Johnson, J. R.; Kah, L. C.; Lemmon, M. T.; Minitti, M.; Olson, T. S.; Parker, T. J.; Rice, M. S.; Rowland, S. K.; Schieber, J.; Sletten, R. S.; Sullivan, R. J.; Sumner, D. Y.; Thomas, P. C.; Yingst, R.; Team, M.

    2013-12-01

    The Mars Science Laboratory Curiosity rover landed in Gale crater on August 6, 2012 and has been enabling the exploration of a variety of geologic terrains between the rover's landing site at Bradbury Rise and the nearby topographic low point known as Yellowknife Bay. Curiosity carries a multispectral imaging system known as Mastcam, which consists of two boresighted CCD cameras, one of which acquires relatively wide field images (34-mm focal length, 18.4x15 degree FOV) and the other of which obtains narrower-angle telephoto images (100-mm focal length, 6.3x5.1 degree FOV). Each of these cameras has an 8-position filter wheel to enable imaging through broadband RGB Bayer filtes, nine specific narrowband filters in the 445 to 1012 nm region to enabled limited detectability of certain ferric, ferrous, and hydrated minerals, and neutral density solar filters for monitoring of atmospheric opacity. The Mastcams acquire images designed primarily to address specific scientific goals in geology, mineralogy, and atmospheric science, but also to support operational decisions related to rover driving, arm instrument placement, and rover subsystems status. Here we provide an overview of the initial scientific imaging results from the Mastcam investigation, from sol 0 (landing sol) through the end of the drilling campaign in Yellowknife Bay and the beginning of the long drive from there to the base of Mt. Sharp. A diversity of materials exposed at the surface have been encountered. This includes angular to sub-angular rock fragments scattered across the surface, boulder to fine gravel in size, variably dusty, and commonly fine grained. Thin outcrops of pebble to gravel conglomerate have been encountered across Bradbury rise. Granular ripples and other fine grained deposits were periodically encountered. In the wind-eroded Yellowknife Bay area, extensive polygonally fractured outcrops of sandstone and mudstone (with light-toned fracture fills) were discovered. The occurrence of

  14. The age of IODP Site 1473, Atlantis Bank: Constraints from initial zircon U-Pb dating and geochemistry by SIMS

    NASA Astrophysics Data System (ADS)

    Cheadle, M. J.; John, B. E.; Coble, M. A.; Koepke, J.; Rioux, M. E.; Liu, C. Z.; Expedition 360 Scientists, I.

    2016-12-01

    IODP Hole U1473A, sited at the top of the Atlantis Bank oceanic core complex (OCC) on the southwest Indian Ridge, extends 809m into gabbroic lower ocean crust. The recovered core consists dominantly of olivine gabbro, with subordinate gabbro, Fe-Ti oxide gabbro and felsic veins. Geochemical data define three magmatic series in the core. Ninety samples of evolved gabbro and felsic veins were collected to carry out a comprehensive geochronologic study of the core. When combined with high-precision data from nearby ODP Holes 735B and 1105A (Rioux et al., 2016), the new data will ultimately provide a 4-D view of the lateral continuity and evolution of the lower crust, and the process of magmatic accretion in space and time. Initial zircon U-Pb dating using the Stanford-USGS SHRIMP-RG ion-microprobe yielded a near solidus ( 850oC), weighted mean 206Pb/238U, age of 11.86±0.09 Ma (MSWD =1.01; n=28) for 4 samples of felsic veins and Fe-Ti gabbro from the lower two magmatic series of Hole U1473A. The difference between this age and the high precision TIMS age for ODP Hole 1105A is consistent with an asymmetric spreading rate of 14.1 mm/yr for the OCC. Dates for felsic veins from magmatic series II and felsic veins and oxide gabbro from magmatic series III overlap within uncertainty, however a felsic vein from the 50m thick uppermost series I has a date of 12.12+/-0.18 Ma, and thus could indicate that magmatic series I predates the underlying series, as observed in core from ODP Hole 735B. The presence of any age differences will be subsequently explored by high-precision ID-TIMS dating. The zircon compositions are similar to those from ODP Hole 735B; Hf and U concentrations range from 7,600-33,200 and 2-5,700 ppm, respectively, and Ti-in-zircon temperatures range from 974-718oC. They are typical ocean zircons as defined by the trace element discrimination plots of Grimes et al. (2015). However, the zircons from Hole U1473A show more evidence for hydrothermal alteration

  15. Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface.

    PubMed

    Monteiro, Ana C; Luissint, Anny-Claude; Sumagin, Ronen; Lai, Caroline; Vielmuth, Franziska; Wolf, Mattie F; Laur, Oskar; Reiss, Kerstin; Spindler, Volker; Stehle, Thilo; Dermody, Terence S; Nusrat, Asma; Parkos, Charles A

    2014-05-01

    Junctional adhesion molecule-A (JAM-A) is a tight junction-associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.

  16. Polyhomologation based on in situ generated boron-thexyl-silaboracyclic initiating sites: a novel strategy towards the synthesis of polyethylene-based complex architectures.

    PubMed

    Zhang, Zhen; Zhang, Hefeng; Gnanou, Yves; Hadjichristidis, Nikos

    2015-06-21

    A novel strategy, based on the in situ generated boron-thexyl-silaboracyclic initiating sites for the polyhomologation of dimethylsulfoxonium methylide, has been developed for the synthesis of complex polyethylene-based architectures. As examples, the synthesis of a 4-arm polyethylene star, three (polystyrene)(polyethylene)2 3-miktoarm stars and a PE-branched double graft copolymer is given.

  17. Changing Transcriptional Initiation Sites and Alternative 5'- and 3'-Splice Site Selection of the First Intron Deploys the Arabidopsis Protein Isoaspartyl Methyltransferase2 Variants to Different Subcellular Compartments

    USDA-ARS?s Scientific Manuscript database

    Arabidopsis thaliana (L.) Heynh. possesses two PROTEIN-L-ISOASPARTATE METHYLTRANSFERASE (PIMT), genes encoding an enzyme (EC 2.1.1.77) capable of converting uncoded, L-isoaspartyl residues, arising spontaneously at L-asparaginyl and L-aspartyl sites in proteins, to L-aspartate. PIMT2 produces at lea...

  18. Evaluating a complex, multi-site, community-based program to improve healthcare quality: the summative research design for the Aligning Forces for Quality initiative.

    PubMed

    Scanlon, Dennis P; Wolf, Laura J; Alexander, Jeffrey A; Christianson, Jon B; Greene, Jessica; Jean-Jacques, Muriel; McHugh, Megan; Shi, Yunfeng; Leitzell, Brigitt; Vanderbrink, Jocelyn M

    2016-08-01

    The Aligning Forces for Quality (AF4Q) initiative was the Robert Wood Johnson Foundation's (RWJF's) signature effort to increase the overall quality of healthcare in targeted communities throughout the country. In addition to sponsoring this 16-site complex program, RWJF funded an independent scientific evaluation to support objective research on the initiative's effectiveness and contributions to basic knowledge in 5 core programmatic areas. The research design, data, and challenges faced during the summative evaluation phase of this near decade-long program are discussed. A descriptive overview of the summative research design and its development for a multi-site, community-based, healthcare quality improvement initiative is provided. The summative research design employed by the evaluation team is discussed. The evaluation team's summative research design involved a data-driven assessment of the effectiveness of the AF4Q program at large, assessments of the impact of AF4Q in the specific programmatic areas, and an assessment of how the AF4Q alliances were positioned for the future at the end of the program. The AF4Q initiative was the largest privately funded community-based healthcare improvement initiative in the United States to date and was implemented at a time of rapid change in national healthcare policy. The implementation of large-scale, multi-site initiatives is becoming an increasingly common approach for addressing problems in healthcare. The summative evaluation research design for the AF4Q initiative, and the lessons learned from its approach, may be valuable to others tasked with evaluating similarly complex community-based initiatives.

  19. Replication in the amplified dihydrofolate reductase domain in CHO cells may initiate at two distinct sites, one of which is a repetitive sequence element.

    PubMed

    Anachkova, B; Hamlin, J L

    1989-02-01

    To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.

  20. Tumor-derived mesenchymal stem cells and orthotopic site increase the tumor initiation potential of putative mouse mammary cancer stem cells derived from MMTV-PyMT mice.

    PubMed

    Lanza, Denise Grant; Ma, Jun; Guest, Ian; Uk-Lim, Chang; Glinskii, Anna; Glinsky, Gennadi; Sell, Stewart

    2012-12-01

    The ability to transplant mammary cancer stem cells, identified by the phenotype CD24(+)CD29(+)CD49f(+)Sca-1(low), is dependent on the microenvironment in which the cells are placed. Using the MMTV-PyMT mouse model of mammary cancer, we now report two methods of tumor growth enhancement: contributions of tumor stroma in the form of tumor-derived mesenchymal stem cells and orthotopic vs. heterotopic transplantation sites. To support evidence of stem cell function, tumor-derived mesenchymal stem cells differentiated into adipocyte- and osteocyte-like cells after culture in specific medium. Co-injection of tumor-initiating cells with tumor-derived mesenchymal stem cells significantly increased tumor initiation compared to subcutaneous injection of TICs alone; co-injection also allowed tumor initiation with a single TIC. Interestingly, we observed the formation of sarcomas after co-injections of tumor-derived mesenchymal stem cells or mouse embryonic fibroblasts with TICs; sarcomas are not observed in spontaneous MMTV-PyMT tumors and rarely observed in injections of TICs alone. Tumor initiation was also significantly increased in the orthotopic injection site compared to heterotopic injections. We conclude that tumor stroma and orthotopic sites both enhance tumor initiation by mammary cancer stem cells.

  1. A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins.

    PubMed

    Jastrzebska, Beata; Comar, William D; Kaliszewski, Megan J; Skinner, Kevin C; Torcasio, Morgan H; Esway, Anthony S; Jin, Hui; Palczewski, Krzysztof; Smith, Adam W

    2017-01-10

    G protein-coupled receptors (GPCRs) detect a wide variety of physical and chemical signals and transmit that information across the cellular plasma membrane. Dimerization is a proposed modulator of GPCR signaling, but the structure and stability of class A GPCR dimerization have been difficult to establish. Here we investigated the dimerization affinity and binding interface of human cone opsins, which initiate and sustain daytime color vision. Using a time-resolved fluorescence approach, we found that human red cone opsin exhibits a strong propensity for dimerization, whereas the green and blue cone opsins do not. Through mutagenesis experiments, we identified a dimerization interface in the fifth transmembrane helix of human red cone opsin involving amino acids I230, A233, and M236. Insights into this dimerization interface of red cone opsin should aid ongoing investigations of the structure and function of GPCR quaternary interactions in cell signaling. Finally, we demonstrated that the same residues needed for dimerization are also partially responsible for the spectral tuning of red cone opsin. This last observation has the potential to open up new lines of inquiry regarding the functional role of dimerization for red cone opsin.

  2. Oxaliplatin Binding to Human Copper Chaperone Atox1 and Protein Dimerization.

    PubMed

    Belviso, Benny D; Galliani, Angela; Lasorsa, Alessia; Mirabelli, Valentina; Caliandro, Rocco; Arnesano, Fabio; Natile, Giovanni

    2016-07-05

    Copper trafficking proteins have been implicated in the cellular response to platinum anticancer drugs. We investigated the reaction of the chaperone Atox1 with an activated form of oxaliplatin, the third platinum drug to reach worldwide approval. Unlike cisplatin, which contains monodentate ammines, oxaliplatin contains chelated 1,2-diaminocyclohexane (DACH), which is more resistant to displacement by nucleophiles. In solution, one or two {Pt(DACH)(2+)} moieties bind to the conserved CXXC metal-binding motif of Atox1; in the latter case the two sulfur atoms likely bridging the two platinum units. At longer reaction times, a dimeric species is formed whose composition, Atox12·Pt(2+)2, indicates complete loss of the diamine ligands. Such a dimerization process is accompanied by partial unfolding of the protein. Crystallization experiments aiming at the characterization of the monomeric species have afforded, instead, a dimeric species resembling that already obtained by Boal and Rosenzweig in a similar reaction performed with cisplatin. However, while in the latter case there was only one Pt-binding site (0.4 occupancy) made of four sulfur atoms of the CXXC motifs of the two Atox1 chains in a tetrahedral arrangement, we found, in addition, a secondary Pt-binding site involving Cys41 of the B chain (0.25 occupancy). Moreover, both platinum atoms have lost their diamines. Thus, there appears to be little relationship between what is observed in solution and what is formed in the solid state. Since full occupancy of the tetrahedral cavity is a common feature of all Atox1 dimeric structures obtained with other metal ions (Cu(+), Cd(2+), and Hg(2+)), we propose that in the case of platinum, where the occupancy is only 0.4, the remaining cavities are occupied by Cu(+) ions. Experimental evidence is reported in support of the latter hypothesis. Our proposal represents a meeting point between the initial proposal of Boal and Rosenzweig (0.4 Pt occupancy) and the

  3. Cryptic protein priming sites in two different domains of duck hepatitis B virus reverse transcriptase for initiating DNA synthesis in vitro.

    PubMed

    Boregowda, Rajeev K; Lin, Li; Zhu, Qin; Tian, Fang; Hu, Jianming

    2011-08-01

    Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.

  4. Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro▿

    PubMed Central

    Boregowda, Rajeev K.; Lin, Li; Zhu, Qin; Tian, Fang; Hu, Jianming

    2011-01-01

    Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming. PMID:21593164

  5. Direct Observation of Thymine Dimer Repair in DNA by Photolyase

    NASA Astrophysics Data System (ADS)

    Zhong, Dongping

    2006-03-01

    Departments of Physics, Chemistry, and Biochemistry, Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University, Columbus, 191 West Woodruff Avenue, OH 43210. Photolyase uses light energy to split ultraviolet-induced cyclobutane pyrimidine dimers in damaged DNA, but its molecular mechanism has never been directly revealed. We report here the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site with no net electron change in redox states of the flavin cofactor.

  6. GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides

    PubMed Central

    Zhang, Yu; Degen, David; Ho, Mary X; Sineva, Elena; Ebright, Katherine Y; Ebright, Yon W; Mekler, Vladimir; Vahedian-Movahed, Hanif; Feng, Yu; Yin, Ruiheng; Tuske, Steve; Irschik, Herbert; Jansen, Rolf; Maffioli, Sonia; Donadio, Stefano; Arnold, Eddy; Ebright, Richard H

    2014-01-01

    Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance. DOI: http://dx.doi.org/10.7554/eLife.02450.001 PMID:24755292

  7. Antifreeze protein dimer: when two ice-binding faces are better than one.

    PubMed

    Baardsnes, Jason; Kuiper, Michael J; Davies, Peter L

    2003-10-03

    A naturally occurring tandem duplication of the 7-kDa type III antifreeze protein from Antarctic eel pout (Lycodichthys dearborni) is twice as active as the monomer in depressing the freezing point of a solution. We have investigated the basis for this enhanced activity by producing recombinant analogues of the linked dimer that assess the effects of protein size and the number and area of the ice-binding site(s). The recombinant dimer connected by a peptide linker had twice the activity of the monomer. When one of the two ice-binding sites was inactivated by site-directed mutagenesis, the linked dimer was only 1.2 times more effective than the monomer. When the two monomers were linked through a C-terminal disulfide bond in such a way that their two ice-binding sites were opposite each other and unable to engage the same ice surface simultaneously, the dimer was again only 1.2 times as active as the monomer. We conclude from these analyses that the enhanced activity of the dimer stems from the two ice-binding sites being able to engage to ice at the same time, effectively doubling the area of the ice-binding site.

  8. G domain dimerization controls dynamin's assembly-stimulated GTPase activity

    PubMed Central

    Chappie, Joshua S.; Acharya, Sharmistha; Leonard, Marilyn; Schmid, Sandra L.; Dyda, Fred

    2010-01-01

    Dynamin is an atypical GTPase that catalyzes membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin’s basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0Å resolution crystal structure of a minimal GTPase-GED fusion protein (GG) constructed from human dynamin 1, which has dimerized in the presence of the transition state mimic GDP.AlF4−. The structure reveals dynamin’s catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GG dimer provides new insight into the mechanisms underlying dynamin-catalyzed membrane fission. PMID:20428113

  9. Ising anyons in frustration-free Majorana-dimer models

    NASA Astrophysics Data System (ADS)

    Ware, Brayden; Son, Jun Ho; Cheng, Meng; Mishmash, Ryan V.; Alicea, Jason; Bauer, Bela

    2016-09-01

    Dimer models have long been a fruitful playground for understanding topological physics. Here, we introduce a class, termed Majorana-dimer models, wherein bosonic dimers are decorated with pairs of Majorana modes. We find that the simplest examples of such systems realize an intriguing, intrinsically fermionic phase of matter that can be viewed as the product of a chiral Ising theory, which hosts deconfined non-Abelian quasiparticles, and a topological px-i py superconductor. While the bulk anyons are described by a single copy of the Ising theory, the edge remains fully gapped. Consequently, this phase can arise in exactly solvable, frustration-free models. We describe two parent Hamiltonians: one generalizes the well-known dimer model on the triangular lattice, while the other is most naturally understood as a model of decorated fluctuating loops on a honeycomb lattice. Using modular transformations, we show that the ground-state manifold of the latter model unambiguously exhibits all properties of the Ising×(px-i py) theory. We also discuss generalizations with more than one Majorana mode per site, which realize phases related to Kitaev's 16-fold way in a similar fashion.

  10. Nonreciprocal transmission through a saturable nonlinear asymmetric dimer.

    PubMed

    Assunção, T F; Nascimento, E M; Lyra, M L

    2014-08-01

    We investigate the nonreciprocal diodelike behavior of a dimer with an asymmetric on-site potential and a saturable nonlinearity. The dimer is coupled to linear side chains. The spectra of transmission and the rectifying factor are analytically obtained using a backward iteration of the set of discrete nonlinear Schrödinger equations used to model the wave propagation through the nonlinear dimer. We show that the windows of bistable behavior leading to a pronounced nonreciprocal diodelike transmission become wider and displaced to higher input field intensities as the saturation coefficient increases. Further, saturation of the nonlinear response has opposite impacts on the rectifying action over short- and long-wavelength input signals within the second bistability window. In the first window, the rectifying action is not compromised by the saturation, thus showing that a weak contribution of high-order susceptibilities to the nonlinear response can improve the efficiency of the nonreciprocal transmission. The rectifying action of a dimer with an asymmetric nonlinearity is also discussed.

  11. G domain dimerization controls dynamin's assembly-stimulated GTPase activity

    SciTech Connect

    Chappie, Joshua S.; Acharya, Sharmistha; Leonard, Marilyn; Schmid, Sandra L.; Dyda, Fred

    2010-06-14

    Dynamin is an atypical GTPase that catalyses membrane fission during clathrin-mediated endocytosis. The mechanisms of dynamin's basal and assembly-stimulated GTP hydrolysis are unknown, though both are indirectly influenced by the GTPase effector domain (GED). Here we present the 2.0 {angstrom} resolution crystal structure of a human dynamin 1-derived minimal GTPase-GED fusion protein, which was dimeric in the presence of the transition state mimic GDP.AlF{sub 4}{sup -}. The structure reveals dynamin's catalytic machinery and explains how assembly-stimulated GTP hydrolysis is achieved through G domain dimerization. A sodium ion present in the active site suggests that dynamin uses a cation to compensate for the developing negative charge in the transition state in the absence of an arginine finger. Structural comparison to the rat dynamin G domain reveals key conformational changes that promote G domain dimerization and stimulated hydrolysis. The structure of the GTPase-GED fusion protein dimer provides insight into the mechanisms underlying dynamin-catalysed membrane fission.

  12. The dimeric proto-ribosome: Structural details and possible implications on the origin of life.

    PubMed

    Agmon, Ilana

    2009-06-30

    A symmetric pocket-like entity, composed of two L-shaped RNA units, encircles the peptide synthesis site within the contemporary ribosome. This entity was suggested to be the vestige of a dimeric proto-ribosome, which could have formed spontaneously in the prebiotic world, catalyzing non-coded peptide bond formation and elongation. This structural element, beyond offering the initial step in the evolution of translation, is hypothesized here to be linked to the origin of life. By catalyzing the production of random peptide chains, the proto-ribosome could have enabled the formation of primary enzymes, launching a process of co-evolution of the translation apparatus and the proteins, thus presenting an alternative to the RNA world hypothesis.

  13. Pirenzepine Promotes the Dimerization of Muscarinic M1 Receptors through a Three-step Binding Process*

    PubMed Central

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B.; Galzi, Jean-Luc; Mely, Yves

    2009-01-01

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state. PMID:19451648

  14. Pirenzepine promotes the dimerization of muscarinic M1 receptors through a three-step binding process.

    PubMed

    Ilien, Brigitte; Glasser, Nicole; Clamme, Jean-Pierre; Didier, Pascal; Piemont, Etienne; Chinnappan, Raja; Daval, Sandrine B; Galzi, Jean-Luc; Mely, Yves

    2009-07-17

    Ligand binding to G protein-coupled receptors is a complex process that involves sequential receptor conformational changes, ligand translocation, and possibly ligand-induced receptor oligomerization. Binding events at muscarinic acetylcholine receptors are usually interpreted from radioligand binding studies in terms of two-step ligand-induced receptor isomerization. We report here, using a combination of fluorescence approaches, on the molecular mechanisms for Bodipy-pirenzepine binding to enhanced green fluorescent protein (EGFP)-fused muscarinic M1 receptors in living cells. Real time monitoring, under steady-state conditions, of the strong fluorescence energy transfer signal elicited by this interaction permitted a fine kinetic description of the binding process. Time-resolved fluorescence measurements allowed us to identify discrete EGFP lifetime species and to follow their redistribution upon ligand binding. Fluorescence correlation spectroscopy, with EGFP brightness analysis, showed that EGFP-fused muscarinic M1 receptors predominate as monomers in the absence of ligand and dimerize upon pirenzepine binding. Finally, all these experimental data could be quantitatively reconciled into a three-step mechanism, with four identified receptor conformational states. Fast ligand binding to a peripheral receptor site initiates a sequence of conformational changes that allows the ligand to access to inner regions of the protein and drives ligand-receptor complexes toward a high affinity dimeric state.

  15. Distinct DNA binding preferences for the c-Myc/Max and Max/Max dimers.

    PubMed Central

    Solomon, D L; Amati, B; Land, H

    1993-01-01

    The transcription factor c-Myc and its dimerisation partner Max are members of the basic/helix-loop-helix/leucine-zipper (bHLH-Z) family and bind to the DNA core sequence CACGTG. Using a site-selection protocol, we determined the complete 12 base pair consensus binding sites of c-Myc/Max (RACCACGTGGTY) and Max/Max (RANCACGTGNTY) dimers. We find that the c-Myc/Max dimer fails to bind the core when it is flanked by a 5'T or a 3'A, while the Max/Max dimer readily binds such sequences. Furthermore we show that inappropriate flanking sequences preclude transactivation by c-Myc in vivo. In conclusion, Max/Max dimers are less discriminatory than c-Myc/Max and may regulate other genes in addition to c-Myc/Max targets. PMID:8265351

  16. Phosphorylation of Ser8 promotes zinc-induced dimerization of the amyloid-β metal-binding domain.

    PubMed

    Kulikova, Alexandra A; Tsvetkov, Philipp O; Indeykina, Maria I; Popov, Igor A; Zhokhov, Sergey S; Golovin, Andrey V; Polshakov, Vladimir I; Kozin, Sergey A; Nudler, Evgeny; Makarov, Alexander A

    2014-10-01

    Zinc-induced aggregation of the amyloid-β peptide (Aβ) is a hallmark molecular feature of Alzheimer's disease (AD). Recently it was shown that phosphorylation of Aβ at Ser8 promotes the formation of toxic aggregates. In this work, we have studied the impact of Ser8 phosphorylation on the mode of zinc interaction with the Aβ metal-binding domain 1-16 using isothermal titration calorimetry, electrospray ionization mass spectrometry and NMR spectroscopy. We have discovered a novel zinc binding site ((6)HDpS(8)) in the phosphorylated peptide, in which the zinc ion is coordinated by the imidazole ring of His6, the phosphate group attached to Ser8 and a backbone carbonyl group of His6 or Asp7. Interaction of the zinc ion with this site involves His6, thereby withdrawing it from the interaction pattern observed in the non-modified peptide. This event was found to stimulate dimerization of peptide chains through the (11)EVHH(14) site, where the zinc ion is coordinated by the two pairs of Glu11 and His14 in the two peptide subunits. The proposed molecular mechanism of zinc-induced dimerization could contribute to the understanding of initiation of pathological Aβ aggregation, and the (11)EVHH(14) tetrapeptide can be considered as a promising drug target for the prevention of amyloidogenesis.

  17. Dimer geometry, amoebae and a vortex dimer model

    NASA Astrophysics Data System (ADS)

    Nash, Charles; O'Connor, Denjoe

    2017-09-01

    We present a geometrical approach and introduce a connection for dimer problems on bipartite and non-bipartite graphs. In the bipartite case the connection is flat but has non-trivial {Z}2 holonomy round certain curves. This holonomy has the universality property that it does not change as the number of vertices in the fundamental domain of the graph is increased. It is argued that the K-theory of the torus, with or without punctures, is the appropriate underlying invariant. In the non-bipartite case the connection has non-zero curvature as well as non-zero Chern number. The curvature does not require the introduction of a magnetic field. The phase diagram of these models is captured by what is known as an amoeba. We introduce a dimer model with negative edge weights which correspond to vortices. The amoebae for various models are studied with particular emphasis on the case of negative edge weights. Vortices give rise to new kinds of amoebae with certain singular structures which we investigate. On the amoeba of the vortex full hexagonal lattice we find the partition function corresponds to that of a massless Dirac doublet.

  18. Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. I. T4 endonuclease V-initiated excision repair

    SciTech Connect

    Gruskin, E.A.; Lloyd, R.S.

    1988-09-05

    The process by which DNA-interactive proteins locate specific sequences or target sites on cellular DNA within Escherichia coli is a poorly understood phenomenon. In this study, we present the first direct in vivo analysis of the interaction of a DNA repair enzyme, T4 endonuclease V, and its substrate, pyrimidine dimer-containing plasmid DNA, within UV-irradiated E. coli. A pyrimidine dimer represents a small target site within large domains of DNA. There are two possible paradigms by which endonuclease V could locate these small target sites: a processive mechanism in which the enzyme scans DNA for dimer sites or a distributive process in which dimers are located by random three-dimensional diffusion. In order to discriminate between these two possibilities in E. coli, an in vivo DNA repair assay was developed to study the kinetics of plasmid DNA repair and the dimer frequency (i.e. the number of dimer sites on a given plasmid molecule) in plasmid DNA as a function of time during repair. Our results demonstrate that the overall process of plasmid DNA repair initiated by T4 endonuclease V (expressed from a recombinant plasmid within repair-deficient E. coli) occurs by a processive mechanism. Furthermore, by reducing the temperature of the repair incubation, the endonuclease V-catalyzed incision step has been effectively decoupled from the subsequent steps including repair patch synthesis, ligation, and supercoiling. By this manipulation, it was determined that the overall processive mechanism is composed of two phases: a rapid processive endonuclease V-catalyzed incision reaction, followed by a slower processive mechanism, the ultimate product of which is the dimer-free supercoiled plasmid molecule.

  19. D-Dimer elevation and adverse outcomes.

    PubMed

    Halaby, Rim; Popma, Christopher J; Cohen, Ander; Chi, Gerald; Zacarkim, Marcelo Rodrigues; Romero, Gonzalo; Goldhaber, Samuel Z; Hull, Russell; Hernandez, Adrian; Mentz, Robert; Harrington, Robert; Lip, Gregory; Peacock, Frank; Welker, James; Martin-Loeches, Ignacio; Daaboul, Yazan; Korjian, Serge; Gibson, C Michael

    2015-01-01

    D-Dimer is a biomarker of fibrin formation and degradation. While a D-dimer within normal limits is used to rule out the diagnosis of deep venous thrombosis and pulmonary embolism among patients with a low clinical probability of venous thromboembolism (VTE), the prognostic association of an elevated D-dimer with adverse outcomes has received far less emphasis. An elevated D-dimer is independently associated with an increased risk for incident VTE, recurrent VTE, and mortality. An elevated D-dimer is an independent correlate of increased mortality and subsequent VTE across a broad variety of disease states. Therefore, medically ill subjects in whom the D-dimer is elevated constitute a high risk subgroup in which the prospective evaluation of the efficacy and safety of antithrombotic therapy is warranted.

  20. Solitary waves in dimer binary collision model

    NASA Astrophysics Data System (ADS)

    Ahsan, Zaid; Jayaprakash, K. R.

    2017-01-01

    Solitary wave propagation in nonlinear diatomic (dimer) chains is a very interesting topic of research in the study of nonlinear lattices. Such waves were recently found to be supported by the essentially nonlinear granular lattice and Toda lattice. An interesting aspect of this discovery is attributed to the realization of a spectrum of the mass ratio (the only system parameter governing the dynamics) that supports the propagation of such waves corresponding to the considered interaction potential. The objective of this exposition is to explore solitary wave propagation in the dimer binary collision (BC) model. Interestingly, the dimer BC model supports solitary wave propagation at a discrete spectrum of mass ratios similar to those observed in granular and Toda dimers. Further, we report a qualitative and one-to-one correspondence between the spectrum of the mass ratio corresponding to the dimer BC model and those corresponding to granular and Toda dimer chains.

  1. Dimerization of Human Growth Hormone by Zinc

    NASA Astrophysics Data System (ADS)

    Cunningham, Brian C.; Mulkerrin, Michael G.; Wells, James A.

    1991-08-01

    Size-exclusion chromatography and sedimentation equilibrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn2+-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn2+-hGH dimeric complex may be important for storage of hGH in secretory granules.

  2. A short double-stranded RNA motif of Peach latent mosaic viroid contains the initiation and the self-cleavage sites of both polarity strands.

    PubMed

    Delgado, Sonia; Martínez de Alba, Angel E; Hernández, Carmen; Flores, Ricardo

    2005-10-01

    The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5' termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5'-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels.

  3. A Short Double-Stranded RNA Motif of Peach Latent Mosaic Viroid Contains the Initiation and the Self-Cleavage Sites of Both Polarity Strands†

    PubMed Central

    Delgado, Sonia; Martínez de Alba, Ángel E.; Hernández, Carmen; Flores, Ricardo

    2005-01-01

    The transcription initiation sites of viroid RNAs, despite their relevance for replication and in vivo folding, are poorly characterized. Here we have examined this question for Peach latent mosaic viroid (PLMVd), which belongs to the family of chloroplastic viroids with hammerhead ribozymes (Avsunviroidae), by adapting an RNA ligase-mediated rapid amplification of cDNA ends methodology developed for mapping the genuine capped 5′ termini of eukaryotic messenger RNAs. To this aim, the characteristic free 5′-triphosphate group of chloroplastic primary transcripts from PLMVd-infected young fruits was previously capped in vitro with GTP and guanylyltransferase. PLMVd plus and minus initiation sites map at similar double-stranded motifs of 6 to 7 bp that also contain the conserved GUC triplet preceding the self-cleavage site in both polarity strands. Within the branched secondary structures predicted for the two PLMVd strands, this motif is located at the base of a similar long hairpin that presumably contains the promoters for a chloroplastic RNA polymerase. The transcription templates could be the circular viroid RNAs or their most abundant linear counterparts, assuming the involvement of an RNA polymerase able to jump over template discontinuities. Both PLMVd initiation sites were confirmed by applying the same methodology to two purified PLMVd subgenomic RNAs and by primer extension, and they therefore likely reflect the in vivo situation. The location of the PLMVd initiation sites provides a mechanistic view into how the nascent strands may fold and self-cleave during transcription. The approach described here may be extended to other chloroplastic RNA replicons and transcripts accumulating at low levels. PMID:16188995

  4. Monomer-dimer problem on some networks

    NASA Astrophysics Data System (ADS)

    Wu, Ruijuan; Yan, Weigen

    2016-09-01

    Zhang et al. (2012) obtained the exact formula for the number of all possible monomer-dimer arrangements and the asymptotic growth constant on a scale-free small-world network. In this note, we generalize this result and obtain the exact solution on the monomer-dimer model on many networks. Particularly, we prove that these networks have the same asymptotic growth constant of the number of monomer-dimer arrangements.

  5. Hmo1 directs pre-initiation complex assembly to an appropriate site on its target gene promoters by masking a nucleosome-free region

    PubMed Central

    Kasahara, Koji; Ohyama, Yoshifumi; Kokubo, Tetsuro

    2011-01-01

    Saccharomyces cerevisiae Hmo1 binds to the promoters of ∼70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in Δhmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5′- and 3′-boundaries of the PIC assembly zone. PMID:21288884

  6. Functional Roles of the Dimer-Interface Residues in Human Ornithine Decarboxylase

    PubMed Central

    Lee, Chien-Yun; Liu, Yi-Liang; Lin, Chih-Li; Liu, Guang-Yaw; Hung, Hui-Chih

    2014-01-01

    Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the Kd value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis. PMID:25140796

  7. Structural insights into lipid-dependent reversible dimerization of human GLTP

    SciTech Connect

    Samygina, Valeria R.; Ochoa-Lizarralde, Borja; Popov, Alexander N.; Cabo-Bilbao, Aintzane; Goni-de-Cerio, Felipe; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.; Malinina, Lucy

    2013-04-01

    It is shown that dimerization is promoted by glycolipid binding to human GLTP. The importance of dimer flexibility in wild-type protein is manifested by point mutation that ‘locks’ the dimer while diversifying ligand/protein adaptations. Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the ‘portal entrance’ of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization. In all crystal forms, the hsGLTP–SF complexes displayed homodimeric structures supported by similarly organized intermolecular interactions. The dimerization interface always involved the lipid sphingosine chain, the protein C-terminus (C-end) and α-helices 6 and 2, but the D48V mutant displayed a ‘locked’ dimer conformation compared with the hinge-like flexibility of wild-type dimers. Differences in contact angles, areas and residues at the dimer interfaces in the ‘flexible’ and ‘locked’ dimers revealed a potentially important role of the dimeric structure in the C-end conformation of hsGLTP and in the precise positioning of the key residue of the glycolipid recognition centre, His140. ΔY207 and ΔC-end deletion mutants, in which the C-end is shifted or truncated, showed an almost complete loss of transfer activity. The new structural insights suggest that ligand-dependent reversible dimerization plays a role in the function of human GLTP.

  8. Thermalization of a dimerized antiferromagnetic spin chain.

    PubMed

    Konstantinidis, N P

    2016-01-20

    Thermalization is investigated for the one-dimensional anisotropic antiferromagnetic Heisenberg model with dimerized nearest-neighbor interactions that break integrability. For this purpose the time evolution of local operator expectation values after an interacting quench is calculated directly with the Chebyshev polynomial expansion, and the deviation of the diagonal from the canonical thermal ensemble value is calculated for increasing system size for these operators. The spatial and spin symmetries of the Hamiltonian are taken into account to divide it into symmetry subsectors. The rate of thermalization is found to weaken with the dimerization parameter as the Hamiltonian evolves between two integrable limits, the non-dimerized and the fully dimerized where the chain breaks up into isolated dimers. This conclusion is supported by the distribution of the local operator off-diagonal elements between the eigenstates of the Hamiltonian with respect to their energy difference, which determines the strength of temporal fluctuations. The off-diagonal elements have a low-energy peak for small dimerization which facilitates thermalization, and originates in the reduction of spatial symmetry with respect to the non-dimerized limit. For increasing dimerization their distribution changes and develops a single low-energy maximum that relates to the fully dimerized limit and slows down thermalization.

  9. Postlumpectomy Insertion of the MammoSite brachytherapy device using the scar entry technique: initial experience and technical considerations.

    PubMed

    Stolier, Alan J; Fuhrman, George M; Scroggins, Troy G; Boyer, Cynthia I

    2005-01-01

    For women undergoing breast-conserving surgery, recent reports suggest that in selected cases accelerated partial breast irradiation may yield results equal to that of whole breast irradiation. Over 31 months, 19 patients underwent accelerated partial breast irradiation using the MammoSite as the sole radiation treatment following breast-conserving surgery. Seventeen patients had the MammoSite inserted postoperatively using the scar entry technique (SET). Treatments were delivered using high dose rate iridium 192 given twice a day for 5 days. Three complications (two minor, one major) occurred. Late radiation morbidity and overall cosmetic results were evaluated. Eighty percent of patients had either no change from baseline or slight change in skin pigment. More than 90% had good or excellent overall cosmetic outcomes. Patients undergoing accelerated partial breast irradiation with the MammoSite inserted using SET had excellent overall cosmetic results. Advantages of the SET over intraoperative placement are presented.

  10. Debatable aspects of initial human colonization of Siberia and age of the Karama site in the Altai Mountains

    NASA Astrophysics Data System (ADS)

    Zykin, V. S.; Zykina, V. S.; Smolyaninova, L. G.

    2016-05-01

    Debatable aspects of age, stratigraphic position, and natural conditions of the oldest stratified Early Paleolithic Karama site in the Altai Mountains are critically revised. The extensive geological, stratigraphic, and paleontological data allow the sufficiently well-substantiated assumption that accumulation of the Karama Formation and existence of the Early Paleolithic Karama site correspond to a long period of climate warming in the Early Pleistocene correlated with the Tiglian of northwestern Europe lasting from 2.23 to 1.59 Ma. The age model proposed for the formation of the Quaternary sequence in the Anui River valley, which includes the artifact-containing deposits of the Karama site, seems to be the most probable one proceeding from interpretation of available data on the geological structure, stratigraphy, paleomagnetism, and paleontological and lithological properties of Upper Cenozoic sequences observable both in the Anui River valley and in Siberian areas adjacent to the Altai mountainous region.

  11. Structural Characterization of Amyloid β17-42 Dimer by potential of mean force analysis: Insights from Molecular Dynamics Simulations.

    PubMed

    Dutta, Mary; Chutia, Rajkalyan; Mattaparthi, Venkata Satish Kumar

    2017-06-20

    Recent experiments with Amyloid β1-42 peptide have indicated that the initial dimerization of Aβ1-42 monomers to form amyloid dimers stand out as a key event in the generation of toxic oligomers. However, the structural characterization of Aβ1-42 dimer at the atomistic level and the dimerization mechanism by which Aβ1-42 peptides co-aggregate still remains not clear. In the present study, the process of Aβ17-42 peptide dimerization which is known to play an important role in the plaque formation was evaluated in terms of potential of mean force, which provided free energies along the reaction coordinates. The global minima structure of the Aβ17-42 dimer at the minimum distance of separation was isolated from the calculated free energy profile indicating a strong tendency of the monomers to associate and form the dimer. Further, the interactions involved in the formation of the dimer structure were examined and the global minima structure was used for the identification of protein-protein interfaces and the residue-residue interactions vital for generation of the dimer complexes. The simulation results elucidated hydrogen bonding to be a critical factor for the stability of the dimer structure. The results thus provide an atomistic insight into the bonding and non-bonding interactions involved in the dimerization process of Aβ1-42 peptide along with the spontaneous formation of several basic structural units of dimer structure, thereby offering a key contribution to enhance our fundamental knowledge about Alzheimer's disease and to design inhibitors to disrupt the Aβ dimers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Feasibility Study of Anaerobic Digestion of Food Waste in St. Bernard, Louisiana. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Moriarty, K.

    2013-01-01

    The U.S. Environmental Protection Agency (EPA) developed the RE-Powering America's Land initiative to re-use contaminated sites for renewable energy generation when aligned with the community's vision for the site. The former Kaiser Aluminum Landfill in St. Bernard Parish, Louisiana, was selected for a feasibility study under the program. Preliminary work focused on selecting a biomass feedstock. Discussions with area experts, universities, and the project team identified food wastes as the feedstock and anaerobic digestion (AD) as the technology.

  13. A national strategy for identification, prioritisation and management of pollution from abandoned non-coal mine sites in England and Wales. I. Methodology development and initial results.

    PubMed

    Mayes, W M; Johnston, D; Potter, H A B; Jarvis, A P

    2009-10-15

    In regions affected by historic non-coal (principally metal) mining activity, government agencies are often faced with the challenge of deploying limited remedial resources at abandoned mine sites to achieve maximum improvements in the chemical and ecological quality of impacted ground and surface waters. As such, strategies for the defensible allocation of public funds require comprehensive and systematic frameworks by which to identify and prioritise polluting sites for remediation. This paper describes the development and initial findings of such a national initiative in England and Wales which allies catchment-scale environmental impact assessments using existing public archive data, with recognition of the uncertainty in impact appraisals arising from disparities in data availability between sites and regions. The methodology identifies polluting sites and takes account not only of the chemical and ecological impacts of mine water discharges on receiving watercourses, but also of socio-economic factors such as conservation and heritage concerns, which can both impede or complement efforts to remediate mine sites. Using a Geographic Information System database and a suite of spatial analyses employing Boolean operators, both the extent of the pollution problem from abandoned non-coal mines in England and Wales (6% of 7815 surface water bodies are affected nationally) and the insight that can be gleaned from systematic analyses of existing archive data are highlighted. The results of the nationwide survey can be used as a dynamic database to inform future remedial planning, in terms of prioritising impacted river basins and abandoned non-coal mine sites themselves for either remediation or future monitoring efforts. As the assessment framework is built upon existing water quality and ecological data and mine site/geological data, there is considerable scope for the approach to be applied elsewhere where the legacy of historic mining persists through the

  14. Dimerization kinetics of the IgE-class antibodies by divalent haptens. I. The Fab-hapten interactions.

    PubMed Central

    Schweitzer-Stenner, R; Licht, A; Pecht, I

    1992-01-01

    The binding of divalent haptens to IgE-class antibodies leads predominantly to their oligomerization into open and closed dimers. Kinetics of the open dimer formation was investigated by fluorescence titrations of Fab fragments of monoclonal DNP-specific IgE antibodies with divalent haptens having different spacer length (gamma = 14-130 A). Binding was monitored by quenching of intrinsic tryptophan emission of the Fab. Addition of divalent haptens with short spacers (gamma = 14-21 A) to the Fabs at rates larger than a distinct threshold value caused a significant decrease of Fab-binding site occupation in the initial phase of the titration. This finding was interpreted to reflect a nonequilibrium state of hapten-Fab-binding. Such nonequilibrium titrations were analyzed by inserting a kinetic model into a theory of antibody aggregation as presented by Dembo and Golstein (Histamine release due to bivalent penicilloyl haptens. 1978. J. Immunol. 121, 345). Fitting of this model to the fluorescence titrations yielded dissociation rate constants of 7.8 x 10(-3) s-1 and 6 x 10(-3) s-1 for the Fab dimers formed by the flexible divalent haptens N alpha, N epsilon-di(dinitrophenyl)-L-lysine (gamma = 16 A) and bis(N beta-2,4-dinitrophenyl-alanyl)-meso-diamino-succinate (gamma = 21 A). Making the simplifying assumption that a single step binding equilibrium prevails, the corresponding dimer formation rate constants were calculated to be 1.9 x 10(5) M-1 s-1 and 1.1 x 10(4) M-1 s-1, respectively. In contrast, all haptens with spacers longer than 40 A (i.e., bis(N alpha-2,4-dinitrophenyl-tri-D-alanyl)-1,7-diamino-heptane, and di(N epsilon-2,4-dinitrophenyl)-6-aminohexanoate-aspartyl-(prolyl)n-L-l ysyl (n = 24, 27, 33) exhibit a relative fast dimerization rate of the Fab fragments (greater than 7 x 10(6) M-1 s-1). These observations were interpreted as being caused by orientational constraints set by the limited solid angle of the reaction between the macromolecular reactants

  15. Influence of dentures in the initial occurrence site on the prognosis of bisphosphonate-related osteonecrosis of the jaws: a retrospective study.

    PubMed

    Hasegawa, Yoko; Kawabe, Mutuki; Kimura, Hiroto; Kurita, Kenichi; Fukuta, Jinichi; Urade, Masahiro

    2012-09-01

    This retrospective cohort study was conducted to investigate the influence of wearing dentures in the initial occurrence site of bisphosphonate-related osteonecrosis of the jaws (BRONJ). A questionnaire regarding the prevalence, therapy, and outcome of jawbone lesions during 2006-08 was mailed to 248 medical institutions with an oral and maxillofacial surgery department in Japan. Ninety-nine patients wearing dentures had significantly shorter duration until occurrence than 151 patients not wearing dentures. In addition, remission of BRONJ affecting the mandibular canine and premolar region in denture-wearing patients was significantly more difficult. Poor oral hygiene status was found to affect significantly the prognosis of BRONJ in denture-wearing patients. Alcohol habit also delayed remission, but high body mass index promoted remission. Wearing a denture in the initial occurrence site of BRONJ was shown to influence the prognosis of BRONJ, especially in mandibular denture-wearing patients. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates.

    PubMed

    Ghodke, Harshad; Wang, Hong; Hsieh, Ching L; Woldemeskel, Selamawit; Watkins, Simon C; Rapić-Otrin, Vesna; Van Houten, Bennett

    2014-05-06

    How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.

  17. Initial Geochemistry Data of the Lake Ohrid (Macedonia, Albania) DEEP -Site Sediment Record: The ICDP Scopsco Drilling Project

    NASA Astrophysics Data System (ADS)

    Francke, A.; Wagner, B.; Sulpizio, R.; Zanchetta, G.; Leicher, N.; Gromig, R.; Krastel, S.; Lindhorst, K.; Wilke, T.

    2014-12-01

    Ancient lakes, with sediment records spanning >1 million years, are very rare. The UNESCO World Heritage site of Lake Ohrid on the Balkans is thought to be the oldest lake in Europe. With 212 endemic species described to date, it is also a hotspot of evolution. In order to unravel the geological and evolutionary history of the lake, an international group of scientists, conducted a deep drilling campaign in spring 2013 under the umbrella of the ICDP SCOPSCO project (Scientific Collaboration on Past Speciation Conditions in Lake Ohrid). Overall, about 2,100 m of sediments were recovered from four drill sites. At the main drill site (DEEP-site) in central parts of the lake where seismic data indicated a maximum sediment fill of ca. 700 m, a total of more than 1,500 m of sediments were recovered until a penetration depth of 569 m. Currently, core opening, core description, XRF and MSCL scanning, sub-sampling (16 cm resolution), and inorganic and organic geochemical as well as sedimentological analyses of the sediment cores from the DEEP site are in progress at the University of Cologne. Previous studies at Lake Ohrid have shown that interglacial periods are characterized by high TIC and TOC contents, likely associated with high contents of calcite and organic matter in the sediments. In contrast, during glacial periods negligible TIC and low TOC contents correspond to high K counts indicating enhanced supply of clastic material. Similar patterns can be observed in the biogeochemical analyses of the subsamples and in the XRF data of the DEEP site record. Following these variations on a glacial-interglacial time scale, TIC and TOC data obtained from the subsamples and from core catcher samples indicate that the DEEP site sequence provides a 1.2 million year old continuous record of environmental and climatological variability in the Balkan Region. The age control can be further improved by first findings of macroscopic tephra horizons. Peaks in K, Sr, Zr, and magnetic

  18. Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus.

    PubMed Central

    Sedliaková, M; Brozmanová, J; Masek, F; Kleibl, K

    1981-01-01

    In Escherichia coli B/r Her+ irradiated with two separate fluences, dimer excision is prematurely interrupted. The present study was designed to follow tha fate of dimers remaining unexcised. The results imply that these dimers (or distortions containing dimers) are transformed on replication from the state of sensitivity to the state of insensitivity to endonuclease from Micrococcus luteus. This conclusion is based on the following findings: (a) dimers were radiochromatographically detectable in DNA replicated after UV, which indicated that they were tolerated on replication. (b) Similar amounts of dimers were detected radiochromatographically both in DNA remaining unreplicated and DNA twice replicated after UV, This along with the low transfer of parental label into daughter DNA, indicated that dimers remained in situ in parental chains. (c) Immediately after UV, all parental DNA contained numerous sites sensitive to the extract from M. luteus. 2 h after UV, a portion of parental DNA still contained a number of endonuclease-sensitive (Es) sites, while another portion of parental DNA and all daughter DNA were free of Es sites. (d) The occurrence of parental DNA free of Es sites was not temporally correlated with dimer excision, but with the first round of DNA replication. (e) The amount of DNA free of Es sites corresponded to the amount of replicated DNA. (f) Separation of replicated and unreplicated DNA, and detection of Es sites in both portions separately showed that the replicated DNA was almost free of Es sites, whereas unreplicated DNA contained a number of such sites. PMID:7030422

  19. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  20. Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site.

    PubMed

    Papp, P P; Elö, P; Semsey, S; Orosz, L

    2000-10-01

    The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.

  1. Texas Study of Students at Risk: Case Studies of Initiatives Supporting Ninth Graders' Success. Cross-Site Report

    ERIC Educational Resources Information Center

    Shapley, Kelly; Vicknair, Keven; Sheehan, Daniel; Pieper, Amy; Jepson, Dana; Sturges, Keith; Bush, Joan; Vandiver, Sherrie

    2004-01-01

    Researchers conducted case studies of Ninth Grade Success Initiative (NGSI) grants to gain a greater understanding of issues facing large numbers of at-risk students, many of whom, despite potentially receiving services as early as kindergarten, still reach ninth grade unprepared to succeed academically in high school. Case studies focused on NGSI…

  2. A public health Immunization Resource Web site for chiropractors: discussion of current issues and future challenges for evidence-based initiatives for the chiropractic profession.

    PubMed

    Khorsan, Raheleh; Smith, Monica; Hawk, Cheryl; Haas, Mitchell

    2009-01-01

    The Immunization Information Resource Web site is provided as a public service by the Chiropractic Health Care Section of the American Public Health Association. The site compiles annotated bibliographies of citations from the scientific literature, as well as other authoritative peer-reviewed information sources on this topic. Our intent was to create a resource of information for health care professionals that is current, accurate, objective, evidence based, and as user-friendly as possible. This article describes the Internet-based Immunization Information Resource Web site developed and sponsored by the Chiropractic Health Care Section of the American Public Health Association and discusses current issues and future challenges for sustaining and further advancing such evidence-based initiatives for the chiropractic profession.

  3. A mutation creating an out-of-frame alternative translation initiation site in the GRHPR 5'UTR causing primary hyperoxaluria type II.

    PubMed

    Fu, Y; Rope, R; Fargue, S; Cohen, H T; Holmes, R P; Cohen, D M

    2015-11-01

    Primary hyperoxaluria type II is a recessive genetic disorder caused by mutations in the GRHPR gene. Although several dozen mutations have been described, all affect coding or transcript splicing. A man suspected of having primary hyperoxaluria type II was heterozygous for a novel single-nucleotide deletion (c.694delC) in GRHPR affecting Gln(232) , which introduced a pre-mature termination (p.Gln232Argfs*3). Two 5'untranslated region (UTR) variants of unknown significance were also noted. We show that these two variants occur in cis, on the opposite allele, and introduce - immediately upstream of the canonical translation initiation site - a novel out-of-frame translational start site. In vitro studies using the GRHPR 5'UTR fused to a luciferase reporter show that the variant start site pre-empted initiation at the canonical translational start site, and this was corroborated within the broader context of 1.3 kb of the GRHPR proximal promoter. This latter mechanism may be underappreciated in general; reports of clinically significant functional variation of this type are extremely rare.

  4. Initial Response of Pine Seedlings and Weeds to Dried Sewage Sludge in Rehabilitation of an Eroded Forest Site

    Treesearch

    Charles R. Berry

    1977-01-01

    Dried sewage sludge was applied at rates of 0, 17, 34, and 69 metric tons/ha on a badly eroded forest site in the Piedmont region of northeast Georgia. Production of weed bio mass varied directly with amount of sludge applied. Heigh growth for both shortleafand loblolly pine seedlings appeared to be greater on plots receiving 17 metric tons of sludge/ha, bu differences...

  5. Initial success of native grasses is contingent on multiple interactions among exotic grass competition, temporal priority, rainfall and site effects.

    PubMed

    Young, Truman P; Zefferman, Emily P; Vaughn, Kurt J; Fick, Stephen

    2014-12-05

    Ecological communities are increasingly being recognized as the products of contemporary drivers and historical legacies that are both biotic and abiotic. In an attempt to unravel multiple layers of ecological contingency, we manipulated (i) competition with exotic annual grasses, (ii) the timing of this competition (temporal priority in arrival/seeding times) and (iii) watering (simulated rainfall) in a restoration-style planting of native perennial grasses. In addition, we replicated this experiment simultaneously at three sites in north-central California. Native perennial grasses had 73-99 % less cover when planted with exotic annuals than when planted alone, but this reduction was greatly ameliorated by planting the natives 2 weeks prior to the exotics. In a drought year, irrigation significantly reduced benefits of early planting so that these benefits resembled those observed in a non-drought year. There were significant differences across the three sites (site effects and interactions) in (i) overall native cover, (ii) the response of natives to competition, (iii) the strength of the temporal priority effect and (iv) the degree to which supplemental watering reduced priority effects. These results reveal the strong multi-layered contingency that underlies even relatively simple communities.

  6. Initial success of native grasses is contingent on multiple interactions among exotic grass competition, temporal priority, rainfall and site effects

    PubMed Central

    Young, Truman P.; Zefferman, Emily P.; Vaughn, Kurt J.; Fick, Stephen

    2015-01-01

    Ecological communities are increasingly being recognized as the products of contemporary drivers and historical legacies that are both biotic and abiotic. In an attempt to unravel multiple layers of ecological contingency, we manipulated (i) competition with exotic annual grasses, (ii) the timing of this competition (temporal priority in arrival/seeding times) and (iii) watering (simulated rainfall) in a restoration-style planting of native perennial grasses. In addition, we replicated this experiment simultaneously at three sites in north-central California. Native perennial grasses had 73–99 % less cover when planted with exotic annuals than when planted alone, but this reduction was greatly ameliorated by planting the natives 2 weeks prior to the exotics. In a drought year, irrigation significantly reduced benefits of early planting so that these benefits resembled those observed in a non-drought year. There were significant differences across the three sites (site effects and interactions) in (i) overall native cover, (ii) the response of natives to competition, (iii) the strength of the temporal priority effect and (iv) the degree to which supplemental watering reduced priority effects. These results reveal the strong multi-layered contingency that underlies even relatively simple communities. PMID:25480888

  7. Laparoendoscopic single-site surgery for fertility-sparing staging of border line ovarian tumors: initial experience.

    PubMed

    Marocco, Francesco; Fanfani, Francesco; Rossitto, Cristiano; Gallotta, Valerio; Scambia, Giovanni; Fagotti, Anna

    2010-10-01

    To report feasibility of laparoscopic fertility-sparing staging of border line ovarian tumors (BOTs) by using a laparoendoscopic single-site trocar through a unique transumbilical access. This study was conducted in our University Hospital. Four young patients, intended to be submitted to benign adnexal cysts enucleation by an innovative laparoendoscopic single-site approach using a multiport trocar inserted through a unique transumbilical access and straight laparoscopic devices, were found to be affected by border line ovarian tumor at intraoperative frozen analysis. Consequently patients were conservatively staged carrying out all the requested procedures in respect of oncologic guidelines and their child bearing desire, using same single-port access approach. Main outcome measures were conversion rate to multiaccess standard laparoscopic technique or to laparotomic approach and evaluation of intraoperative and postoperative-related complications. All the laparoscopic staging procedures were feasible through a single transumbilical access: no conversion to multiaccess standard laparoscopic technique nor laparotomy and no intraoperative or postoperative complications were observed. Mean operative time was 79 minutes. All patients were discharged home on day 1 with complete satisfaction toward cosmetic outcome. Final pathologic analysis confirmed stage FIGO 1A for all cases. The patients are free from recurrence at 10 months follow-up. Laparoendoscopic single-site staging of border line ovarian tumors with preservation of fertility is feasible and effective with standard laparoscopic instruments. More clinical data are needed to confirm these advantages compared with standard multiaccess laparoscopic technique.

  8. Mechanism for Controlling the Dimer-Monomer Switch and Coupling Dimerization to Catalysis of the Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease

    SciTech Connect

    Shi,J.; Sivaraman, J.; Song, J.

    2008-01-01

    Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Angstroms . Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

  9. Direct visualization of a cycloaddition reaction on frozen asymmetric Si dimers at room temperature

    NASA Astrophysics Data System (ADS)

    Baik, Jaeyoon; Ihm, Kyuwook; Ha, Taekyun; An, Ki-Seok; Ahn, Joung Real; Park, Chong-Yun

    2016-07-01

    We firstly report an experimental visualization of a cycloaddition reaction on RT frozen asymmetric Si dimers. The frozen Si dimers with a local c(4 × 2) order were prepared by pinning flip-flopping Si dimers by using molecules. This RT pristine c(4 × 2) structure was used to determine what Si atom of an asymmetric Si dimer bonds to a molecule at the initial stage of the RT cycloaddition reaction, which has been a long-standing puzzling issue. This made it possible to compare directly experimental cycloaddition reactions with theoretical ones. As a prototype for the experiment, a 1,3-butadiene molecule adsorbed between Si dimer rows was used. The 1,3-butadiene molecule was found to prefer a symmetric Si pair on the frozen Si dimers, i.e., two electrophilic lower atoms of asymmetric Si dimers. This result is consistent with the theoretical prediction that a 1,3-diene molecule prefers a symmetric Si pair on the Si(001)c(4 × 2) surface. This experimental approach can also be applied to other studies for the adsorption of a molecule on a Si(001) surface at room temperature.

  10. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Price Landfill Site in Pleasantville, New Jersey. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Salasovich, J.; Geiger, J.; Mosey, G.; Healey, V.

    2013-05-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Price Landfill site in Pleasantville, New Jersey, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) provided technical assistance for this project. The purpose of this report is to assess the site for a possible photovoltaic (PV) system installation and estimate the cost, performance, and site impacts of different PV options. In addition, the report recommends financing options that could assist in the implementation of a PV system at the site. This study did not assess environmental conditions at the site.

  11. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Former Chicago, Milwaukee, and St. Paul Rail Yard Company Site in Perry, Iowa. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Salasovich, J.; Geiger, J.; Healey, V.; Mosey, G.

    2013-03-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Former Chicago, Milwaukee & St. Paul Rail Yard Company site in Perry, Iowa, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) provided technical assistance for this project. The purpose of this report is to assess the site for a photovoltaic (PV) system installation and estimate the cost, performance, and site impacts of different PV options. In addition, the report recommends financing options that could assist in the implementation of a PV system at the site. This study did not assess environmental conditions at the site.

  12. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Tower Road Site in Aurora, Colorado. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Van Geet, O.; Mosey, G.

    2013-03-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Tower Road site in Aurora, Colorado, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) provided technical assistance for this project. The purpose of this report is to assess the site for a possible photovoltaic (PV) system installation and estimate the cost, performance, and site impacts of different PV options. In addition, the report recommends financing options that could assist in the implementation of a PV system at the site. This study did not assess environmental conditions at the site.

  13. Potassium Hexacyanoferrate (III)-Catalyzed Dimerization of Hydroxystilbene: Biomimetic Synthesis of Indane Stilbene Dimers.

    PubMed

    Xie, Jing-Shan; Wen, Jin; Wang, Xian-Fen; Zhang, Jian-Qiao; Zhang, Ji-Fa; Kang, Yu-Long; Hui, You-Wei; Zheng, Wen-Sheng; Yao, Chun-Suo

    2015-12-18

    Using potassium hexacyanoferrate (III)-sodium acetate as oxidant, the oxidative coupling reaction of isorhapontigenin and resveratrol in aqueous acetone resulted in the isolation of three new indane dimers 4, 6, and 7, together with six known stilbene dimers. Indane dimer 5 was obtained for the first time by direct transformation from isorhapontigenin. The structures and relative configurations of the dimers were elucidated using spectral analysis, and their possible formation mechanisms were discussed. The results indicate that this reaction could be used as a convenient method for the semi-synthesis of indane dimers because of the mild conditions and simple reaction products.

  14. Combining bulk sediment OSL and meteoric 10Be fingerprinting techniques to identify gully initiation sites and erosion depths

    NASA Astrophysics Data System (ADS)

    Portenga, E. W.; Bishop, P.; Rood, D. H.; Bierman, P. R.

    2017-02-01

    Deep erosional gullies dissect landscapes around the world. Existing erosion models focus on predicting where gullies might begin to erode, but identifying where existing gullies were initiated and under what conditions is difficult, especially when historical records are unavailable. Here we outline a new approach for fingerprinting alluvium and tracing it back to its source by combining bulk sediment optically stimulated luminescence (bulk OSL) and meteoric 10Be (10Bem) measurements made on gully-derived alluvium samples. In doing so, we identify where gully erosion was initiated and infer the conditions under which such erosion occurred. As both 10Bem and bulk OSL data have distinctive depth profiles in different uneroded and depositional settings, we are able to identify the likely incision depths in potential alluvium source areas. We demonstrate our technique at Birchams Creek in the southeastern Australian Tablelands—a well-studied and recent example of gully incision that exemplifies a regional landscape transition from unchanneled swampy meadow wetlands to gully incision and subsequent wetland burial by post-European settlement alluvium. We find that such historic alluvium was derived from a shallow erosion of valley fill upstream of former swampy meadows and was deposited down the center of the valley. Incision likely followed catchment deforestation and the introduction of livestock, which overgrazed and congregated in valley bottoms in the early 20th century during a period of drought. As a result, severe gully erosion was likely initiated in localized, compacted, and oversteepened reaches of the valley bottom.

  15. Electronic transitions of palladium dimer

    SciTech Connect

    Qian, Yue; Ng, Y. W.; Chen, Zhihua; Cheung, A. S.-C.

    2013-11-21

    The laser induced fluorescence spectrum of palladium dimer (Pd{sub 2}) in the visible region between 480 and 700 nm has been observed and analyzed. The gas-phase Pd{sub 2} molecule was produced by laser ablation of palladium metal rod. Eleven vibrational bands were observed and assigned to the [17.1] {sup 3}II{sub g} - X{sup 3}Σ{sub u}{sup +} transition system. The bond length (r{sub o}) and vibrational frequency (ΔG{sub 1/2}) of the ground X{sup 3}Σ{sub u}{sup +} state were determined to be 2.47(4) Å and 211.4(5) cm{sup −1}, respectively. A molecular orbital energy level diagram was used to understand the observed ground and excited electronic states. This is the first gas-phase experimental investigation of the electronic transitions of Pd{sub 2}.

  16. Structural characterization of dimeric murine aminoacylase III.

    PubMed

    Ryazantsev, Sergey; Abuladze, Natalia; Newman, Debra; Bondar, Galyna; Kurtz, Ira; Pushkin, Alexander

    2007-05-01

    Aminoacylase III (AAIII) plays an important role in deacetylation of acetylated amino acids and N-acetylated S-cysteine conjugates of halogenated alkenes and alkanes. AAIII, recently cloned from mouse kidney and partially characterized, is a mixture of tetramers and dimers. In the present work, AAIII dimers were purified and shown to be enzymatically active. Limited trypsinolysis showed two domains of approximately 9 and 25 kDa. The three-dimensional structure of the dimer was studied by electron microscopy of negative stained samples and by single-particle reconstruction. A 16A resolution model of the AAIII dimer was created. It has an unusual, cage-like, structure. A realistic AAIII tetramer model was built from two dimers.

  17. Dimer statistics on the Klein bottle

    NASA Astrophysics Data System (ADS)

    Lu, Fuliang; Zhang, Lianzhu; Lin, Fenggen

    2011-06-01

    The problem of enumerating close-packed dimers, or perfect matchings, on a quadratic lattice embedded on the Klein bottle is considered. Thomassen [C. Thomassen, Tilings of the torus and the Klein Bottle and vertex-transitive graphs on a fixed surface, Trans. Amer. Math. Soc. 323(2) (1991) 605-635] characterized that there are six quadrilateral lattices embedded on the Klein bottle. Lu and Wu [W. T. Lu and F. Y. Wu, Close-packed dimers on nonorientable surfaces, Phys. Lett. A 293 (2002) 235-246] had obtained a expression for the number of close-packed dimers on one of them. In this paper we investigate four other embeddings and obtain explicit expressions of the numbers of close-packed dimers and free energy per dimer by enumerating Pfaffians.

  18. Statistical transmutation in doped quantum dimer models.

    PubMed

    Lamas, C A; Ralko, A; Cabra, D C; Poilblanc, D; Pujol, P

    2012-07-06

    We prove a "statistical transmutation" symmetry of doped quantum dimer models on the square, triangular, and kagome lattices: the energy spectrum is invariant under a simultaneous change of statistics (i.e., bosonic into fermionic or vice versa) of the holes and of the signs of all the dimer resonance loops. This exact transformation enables us to define the duality equivalence between doped quantum dimer Hamiltonians and provides the analytic framework to analyze dynamical statistical transmutations. We investigate numerically the doping of the triangular quantum dimer model with special focus on the topological Z(2) dimer liquid. Doping leads to four (instead of two for the square lattice) inequivalent families of Hamiltonians. Competition between phase separation, superfluidity, supersolidity, and fermionic phases is investigated in the four families.

  19. Robotic Laparoendoscopic Single-site Retroperitioneal Renal Surgery: Initial Investigation of a Purpose-built Single-port Surgical System.

    PubMed

    Maurice, Matthew J; Ramirez, Daniel; Kaouk, Jihad H

    2017-04-01

    Robotic single-site retroperitoneal renal surgery has the potential to minimize the morbidity of standard transperitoneal and multiport approaches. Traditionally, technological limitations of non-purpose-built robotic platforms have hindered the application of this approach. To assess the feasibility of retroperitoneal renal surgery using a new purpose-built robotic single-port surgical system. This was a preclinical study using three male cadavers to assess the feasibility of the da Vinci SP1098 surgical system for robotic laparoendoscopic single-site (R-LESS) retroperitoneal renal surgery. We used the SP1098 to perform retroperitoneal R-LESS radical nephrectomy (n=1) and bilateral partial nephrectomy (n=4) on the anterior and posterior surfaces of the kidney. Improvements unique to this system include enhanced optics and intelligent instrument arm control. Access was obtained 2cm anterior and inferior to the tip of the 12th rib using a novel 2.5-cm robotic single-port system that accommodates three double-jointed articulating robotic instruments, an articulating camera, and an assistant port. The primary outcome was the technical feasibility of the procedures, as measured by the need for conversion to standard techniques, intraoperative complications, and operative times. All cases were completed without the need for conversion. There were no intraoperative complications. The operative time was 100min for radical nephrectomy, and the mean operative time was 91.8±18.5min for partial nephrectomy. Limitations include the preclinical model, the small sample size, and the lack of a control group. Single-site retroperitoneal renal surgery is feasible using the latest-generation SP1098 robotic platform. While the potential of the SP1098 appears promising, further study is needed for clinical evaluation of this investigational technology. In an experimental model, we used a new robotic system to successfully perform major surgery on the kidney through a single small

  20. UVA Generates Pyrimidine Dimers in DNA Directly

    PubMed Central

    Jiang, Yong; Rabbi, Mahir; Kim, Minkyu; Ke, Changhong; Lee, Whasil; Clark, Robert L.; Mieczkowski, Piotr A.; Marszalek, Piotr E.

    2009-01-01

    There is increasing evidence that UVA radiation, which makes up ∼95% of the solar UV light reaching the Earth's surface and is also commonly used for cosmetic purposes, is genotoxic. However, in contrast to UVC and UVB, the mechanisms by which UVA produces various DNA lesions are still unclear. In addition, the relative amounts of various types of UVA lesions and their mutagenic significance are also a subject of debate. Here, we exploit atomic force microscopy (AFM) imaging of individual DNA molecules, alone and in complexes with a suite of DNA repair enzymes and antibodies, to directly quantify UVA damage and reexamine its basic mechanisms at a single-molecule level. By combining the activity of endonuclease IV and T4 endonuclease V on highly purified and UVA-irradiated pUC18 plasmids, we show by direct AFM imaging that UVA produces a significant amount of abasic sites and cyclobutane pyrimidine dimers (CPDs). However, we find that only ∼60% of the T4 endonuclease V-sensitive sites, which are commonly counted as CPDs, are true CPDs; the other 40% are abasic sites. Most importantly, our results obtained by AFM imaging of highly purified native and synthetic DNA using T4 endonuclease V, photolyase, and anti-CPD antibodies strongly suggest that CPDs are produced by UVA directly. Thus, our observations contradict the predominant view that as-yet-unidentified photosensitizers are required to transfer the energy of UVA to DNA to produce CPDs. Our results may help to resolve the long-standing controversy about the origin of UVA-produced CPDs in DNA. PMID:19186150

  1. Barite chimneys from two hydrothermal sites along the slow-spreading Arctic Ridge system: Initial isotope and mineralogical results

    NASA Astrophysics Data System (ADS)

    Eickmann, B.; van Zuilen, M. A.; Thorseth, I. H.; Pedersen, R.

    2010-12-01

    Two hydrothermal sites along the slow-spreading Arctic Ridge systems, the Jan Mayen vent fields (JMVFs) and the recently discovered Loki’s Castle hydrothermal field (LCHF) contains numerous barite chimneys partially covered by microbial mats. The JMVFs are located at 71°N on the south-western Mohns Ridge, approximately 50 km north of the Jan Mayen fracture zone. The LCHF is located at 73.5°N on an axial volcanic ridge where the Mohns Ridge transitions into the Knipovich Ridge and consists of two venting areas. Active hydrothermal venting at both sites is confirmed by elevated hydrogen sulphide concentrations and discharge of high-temperature fluids, reaching 270°C in the JMVFs and 317°C in the LCHF. Barite chimneys from the JMVFs are composed of barite, silica and abundant pyrite-dominated sulphide minerals that display a conspicuous concentric morphology. Raman spectroscopic analysis of the central regions of these concentric sulphide minerals points to the existence of mackinawite (FeS). Furthermore, the existence of greigite (Fe3S4) surrounding the mackinawite is suggested. This observation confirms the general conclusion of earlier experimental studies that these phases act as the metastable precursors of pyrite. In contrast, the barite chimneys of the LCHF consist mainly of pure barite with lesser amounts of sulphide minerals. The difference in the mineralogical composition between the two sites is also expressed in its sulphur isotopic composition. δ34Ssulphate values of the barite chimneys from the JMVFs are lower than δ34S of seawater sulphate (δ34S = +21‰) and δ34Ssulphide values point to a magmatic sulphur source (δ34S = 0‰). This implies that the JMHFs barite chimneys have been formed by a mixture of seawater and hydrothermal fluids, similar to the origin of black smokers. In contrast to the JMVFs, the δ34Ssulphate values from the LCHF barite chimneys are higher than δ34S values for seawater sulphate, but show remarkable differences

  2. Implementation of DOE/NFDI D&D Cost Estimating Tool (POWERtool) for Initiative Facilities at the Savannah River Site

    SciTech Connect

    Austin, W. E.; WSRC; Baker, S. B. III, Cutshall, C. M.; Crouse, J. L.

    2003-02-26

    The Savannah River Site (SRS) has embarked on an aggressive D&D program to reduce the footprint of excess facilities. Key to the success of this effort is the preparation of accurate cost estimates for decommissioning. SRS traditionally uses ''top-down'' rough order-of-magnitude (ROM) estimating for decommissioning cost estimates. A second cost estimating method (POWERtool) using a ''bottoms-up'' approach has been applied to many of the SRS excess facilities in the T and D-area. This paper describes the use of both estimating methods and compares the estimated costs to actual costs of 5 facilities that were decommissioned in 2002.

  3. Feasibility Study of Economics and Performance of Biomass Power Generation at the Former Farmland Industries Site in Lawrence, Kansas. A Study Prepared in Partnership with the Environmental Protection Agency for the RE-Powering America's Land Initiative: Siting Renewable Energy on Potentially Contaminated Land and Mine Sites

    SciTech Connect

    Tomberlin, G.; Mosey, G.

    2013-03-01

    Under the RE-Powering America's Land initiative, the U.S. Environmental Protection Agency (EPA) provided funding to the National Renewable Energy Laboratory (NREL) to support a feasibility study of biomass renewable energy generation at the former Farmland Industries site in Lawrence, Kansas. Feasibility assessment team members conducted a site assessment to gather information integral to this feasibility study. Information such as biomass resources, transmission availability, on-site uses for heat and power, community acceptance, and ground conditions were considered.

  4. Structure of a Rabbit Muscle Fructose-1,6-Bisphosphate Aldolase A Dimer Variant

    SciTech Connect

    Sherawat,M.; Tolan, D.; Allen, K.

    2008-01-01

    Fructose-1,6-bisphosphate aldolase (aldolase) is an essential enzyme in glycolysis and gluconeogenesis. In addition to this primary function, aldolase is also known to bind to a variety of other proteins, a property that may allow it to perform 'moonlighting' roles in the cell. Although monomeric and dimeric aldolases possess full catalytic activity, the enzyme occurs as an unusually stable tetramer, suggesting a possible link between the oligomeric state and these noncatalytic cellular roles. Here, the first high-resolution X-ray crystal structure of rabbit muscle D128V aldolase, a dimeric form of aldolase mimicking the clinically important D128G mutation in humans associated with hemolytic anemia, is presented. The structure of the dimer was determined to 1.7 Angstroms resolution with the product DHAP bound in the active site. The turnover of substrate to produce the product ligand demonstrates the retention of catalytic activity by the dimeric aldolase. The D128V mutation causes aldolase to lose intermolecular contacts with the neighboring subunit at one of the two interfaces of the tetramer. The tertiary structure of the dimer does not significantly differ from the structure of half of the tetramer. Analytical ultracentrifugation confirms the occurrence of the enzyme as a dimer in solution. The highly stable structure of aldolase with an independent active site is consistent with a model in which aldolase has evolved as a multimeric scaffold to perform other noncatalytic functions.

  5. Targeting TLR4 Signaling by TLR4 TIR-derived Decoy Peptides: Identification of the TLR4 TIR Dimerization Interface

    PubMed Central

    Toshchakov, Vladimir Y.; Szmacinski, Henryk; Couture, Leah A.; Lakowicz, Joseph R.; Vogel, Stefanie N.

    2011-01-01

    Agonist-induced dimerization of TLR4 TIR domains initiates intracellular signaling. Therefore, identification of the TLR4 TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating “decoy peptides,” each of which represents a non-fragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides, 4R1, 4R3, 4BB, 4R9, and 4αE, potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by FRET using time-resolved fluorescence spectroscopy, Bodipy-TMR-X (BTX)-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean (Cer) expressed in HeLa or HEK293T cells, while 4R3 was partially active and 4R9 was least active. These findings suggest that the area between BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the “decoy peptide approach,” in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function and then their specific targets are identified by FRET, to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions. PMID:21402890

  6. Local energy gap opening induced by hemin dimerization in aqueous solution.

    PubMed

    Golnak, Ronny; Xiao, Jie; Atak, Kaan; Khan, Munirah; Suljoti, Edlira; Aziz, Emad F

    2015-02-19

    The local electronic structure of the hemin Fe center has been investigated by X-ray absorption and emission spectroscopy (XAS/XES) for hemin in aqueous solution where hemin dimerization occurs. The XAS and XES spectra of the hemin dimer were then compared with those of the hemin monomer we previously studied in dimethyl sulfoxide solution. A local energy gap opening at the Fe sites was observed for the hemin dimer, with the occupied valence states shifted to lower binding energies, while the unoccupied valence states share the same energies as the hemin monomer. Such a gap opening is argued to originate from the Fe 3d orbital localization induced by hemin dimerization in aqueous solution.

  7. TSPA 1991: An initial total-system performance assessment for Yucca Mountain; Yucca Mountain Site Characterization Project

    SciTech Connect

    Barnard, R.W.; Wilson, M.L.; Dockery, H.A.; Kaplan, P.G.; Eaton, R.R.; Bingham, F.W.; Gauthier, J.H.; Robey, T.H.

    1992-07-01

    This report describes an assessment of the long-term performance of a repository system that contains deeply buried highly radioactive waste; the system is assumed to be located at the potential site at Yucca Mountain, Nevada. The study includes an identification of features, events, and processes that might affect the potential repository, a construction of scenarios based on this identification, a selection of models describing these scenarios (including abstraction of appropriate models from detailed models), a selection of probability distributions for the parameters in the models, a stochastic calculation of radionuclide releases for the scenarios, and a derivation of complementary cumulative distribution functions (CCDFs) for the releases. Releases and CCDFs are calculated for four categories of scenarios: aqueous flow (modeling primarily the existing conditions at the site, with allowances for climate change), gaseous flow, basaltic igneous activity, and human intrusion. The study shows that models of complex processes can be abstracted into more simplified representations that preserve the understanding of the processes and produce results consistent with those of more complex models.

  8. Environmental Impacts of Petroleum Production: Initial Results from the Osage-Skiatook Petroleum Environmental Research Sites, Osage County, Oklahoma

    USGS Publications Warehouse

    Kharaka, Yousif K.; Otton, James K.

    2003-01-01

    Exploration for and production of petroleum have caused major detrimental impacts to soils, surface and ground waters, and the local