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Sample records for dimerization initiation site

  1. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

    PubMed

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

  2. Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

    PubMed Central

    2011-01-01

    Background During the replication process of bacteria with circular chromosomes, an odd number of homologous recombination events results in concatenated dimer chromosomes that cannot be partitioned into daughter cells. However, many bacteria harbor a conserved dimer resolution machinery consisting of one or two tyrosine recombinases, XerC and XerD, and their 28-bp target site, dif. Results To study the evolution of the dif/XerCD system and its relationship with replication termination, we report the comprehensive prediction of dif sequences in silico using a phylogenetic prediction approach based on iterated hidden Markov modeling. Using this method, dif sites were identified in 641 organisms among 16 phyla, with a 97.64% identification rate for single-chromosome strains. The dif sequence positions were shown to be strongly correlated with the GC skew shift-point that is induced by replicational mutation/selection pressures, but the difference in the positions of the predicted dif sites and the GC skew shift-points did not correlate with the degree of replicational mutation/selection pressures. Conclusions The sequence of dif sites is widely conserved among many bacterial phyla, and they can be computationally identified using our method. The lack of correlation between dif position and the degree of GC skew suggests that replication termination does not occur strictly at dif sites. PMID:21223577

  3. Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.

    PubMed

    Kripke, M L; Cox, P A; Alas, L G; Yarosh, D B

    1992-08-15

    Exposing the skin of mice to UV radiation interferes with the induction of delayed and contact hypersensitivity immune responses initiated at nonirradiated sites. The identity of the molecular target in the skin for these immunosuppressive effects of UV radiation remains controversial. To test the hypothesis that DNA is the target for UV-induced systemic immunosuppression, we exposed C3H mice to UV radiation and then used liposomes to deliver a dimer-specific excision repair enzyme into the epidermis in situ. The application of T4 endonuclease V encapsulated in liposomes to UV-irradiated mouse skin decreased the number of cyclobutane pyrimidine dimers in the epidermis and prevented suppression of both delayed and contact hypersensitivity responses. Moreover, the formation of suppressor lymphoid cells was inhibited. Control, heat-inactivated endonuclease encapsulated in liposomes had no effect. These studies demonstrate that DNA is the major target of UV radiation in the generation of systemic immunosuppression and suggest that the primary molecular event mediating these types of immunosuppression by UV radiation is the formation of pyrimidine dimers. Furthermore, they illustrate that the delivery of lesion-specific DNA repair enzymes to living skin after UV irradiation is an effective tool for restoring immune function and suggest that this approach may be broadly applicable to preventing other alterations caused by DNA damage. PMID:1502162

  4. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    PubMed Central

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

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

    SciTech Connect

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

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer aIF5B forms maximum 5.0-6.8% irreversible dimers in solution. Black-Right-Pointing-Pointer Sedimentation coefficients for monomer and dimer are 3.64 and 5.51 {+-} 0.29 S. Black-Right-Pointing-Pointer Adding only 2% glycerol prevents dimerization. Black-Right-Pointing-Pointer SAXS on aIF5B monomer gave an R{sub g} of 37.5 {+-} 0.2 A and a D{sub max} of {approx}130 A. Black-Right-Pointing-Pointer There are universal structural differences between aIF5B and Escherichia coli IF2. -- Abstract: 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. ) 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 A

  6. 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.

  7. A search for site-filling ligands in the Mcg Bence-Jones dimer: crystal binding studies of fluorescent compounds.

    PubMed

    Edmundson, A B; Ely, K R; Herron, J N

    1984-07-01

    In trigonal crystals grown in 1.9 M ammonium sulfate buffered at pH 6.2, the Mcg light-chain (Bence-Jones) dimer has a highly aromatic binding cavity accessible to a wide range of hydrophobic and aromatic ligands. A search was made for site-filling ligands by diffusing compounds into the crystals and determining their locations, orientations and relative occupancies by difference Fourier analysis at 2.7-A resolution. 1-Anilinonaphthalene-8-sulfonate, a small ligand in comparison with the rest of the series, initially occupied a site in the main binding cavity. With time, however, this ligand changed its position to the deep binding pocket beyond the floor of the main cavity. The original binding site remained vacant, despite the presence of a large excess of ligand in the soaking solution. Ligands increasing in size from fluorescein to bis(N-methyl)acridine (lucigenin) to dimers of carboxytetramethylrhodamine were found to bind with stringent stereospecificity in the main cavity, but the mode of binding was different in each case. The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand. The differences in the binding properties of dimers of 5- and 6-carboxytetramethylrhodamine led to an explanation for isomeric discrimination in the binding site. There were extensive conformational changes in the binding cavity to accommodate the ligands, particularly 6-carboxytetramethylrhodamine. The second and third hypervariable loops proved very flexible, and moved in ways to expand the binding site. The side chains of key tyrosine and phenylalanine residues in the site were also highly mobile. Their orientations adjusted to optimize complementarity with the ligands. These conformational adjustments are consistent with the tenets of a limited neo-instructive theory of ligand binding.

  8. Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer.

    PubMed

    Fox, Jamie C; Tyler, Robert C; Peterson, Francis C; Dyer, Douglas P; Zhang, Fuming; Linhardt, Robert J; Handel, Tracy M; Volkman, Brian F

    2016-03-01

    Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition. PMID:26836755

  9. Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2003-11-11

    Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. PMID:14596623

  10. 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.

  11. Intrinsic site-selectivity of ubiquitin dimer formation

    PubMed Central

    Andersen, Kristen A; Martin, Langdon J; Prince, Joel M; Raines, Ronald T

    2015-01-01

    The post-translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme-catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme-free system by measuring the rate constants for thiol–disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles. PMID:25401704

  12. 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.

  13. ATP binding to two sites is necessary for dimerization of nucleotide-binding domains of ABC proteins.

    PubMed

    Zoghbi, Maria E; Altenberg, Guillermo A

    2014-01-01

    ATP binding cassette (ABC) transporters have a functional unit formed by two transmembrane domains and two nucleotide binding domains (NBDs). ATP-bound NBDs dimerize in a head-to-tail arrangement, with two nucleotides sandwiched at the dimer interface. Both NBDs contribute residues to each of the two nucleotide-binding sites (NBSs) in the dimer. In previous studies, we showed that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii forms ATP-bound dimers that dissociate completely following hydrolysis of one of the two bound ATP molecules. Since hydrolysis of ATP at one NBS is sufficient to drive dimer dissociation, it is unclear why all ABC proteins contain two NBSs. Here, we used luminescence resonance energy transfer (LRET) to study ATP-induced formation of NBD homodimers containing two NBSs competent for ATP binding, and NBD heterodimers with one active NBS and one binding-defective NBS. The results showed that binding of two ATP molecules is necessary for NBD dimerization. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dissociation, but two binding sites are required to form the ATP-sandwich NBD dimer necessary for hydrolysis.

  14. Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.

    PubMed

    He, Yan; Wang, Minghui; Sun, Qi; Pawlowski, Wojciech P

    2016-01-01

    Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi. PMID:27511175

  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. 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.

  17. Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

    PubMed

    Pasquo, A; Britton, K L; Stillman, T J; Rice, D W; Cölfen, H; Harding, S E; Scandurra, R; Engel, P C

    1996-10-17

    By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the

  18. Effect of Spatial Inhomogeneities on the Membrane Surface on Receptor Dimerization and Signal Initiation

    PubMed Central

    Kerketta, Romica; Halász, Ádám M.; Steinkamp, Mara P.; Wilson, Bridget S.; Edwards, Jeremy S.

    2016-01-01

    Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as “clusters” by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (“domains”) for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems. PMID:27570763

  19. Effect of Spatial Inhomogeneities on the Membrane Surface on Receptor Dimerization and Signal Initiation.

    PubMed

    Kerketta, Romica; Halász, Ádám M; Steinkamp, Mara P; Wilson, Bridget S; Edwards, Jeremy S

    2016-01-01

    Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as "clusters" by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones ("domains") for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems. PMID:27570763

  20. 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

  1. Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

    PubMed

    Moya-García, Aurelio A; Rodríguez-Agudo, Daniel; Hayashi, Hideyuki; Medina, Miguel Angel; Urdiales, José Luis; Sánchez-Jiménez, Francisca

    2011-06-14

    Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases. PMID:26596454

  2. 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

  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. 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

  5. A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication ▿ †

    PubMed Central

    Oke, Muse; Kerou, Melina; Liu, Huanting; Peng, Xu; Garrett, Roger A.; Prangishvili, David; Naismith, James H.; White, Malcolm F.

    2011-01-01

    The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5′ end of the DNA, releasing a 3′ DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed. PMID:21068244

  6. Thioredoxin A Active-Site Mutants Form Mixed Disulfide Dimers That Resemble Enzyme–Substrate Reaction Intermediates

    PubMed Central

    Kouwen, Thijs R.H.M.; Andréll, Juni; Schrijver, Rianne; Dubois, Jean-Yves F.; Maher, Megan J.; Iwata, So; Carpenter, Elisabeth P.; van Dijl, Jan Maarten

    2008-01-01

    Thioredoxin functions in nearly all organisms as the major thiol–disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme–substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes. PMID:18455736

  7. 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...

  8. Hanford tank initiative test facility site selection study

    SciTech Connect

    Staehr, T.W.

    1997-04-03

    The Hanford Tanks Initiative (HTI) project is developing equipment for the removal of hard heel waste from the Hanford Site underground single-shell waste storage tanks. The HTI equipment will initially be installed in the 241-C-106 tank where its operation will be demonstrated. This study evaluates existing Hanford Site facilities and other sites for functional testing of the HTI equipment before it is installed into the 241-C-106 tank.

  9. 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.

  10. 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

  11. 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. PMID:26354431

  12. 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.

  13. 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

    2012-02-01

    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

  14. 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

  15. 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.

  16. 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…

  17. Somitogenesis clock-wave initiation requires differential decay and multiple binding sites for clock protein.

    PubMed

    Campanelli, Mark; Gedeon, Tomás

    2010-04-01

    Somitogenesis is a process common to all vertebrate embryos in which repeated blocks of cells arise from the presomitic mesoderm (PSM) to lay a foundational pattern for trunk and tail development. Somites form in the wake of passing waves of periodic gene expression that originate in the tailbud and sweep posteriorly across the PSM. Previous work has suggested that the waves result from a spatiotemporally graded control protein that affects the oscillation rate of clock-gene expression. With a minimally constructed mathematical model, we study the contribution of two control mechanisms to the initial formation of this gene-expression wave. We test four biologically motivated model scenarios with either one or two clock protein transcription binding sites, and with or without differential decay rates for clock protein monomers and dimers. We examine the sensitivity of wave formation with respect to multiple model parameters and robustness to heterogeneity in cell population. We find that only a model with both multiple binding sites and differential decay rates is able to reproduce experimentally observed waveforms. Our results show that the experimentally observed characteristics of somitogenesis wave initiation constrain the underlying genetic control mechanisms.

  18. Dimeric Cinchona alkaloids.

    PubMed

    Boratyński, Przemysław J

    2015-05-01

    Nature is full of dimeric alkaloids of various types from many plant families, some of them with interesting biological properties. However, dimeric Cinchona alkaloids were not isolated from any species but were products of designed partial chemical synthesis. Although the Cinchona bark is amongst the sources of oldest efficient medicines, the synthetic dimers found most use in the field of asymmetric synthesis. Prominent examples include the Sharpless dihydroxylation and aminohydroxylation ligands, and dimeric phase transfer catalysts. In this article the syntheses of Cinchona alkaloid dimers and oligomers are reviewed, and their structure and applications are outlined. Various synthetic routes exploit reactivity of the alkaloids at the central 9-hydroxyl group, quinuclidine, and quinoline rings, as well as 3-vinyl group. This availability of reactive sites, in combination with a plethora of linker molecules, contributes to the diversity of the products obtained.

  19. 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

  20. Structure of the human dimeric ATM kinase.

    PubMed

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

    2016-01-01

    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

  1. 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.

    PubMed

    Dellarole, Mariano; Sánchez, Ignacio E; Freire, Eleonora; de Prat-Gay, Gonzalo

    2007-10-30

    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.

  2. 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.

  3. Site Specific Advisory Board initiative, evaluation survey results supplementary appendix: Summary of individual site results

    SciTech Connect

    1996-08-01

    This Appendix presents results of the Site-Specific Advisory Board (SSAB) Initiative for each of the 11 sites that participated in the survey. These individual results are a supplement to the June 1996 Summary Report which presented overall survey results. Results are presented in 11 sections, arranged alphabetically by site. Each section includes a series of figures and tables that parallel those presented in the Summary Report. To facilitate comparison, figures are presented both for the individual site and for the overall long survey. The sequence of sections is: Fernald, Hanford, Idaho, Los Alamos, Monticello, Nevada, Pantex, Rocky Flats, St. Louis, Sandia, and Savannah River.

  4. Specific Initiation Site for Simian Virus 40 Deoxyribonucleic Acid Replication

    PubMed Central

    Thoren, Marilyn M.; Sebring, Edwin D.; Salzman, Norman P.

    1972-01-01

    Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with 3H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication. PMID:4342054

  5. 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.

  6. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites

    PubMed Central

    Sathyanarayana, Bangalore K.; Li, Peng; Lin, Jian-Xin; Leonard, Warren J.

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  7. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites.

    PubMed

    Sathyanarayana, Bangalore K; Li, Peng; Lin, Jian-Xin; Leonard, Warren J; Lee, Byungkook

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  8. 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

  9. 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. PMID:27550720

  10. 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.

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

    PubMed Central

    Walsh, Martin A; Otwinowski, Zbyszek; Perrakis, Anatassis; Anderson, Paul M; Joachimiak, Andrzej

    2011-01-01

    Background 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. Results We have determined the crystal structure of cyanase at 1.65 Å 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. Conclusions The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-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. PMID:10801492

  12. Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer resolution only when located between long stretches of opposite polarity.

    PubMed

    Pérals, K; Cornet, F; Merlet, Y; Delon, I; Louarn, J M

    2000-04-01

    In Escherichia coli, chromosome dimers are generated by recombination between circular sister chromosomes. Dimers are lethal unless resolved by a system that involves the XerC, XerD and FtsK proteins acting at a site (dif) in the terminus region. Resolution fails if dif is moved from its normal position. To analyse this positional requirement, dif was transplaced to a variety of positions, and deletions and inversions of portions of the dif region were constructed. Resolution occurs only when dif is located at the convergence of multiple, oppositely polarized DNA sequence elements, inferred to lie in the terminus region. These polar elements may position dif at the cell septum and be general features of chromosome organization with a role in nucleoid dynamics.

  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. PMID:26732804

  14. 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.

  15. Redesigning the procaspase-8 dimer interface for improved dimerization

    PubMed Central

    Ma, Chunxiao; MacKenzie, Sarah H; Clay Clark, A

    2014-01-01

    Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis. PMID:24442640

  16. 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.

  17. Single-site video endoscopic inguinal lymphadenectomy: initial report.

    PubMed

    Tobias-Machado, Marcos; Correa, Walter F; Reis, Leonardo Oliveira; Starling, Eduardo S; de Castro Neves, Oseas; Juliano, Roberto V; Pompeo, Antonio C L

    2011-04-01

    Techniques that attempt to further reduce the morbidity and improve cosmesis of laparoscopic surgery have particularly generated interest. Since its initial urologic description in 2007, there has been a surge of interest in laparoendoscopic single-site surgery, which is now an emerging technique within the field of minimally invasive urologic surgery. This report describes a preliminary experience with single-site video endoscopic inguinal lymphadenectomy (SSVEIL) compared with conventional video endoscopic inguinal lymphadenectomy (VEIL) on inguinal nodes management in a 45-year-old man with pT(2) grade 2 squamous cell penile carcinoma and impalpable inguinal nodes. VEIL with saphenous vein preservation in the left leg and SSVEIL on the other side presented no difference concerning operative time (100 vs 120 min), blood loss (50 mL), drainage volume, number of nodes retrieved (8), pain, and oncologic outcome. The patient had an uneventful postoperative course, was discharged 12 hours after the procedure, and preferred the aesthetic result of SSVEIL. Further refinements in technology will likely alleviate many of the persistent technical problems. Additional rigorous comparison studies are needed to evaluate the true benefits of the technique and the extent of its clinical application, mainly oncologic results, before the widespread adoption of SSVEIL. Ultimately, advance breakthroughs in fields of in-vivo instrumentation, robotics, and purpose-built robotic platforms will bring its potential to full clinical realization. PMID:21226622

  18. 40 CFR 280.63 - Initial site characterization.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... site investigations concerning the following factors: surrounding populations, water quality, use and... subsurface sewers, climatological conditions, and land use; (3) Results of the site check required...

  19. The low-affinity ATP binding site of the Escherichia coli SecA dimer is localized at the subunit interface.

    PubMed

    van der Wolk, J P; Boorsma, A; Knoche, M; Schäfer, H J; Driessen, A J

    1997-12-01

    The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosine 5'-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer. PMID:9398216

  20. 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

  1. 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.

  2. 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

  3. Hanford Tanks Initiative cone penetrometer siting plan and progress report

    SciTech Connect

    IWATATE, D.F.

    1998-10-15

    The HTI subsurface characterization task will use the Hanford Cone Penetrometer platform (CPP) to deploy soil sensor and sampling probes into the vadose zone/soils around AX-104 during FY-99. This Siting Plan describes activities and actions undertaken in support of CPP deployment: deployment goals, maps of the deployment sites/locations, pre-activity (siting-related) documentation tasks, a summary of activities that have been completed to date, and an estimated schedule of additional planned activities.

  4. High-affinity Manganese Coordination by Human Calprotectin is Calcium-dependent and Requires the Histidine-rich Site Formed at the Dimer Interface

    PubMed Central

    Hayden, Joshua A.; Brophy, Megan Brunjes; Cunden, Lisa S.; Nolan, Elizabeth M.

    2013-01-01

    Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of calprotectin. We demonstrate that the unusual His4 motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn(II) coordination. We identify a low-temperature Mn(II) spectroscopic signal for this site consistent with an octahedral Mn(II) coordination sphere with simulated zero-field splitting parameters D = 270 MHz and E/D = 0.33 (E = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn(II) in addition to two as-yet unidentified ligands. The His3Asp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn(II) affinity of the His4 site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn(II) over Mn(II). Nevertheless, the specificity of Mn(II) for the His4 site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn(II) ion occupies site 1 and one Mn(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth. PMID:23276281

  5. 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.

  6. Sequence-specific initiator elements focus initiation of transcription to distinct sites in the yeast TRP4 promoter.

    PubMed Central

    Mösch, H U; Graf, R; Braus, G H

    1992-01-01

    Transcription from the yeast TRP4 promoter initiates at two basal (i127 and i76) and three GCN4 dependent (i31, i25 and i12) initiator elements. All of these elements contain not more than one deviation from the earlier proposed initiator consensus sequence PuPuPyPuPu, a pyrimidine nucleotide flanked on either side by two purine nucleotides. A point mutation analysis of these elements in various combinations was performed and revealed that the central pyrimidine nucleotide and at least one of the 3' flanking purine nucleotides of the PuPuPyPuPu consensus sequence are essential but alone not sufficient to define a functional initiator element. Multiple cryptic transcription start sites, which function independently whether they are located on the coding or the non-coding strand, can replace the function of mutated initiator elements and therefore the overall level of transcription initiation is not affected. The sequence specificity is identical for basal and GCN4 dependent initiator elements demonstrating that they are functionally homologous. These findings imply that the role of initiator elements is to 'focus' the start point(s) of transcription to distinct sites located in the region between the site(s) of the assembly of the transcriptional complex and the start codon of translation. Images PMID:1425591

  7. Utilizing the fluidized bed to initiate water treatment on site

    SciTech Connect

    Ahmadvand, H.; Germann, G.; Gandee, J.P.; Buehler, V.T.

    1995-12-31

    Escalating wastewater disposal costs coupled with enforcement of stricter regulations push industrial sites previously without water treatment to treat on site. These sites, inexperienced in water treatment, require a treatment technology that is easily installed, operated, and maintained. The aerobic granular activated carbon (GAC) fluidized bed incorporates biological and adsorptive technologies into a simple, cost-effective process capable of meeting strict effluent requirements. Two case studies at industrial sites illustrate the installation and operation of the fluidized bed and emphasize the ability to use the fluidized bed singularly or as an integral component of a treatment system capable of achieving treatment levels that allow surface discharge and reinjection. Attention is focused on BTEX (benzene, toluene, ethylbenzene, and xylenes).

  8. Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation.

    PubMed

    Szymanski, Michal R; Jezewska, Maria J; Bujalowski, Wlodzimierz

    2011-08-01

    A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ϕX174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.

  9. Geometric Frustration of Colloidal Dimers on a Honeycomb Magnetic Lattice

    NASA Astrophysics Data System (ADS)

    Tierno, Pietro

    2016-01-01

    We study the phase behavior and the collective dynamics of interacting paramagnetic colloids assembled above a honeycomb lattice of triangular shaped magnetic minima. A frustrated colloidal molecular crystal is realized when filling these potential minima with exactly two particles per pinning site. External in-plane rotating fields are used to anneal the system into different phases, including long range ordered stripes, random fully packed loops, labyrinth and disordered states. At a higher amplitude of the annealing field, the dimer lattice displays a two-step melting transition where the initially immobile dimers perform first localized rotations and later break up by exchanging particles across consecutive lattice minima.

  10. 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.

  11. Retrotransposon profiling of RNA polymerase III initiation sites

    PubMed Central

    Qi, Xiaojie; Daily, Kenneth; Nguyen, Kim; Wang, Haoyi; Mayhew, David; Rigor, Paul; Forouzan, Sholeh; Johnston, Mark; Mitra, Robi David; Baldi, Pierre; Sandmeyer, Suzanne

    2012-01-01

    Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III–transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III–transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes. PMID:22287102

  12. Retrotransposon profiling of RNA polymerase III initiation sites.

    PubMed

    Qi, Xiaojie; Daily, Kenneth; Nguyen, Kim; Wang, Haoyi; Mayhew, David; Rigor, Paul; Forouzan, Sholeh; Johnston, Mark; Mitra, Robi David; Baldi, Pierre; Sandmeyer, Suzanne

    2012-04-01

    Although retroviruses are relatively promiscuous in choice of integration sites, retrotransposons can display marked integration specificity. In yeast and slime mold, some retrotransposons are associated with tRNA genes (tDNAs). In the Saccharomyces cerevisiae genome, the long terminal repeat retrotransposon Ty3 is found at RNA polymerase III (Pol III) transcription start sites of tDNAs. Ty1, 2, and 4 elements also cluster in the upstream regions of these genes. To determine the extent to which other Pol III-transcribed genes serve as genomic targets for Ty3, a set of 10,000 Ty3 genomic retrotranspositions were mapped using high-throughput DNA sequencing. Integrations occurred at all known tDNAs, two tDNA relics (iYGR033c and ZOD1), and six non-tDNA, Pol III-transcribed types of genes (RDN5, SNR6, SNR52, RPR1, RNA170, and SCR1). Previous work in vitro demonstrated that the Pol III transcription factor (TF) IIIB is important for Ty3 targeting. However, seven loci that bind the TFIIIB loader, TFIIIC, were not targeted, underscoring the unexplained absence of TFIIIB at those sites. Ty3 integrations also occurred in two open reading frames not previously associated with Pol III transcription, suggesting the existence of a small number of additional sites in the yeast genome that interact with Pol III transcription complexes. PMID:22287102

  13. 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.

  14. 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.

  15. Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra.

    PubMed

    Vreeswijk, Maaike P G; Meijers, Caro M; Giphart-Gassler, Micheline; Vrieling, Harry; van Zeeland, Albert A; Mullenders, Leon H F; Loenen, Wil A M

    2009-04-26

    Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4 PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed strand. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C>T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.

  16. Initiation sites of protein folding by NMR analysis.

    PubMed Central

    Freund, S M; Wong, K B; Fersht, A R

    1996-01-01

    Detailed characterization of denatured states of proteins is necessary to understand the interactions that funnel the large number of possible conformations along fast routes for folding. Nuclear magnetic resonance experiments based on the nuclear Overhauser effect (NOE) detect hydrogen atoms close in space and provide information about local structure. Here we present an NMR procedure that detects almost all sequential NOEs between amide hydrogen atoms (HN-HN NOE), including those in random coil regions in a protein, barnase, in urea solutions. A semi-quantitative analysis of these HN-HN NOEs identified partly structured regions that are in remarkable agreement with those found to form early on the reaction pathway. Our results strongly suggest that the folding of barnase initiates at the first helix and the beta-turn between the third and the fourth strands. This strategy of defining residual structure has also worked for cold-denatured barstar and guanidinium hydrochloride-denatured chymotrypsin inhibitor 2 and so should be generally applicable. Images Fig. 1 Fig. 3 PMID:8855224

  17. 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.

  18. 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. PMID:21889542

  19. 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.

  20. Vinculin Tail Dimerization and Paxillin Binding

    NASA Astrophysics Data System (ADS)

    Campbell, Sharon

    2006-03-01

    Vinculin is a highly conserved cytoskeletal protein that is essential for regulation of cell morphology and migration, and is a critical component of both cell-cell and cell-matrix complexes. The tail domain of vinculin (Vt) was crystallized as a homodimer and is believed to bind F-actin as a dimer. We have characterized Vt dimerization by Nuclear Magnetic Resonance (NMR) Spectroscopy and identified the dimer interface in solution by chemical shift perturbation. The Vt dimer interface in solution is similar to the crystallographic dimer interface. Interestingly, the Vt dimer interface determined by NMR partially overlaps the paxillin binding region previously defined coarsely by deletion mutagenesis and gel-blot assays. To further characterize the paxillin binding site in Vt and probe relationship between paxillin binding and dimerization, we conducted chemical shift perturbations experiments using a paxillin derived peptide, LD2. Our NMR experiments have confirmed that the paxillin binding site and the Vt dimerization site partially overlap, and we have further characterized both of these two binding interfaces. Information derived from these studies was used to identify mutations in Vt that selectively perturb paxillin binding and Vt self-association. These mutants are currently being characterized for their utility in structural and biological analyses to elucidate the role of paxillin binding and Vt dimerization in vinculin function.

  1. Dynamical DMRG study of non-linear optical response in one-dimensional dimerized Hubbard model with nearest neighbor Coulomb interaction and alternating on-site potential

    NASA Astrophysics Data System (ADS)

    Sota, Shigetoshi; Tohyama, Takami; Brazovskii, Serguei

    2012-02-01

    The optical response of organic compounds has been attracting much attention. The one of the reasons is the huge non-linear and ultrafast optical response [K. Yamamoto et. al., J. Phys. Soc. Jpn. 77, 074709(2008)]. In order to investigate such optical properties, we carry out dynamical DMRG calculations to obtain optical responses in the 1/4-filled one-dimensional Hubbard model including the nearest neighbor Coulomb interaction and the alternating electron hopping. The charge gap [S. Nishimoto, M. Takahashi, and Y. Ohta, J. Phys. Soc. Jpn. 69, 1594(2000)] and the bound state [H. Benthien and E. Jeckelmann, Eur. Phys. J. B 44, 287(2005)] in this model have been discussed based on DMRG calculations. In the present study, we introduce an alternating on-site potential giving the polarization in the system into the dimerized Hubbard model, which breaks the reflection symmetry of the system. In this talk, we discuss the obtained linear and the 2nd order non-linear optical susceptibility in order to make a prediction for non-linear optical experiments in the future.

  2. Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts

    PubMed Central

    1996-01-01

    Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase- arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure. PMID:8947545

  3. Mechanisms of Monomeric and Dimeric Glycogenin Autoglucosylation*

    PubMed Central

    Issoglio, Federico M.; Carrizo, María E.; Romero, Jorge M.; Curtino, Juan A.

    2012-01-01

    Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization. PMID:22128147

  4. D-dimer test

    MedlinePlus

    D-dimer tests are used to check for blood clotting problems. Blood clots can cause health problems, such ... that you probably do not have problems with blood clotting. If you are getting the D-dimer test ...

  5. Mechanisms underlying strong-field double ionization of argon dimers

    SciTech Connect

    Manschwetus, B.; Rottke, H.; Steinmeyer, G.; Sandner, W.; Foucar, L.; Czasch, A.; Schmidt-Boecking, H.

    2010-07-15

    We investigate double ionization of argon dimers in high-intensity ultrashort Ti:sapphire laser pulses. We are able to identify several strong-field excitation pathways of the dimer that terminate in atomic ion pairs from a Coulomb explosion. The explosion starts from two-site double-ionized dimers and from one-site double-ionized ones after radiative charge transfer at small internuclear separation. One-site double ionization is accomplished by laser-induced charge transfer in the high-intensity laser pulse following two-site double ionization. The highest energy ion pairs we observed can be attributed to ''frustrated triple ionization'' of the argon dimer.

  6. 78 FR 73518 - Notice Inviting Suggestions for New Experiments for the Experimental Sites Initiative; Federal...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-06

    ... and academic success, result in shorter time to degree, and reduce student loan indebtedness. Based on... reduce reliance on student loans. Subject to the statutory restrictions and limitations of the Secretary... Notice Inviting Suggestions for New Experiments for the Experimental Sites Initiative; Federal...

  7. 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.

  8. Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair.

    PubMed Central

    Gaillard, P H; Moggs, J G; Roche, D M; Quivy, J P; Becker, P B; Wood, R D; Almouzni, G

    1997-01-01

    To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell-free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed-circular DNA containing a uniquely placed cisplatin-DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited. PMID:9321407

  9. The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

    PubMed Central

    Purzycka, Katarzyna J.; Pachulska-Wieczorek, Katarzyna; Adamiak, Ryszard W.

    2011-01-01

    RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1–560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization. PMID:21622659

  10. Analysis of transcript initiation sites in the mitochondrial genome of Zea mays

    SciTech Connect

    McCarty, D.M.

    1987-01-01

    Heterologous probes from the bovine mitochondrial genome were used to identify and clone the genes encoding cytochrome oxidase subunits 1 and 3 (COX-I and III) and the 26S and 18S rRNA genes from maize mitochondria. The nucleotide sequence of the maize COX-III gene and upstream sequences were determined. The clones were used to locate the transcript initiation sites for these genes by S1 nuclease and primer extension mapping. Sequences associated with the 5' ends of these transcripts were analyzed and found to fall into 3 distinct motifs. The sequence AGAAA occurred upstream of all of the promoter sites examined. Two distinct sequence elements, CGT(A)/sub n/TC or CCCT-TNNTT, could be found downstream of the AGAAA and immediately preceded the initiating nucleotide. In vitro capping with guanylyltransferase demonstrated that these sequences represented true initiation sites and not post-transcriptional processing sites. Preliminary to the development of a homologous in vitro transcription system for maize mitochondria, an RNA polymerase activity capable of incorporating /sup 32/P UTP into acid precipitable material given a non-specific DNA template was isolated from maize mitochondria by heparin-sepharose chromatography.

  11. Alternative translation initiation site in the DA strain of Theiler's murine encephalomyelitis virus.

    PubMed Central

    Kong, W P; Roos, R P

    1991-01-01

    Polyprotein processing studies of Theiler's murine encephalomyelitis virus (TMEV), a group of mouse picornaviruses, demonstrated synthesis of a protein we have called l during in vitro translations from the RNA of DA, a demyelinating strain of TMEV, but not GDVII, an acute neurovirulent strain. We have proposed that l is synthesized from an alternative initiation site in the DA leader (L) coding area out of phase with the polyprotein reading frame (R. P. Roos, W.-P. Kong, B. L. Semler, J. Virol. 63:5344-5353, 1989). We now provide support for this proposal from experiments involving in vitro translation of three separate mutations of an infectious DA cDNA clone: DA"l"-1, which contains a base mismatch at the putative initiation codon of l, DAL-1, which contains a base mismatch at the presumed authentic initiation site of L at the beginning of the polyprotein; and DAL:NheI, which contains nucleotides coding for a four-amino-acid insertion in the L coding area with a termination codon in the l reading frame. Our results demonstrate that the DA strain uses an alternative initiation site and reading frame to in vitro synthesize l. l may have a role in the biological activity of the virus. Images PMID:2033677

  12. 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

  13. 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.

  14. 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. PMID:27422118

  15. 20,000 sites removed from Superfund tracking under brownfield initiative

    SciTech Connect

    1995-05-01

    Admitting that {open_quotes}Superfund has unintentionally become an obstacle to redevelopment,{close_quotes} EPA Administrator Carol M. Browner announced the implementation of Phase 2 of the Superfund administrative improvements initiative on March 29, 1995. This phase of improvements focuses on redeveloping abandoned, potentially contaminated urban sites-so called {open_quotes}brownfields.{close_quotes} Under the brownfield redevelopment program. EPA has committed to the following: (1) Removing 20,000 sites from the Comprehensive Environmental Response. Compensation, and Liability Information System (CERCLIS), the Superfund tracking system list; (2) Clarifying issues of liability for these sites; and (3) Awarding up to $10 million in funds over the next two years to 50 brownfield redevelopment pilot projects. 2 refs.

  16. Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

    PubMed Central

    Jung, Joo In; Premraj, Sasha; Cruz, Pedro E.; Ladd, Thomas B.; Kwak, Yewon; Koo, Edward H.; Felsenstein, Kevin M.; Golde, Todd E.; Ran, Yong

    2014-01-01

    Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity. PMID:25350374

  17. 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.

  18. 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

    2015-01-01

    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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  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. The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

    PubMed

    Pedersen, L B; Birkelund, S; Holm, A; Ostergaard, S; Christiansen, G

    1996-02-01

    The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1. PMID:8576073

  9. 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

  10. 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

  11. 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

  12. Dimerization of lipocalin allergens

    PubMed Central

    Niemi, Merja H.; Rytkönen-Nissinen, Marja; Miettinen, Ilja; Jänis, Janne; Virtanen, Tuomas; Rouvinen, Juha

    2015-01-01

    Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution. PMID:26346541

  13. 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.

  14. 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

  15. 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. PMID:27635237

  16. Adsorption behaviors of monomer and dimer of formic acid on Pt(111) in the absence and presence of water.

    PubMed

    Qi, Yuanyuan; Zhu, Rongxiu; Zhang, Dongju

    2014-06-01

    By performing density functional theory (DFT) theory calculations, we studied the adsorption behaviors of the monomer and dimer of formic acid (HCOOH, FA) on the Pt(111) surface with and without the presence of water molecules. The monomer prefers to stand on the surface of Pt(111), and in the most stable adsorption configuration the carbonyl O of HCOOH binds to the atop site of a Pt atom and the hydroxyl H points asymmetrically to two neighboring Pt atoms. The dimer of HCOOH not only exists in the gas-phase but also on Pt(111) surface, and the eight-membered ring dimer is identified as the energetically most favorable dimeric structure of HCOOH both in gas-phase and on Pt(111) surface. With the presence of water molecules, both the monomer and dimer of HCOOH prefer to lie parallel to the surface so as to maximize the number of H-bonds to adjacent water molecules. These results indicate that water molecules significantly influence the initial adsorption manner of HCOOH and further its decomposition reactivity on Pt(111) surface. The present work shows the adsorption behavior of HCOOH dimer on Pt(111) for the first time and also gives several new adsorption configurations of the monomer that are not reported in literature. The theoretical results are expected to provide a valuable input to understand the reactivity of HCOOH on Pt(111). PMID:24827612

  17. The lung in ACPA-positive rheumatoid arthritis: an initiating site of injury?

    PubMed

    Perry, Elizabeth; Kelly, Clive; Eggleton, Paul; De Soyza, Anthony; Hutchinson, David

    2014-11-01

    Recent findings have highlighted the potential initiation of ACPA in sites away from the joint. Periodontitis is an example of this concept. This process in the gums appears to be independent of smoking, the main environmental risk factor for ACPA-positive RA. There is extensive literature regarding the potential role of smoking in the pathogenesis of ACPA-positive RA. As a consequence of this strong association, the lung has become the focus of research to determine whether processes within the lung are linked to the generation of ACPA. Here we outline the current body of evidence and explore the hypothesis that the lung as an organ of immune defence has a role in the pathogenesis of the autoimmune disease ACPA-positive RA.

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

    NASA Astrophysics Data System (ADS)

    Bodi, Andras

    2013-10-01

    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.

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

    PubMed

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

    2016-09-01

    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. PMID:27316311

  20. 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

  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 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.

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

    DOE PAGES

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

    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

  4. 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

  5. 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.

  6. Superbackscattering nanoparticle dimers

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

    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.

  7. 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

  8. Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA.

    PubMed Central

    Teruya, J H; Kutsunai, S Y; Spear, D H; Edwards, P A; Clarke, C F

    1990-01-01

    A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts. Images PMID:2325654

  9. 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.

  10. 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.

  11. A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication*

    PubMed Central

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

    2014-01-01

    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. PMID:25368321

  12. Initial Experience with Retroperitoneal Laparoendoscopic Single-Site Surgery for Upper Urinary Tract Surgery

    PubMed Central

    Pak, Chul-Ho; Baik, Seung

    2011-01-01

    Purpose To report our initial clinical experience and perioperative outcomes of retroperitoneal laparoendoscopic single-site surgery (RLESS) for upper urinary tract surgery. Materials and Methods Between June 2009 and October 2010, we performed RLESS in 23 patients for various indications including radical nephrectomy (n=4), nephroureterectomy (n=2), simple nephrectomy (n=10), and renal cyst ablation (n=7). RLESS was performed with a homemade single-port device with a conventional rigid laparoscopic instrument and laparoscope. The parameters analyzed were age, body mass index, operative time, estimated blood loss, transfusion, time of oral intake, visual analogue pain scale score (VAPS), length of hospital stay, and complications. Results One case of simple nephrectomy was converted to open nephrectomy because of severe adhesion and inadequate surgical exposure. RLESS was completed in 23 patients. Mean operative time was 168.7±29.2, 227.5±50.0, 230.0±56.5, and 70.5±8.9 minutes for simple nephrectomy, radical nephrectomy, nephroureterectomy, and renal cyst ablation, respectively. Estimated blood loss was 113.0±149.8, 170.0±156.8, 400.0±141.4, and 22.8±16.0 ml. The time to oral intake after surgery was 1.4±0.5, 1.2±0.5, 1.5±0.7, and 1.1±0.3 days. The mean VAPS score was 1.1±0.2, 2.1±0.5, 2.0±0.5, and 1.0±0.0 of 10 (range, 0.8 to 2.6). The hospital stay was 4.6±1.5, 3.7±0.5, 6.0±1.4, and 3.2±1.7 days. No major perioperative complications were observed. Conclusions The initial outcomes of our experience suggest that RLESS is a technically feasible and safe procedure for upper urinary tract surgery. Prospective comparative studies with conventional retroperitoneal laparoscopic surgery are needed to confirm the potential benefits of RLESS. PMID:22216397

  13. 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

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

    PubMed

    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.

  15. 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

  16. 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

  17. 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.

  18. Spectroscopy of dimers.

    NASA Technical Reports Server (NTRS)

    Levine, H. B.

    1972-01-01

    The method of Stogryn and Hirschfelder, which divides two-particle phase space into bound, metastable, and free particle parts, is applied to the statistical averages which determine the first three spectroscopic moments of 'collision-induced' far-infrared and Raman spectra. Numerical results are presented for a number of models of noble gas systems. It is shown that for some systems, but not all, dimers make important, although not necessarily well-separated, contributions to the spectrum.

  19. Metalloporphines: Dimers and Trimers.

    PubMed

    Jentzen, Walter; Shelnutt, John A; Scheidt, W Robert

    2016-06-20

    Procedures for the purification and subsequent crystallization of the slightly soluble four-coordinate metallporphines, the simplest possible porphyrin derivatives, are described. Crystals of the porphine derivatives of cobalt(II), copper(II), platinum(II), and two polymorphs of zinc(II) were obtained. Analysis of the crystal and molecular structures shows that all except the platinum(II) derivative form an unusual trimeric species in the solid state. The isomorphous cobalt(II), copper(II), and one zinc(II) polymorph pack in the unit cell to form dimers as well as the trimers. Interplanar spacings between porphine rings are similar in both the dimers and trimers and range between 3.24 and 3.37 Å. Porphine rings are strongly overlapped with lateral shifts between ring centers in both the dimers and trimers with values between 1.52 and 1.70 Å or in Category S as originally defined by Scheidt and Lee. Periodic trends in the M-Np bond distances parallel those observed previously for tetraphenyl- and octaethylporphyrin derivatives. PMID:27276239

  20. A helix-to-coil transition at the ε-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis

    PubMed Central

    Sato, Takeshi; Tang, Tzu-chun; Reubins, Gabriella; Fei, Jeffrey Z.; Fujimoto, Taiki; Kienlen-Campard, Pascal; Constantinescu, Stefan N.; Octave, Jean-Noel; Aimoto, Saburo; Smith, Steven O.

    2009-01-01

    Processing of amyloid precursor protein (APP) by γ-secretase is the last step in the formation of the Aβ peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618–660), we show that the TM domain forms an α-helical homodimer mediated by consecutive GxxxG motifs. We find that the APP TM helix is disrupted at the intracellular membrane boundary near the ε-cleavage site. This helix-to-coil transition is required for γ-secretase processing; mutations that extend the TM α-helix inhibit ε cleavage, leading to a low production of Aβ peptides and an accumulation of the α- and β-C-terminal fragments. Our data support a progressive cleavage mechanism for APP proteolysis that depends on the helix-to-coil transition at the TM-JM boundary and unraveling of the TM α-helix. PMID:19164538

  1. 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...

  2. 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...

  3. 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...

  4. Study of New Youth Initiatives in Apprenticeship. Interim Report. Volume 2: Site Visit Reports.

    ERIC Educational Resources Information Center

    CSR, Inc., Washington, DC.

    This second volume of the interim report provides detailed case study reports on each of the eight Youth Apprenticeship Projects. (Volume 1, an overview of data from the site visits, is available separately as CE 032 791.) Discussion areas covered in each site visit report are local context/operational environment, administrative information,…

  5. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 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....145, and 52.171. (b) While a partial decision on site suitability is in effect under 10 CFR...

  6. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 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....145, and 52.171. (b) While a partial decision on site suitability is in effect under 10 CFR...

  7. 10 CFR 52.83 - Finality of referenced NRC approvals; partial initial decision on site suitability.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 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....145, and 52.171. (b) While a partial decision on site suitability is in effect under 10 CFR...

  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

    ... 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....145, and 52.171. (b) While a partial decision on site suitability is in effect under 10 CFR...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 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....145, and 52.171. (b) While a partial decision on site suitability is in effect under 10 CFR...

  10. Why are we where we are? Understanding replication origins and initiation sites in eukaryotes using ChIP-approaches.

    PubMed

    Schepers, Aloys; Papior, Peer

    2010-01-01

    DNA replication initiates from origins of replication following a strict sequential activation programme and a conserved temporal order of activation. The number of replication initiation sites varies between species, according to the complexity of the genomes, with an average spacing of 100,000 bp. In contrast to yeast genomes, the location and definition of origins in mammalian genomes has been elusive. Historically, mammalian replication initiation sites have been mapped in situ by systematically searching specific genomic loci for sites that preferentially initiated DNA replication, potential origins by start-site mapping and autonomously replicating sequence experiments, and potential ORC and pre-replicative complex (pre-RC) sites by chromatin immunoprecipitation (ChIP) using antibodies for pre-RC proteins. In the past decade, ChIP has become an important method for analyzing protein/DNA interactions. Classically, ChIP is combined with Southern blotting or PCR. Recently, whole genome-ChIP methods have been very successful in unicellular eukaryotes to understand molecular mechanisms coordinating replication initiation and its flexibility in response to environmental changes. However, in mammalian systems, ChIP with pre-RC antibodies has often been challenging and genome-wide studies are scarce. In this review, we will appraise the progress that has been made in understanding replication origin organization using immunoprecipitation of the ORC and Mcm2-7 complexes. A special focus will be on the advantages and disadvantages of genome-wide ChIP-technologies and their potential impact on understanding metazoan replicators. PMID:19904620

  11. 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

  12. 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

    2014-01-01

    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.

  13. A novel antibody light chain dimer: Implications for T-cell receptor structure

    SciTech Connect

    Schiffer, M.; Chang, Chong-Hwan; Solomon, A.; Stevens, F.J.

    1989-01-01

    The dimeric structures of antibody light chains produced in patients with multiple myeloma (Bence Jones proteins) have for some time been studied chemically and crystallographically as models of the antigen binding fragment (Fab) of an antibody. The conformational concordance of Fabs and a Bence Jones dimer was demonstrated by the initial immunoglobulin crystallographic structures. We have recently described the structure of a second intact light chain, the lambda-type protein Loc. The Loc protein exhibits an unanticipated protruding arrangement of its complementarity-determining residues. Grooves on each side of the protrusion may function as separate binding sites. In this report, we examine the Loc structure and its intracrystalline interactions in more detail and consider aspects of this structure that may possess implications for models of a nonantibody constituent of the immunoglobulin superfamily, the T-cell antigen receptor. 26 refs., 3 figs., 1 tab.

  14. 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.

  15. 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.

  16. 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 ...

  17. 40 CFR 280.62 - Initial abatement measures and site check.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... WASTES (CONTINUED) TECHNICAL STANDARDS AND CORRECTIVE ACTION REQUIREMENTS FOR OWNERS AND OPERATORS OF UNDERGROUND STORAGE TANKS (UST) Release Response and Corrective Action for UST Systems Containing Petroleum or... release confirmation, site investigation, abatement, or corrective action activities. If these...

  18. Initial characterization and autoradiographic localization of a novel sigma/opioid binding site in immune tissues.

    PubMed

    Whitlock, B B; Liu, Y; Chang, S; Saini, P; Ha, B K; Barrett, T W; Wolfe, S A

    1996-07-01

    High concentrations of novel, haloperidol- and DTG-inaccessible (+)-[3H]-3-PPP binding sites were found in human peripheral blood leukocytes rat spleen and splenocytes, but not in rat brain. Splenic sites were localized in a course punctate pattern in the marginal zones and red pulp. The pharmacology of the splenic sites was: (-)-SKF 10,047 > or = naltrexone = (-)-pentazocine > (+)-pentazocine = (-)-3-PPP = (+)-SKF 10,047 > or = (+)-3-PPP > or = dextrorphan > dextromethorphan > PCP > clorgyline. DTG, haloperidol, TCP, (-)-deprenyl and SKF 525-A did not complete. Binding activity was destroyed by heating and phospholipase C, but not by proteases or glycosidases. These sites may be involved in immunomodulation by opiate and sigma receptor agonists.

  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. Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

    PubMed Central

    Coey, Aaron; Larsen, Kevin; Puglisi, Joseph D.; Viani Puglisi, Elisabetta

    2016-01-01

    Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3. Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription. PMID:27613581

  1. 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

  2. Facility preparations for the initial International Atomic Energy Agency Inpsection of Hanford Site excess material

    SciTech Connect

    Johnson, W.C.; Scott, D.D.; Bartlett, W.D.; Delegard, C.H.; McRae, L.P.; Six, D.E.; Amacker, O.P.

    1995-09-01

    In September 1993 President Clinton offered to place excess US nuclear materials under IAEA safeguards. In January 1994, the Hanford Site was identified as the second site in the US to be prepared for placement on the eligibility list for LAEA safeguards selection. Planning and preparation started at Hanford in February 1994. The PFP mission is to provide safe storage of Category 1 and 2 special nuclear material (SNM) and laboratory support to the Hanford Site. The mission includes the stabilizing and packaging of SNM for temporary storage sufficient to support the deactivation and cleanup function of the facility. The storage of Category 1 and 2 SNM at this facility indirectly supports national security interests, and safe storage is accomplished in a manner that ensures the health and safety of the public and employees are not compromised. The PFP is located in the approximate center of the Hanford Site inside the 200 West Area. The PFP is within a designated protected area (PA) and is located approximately 10.5 km from the Columbia River and 34 km northwest of the Richland city limits. The, Hanford Site is located in Southeastern Washington and has been associated with plutonium production since the mid 1940s. Excess plutonium oxide has been placed under IAEA safeguards in a phased approach at the PFP`s Plutonium Storage Vault. This paper is an overview and summary of the many tasks required to meet IAEA safeguards requirements.

  3. Cellular or viral protein binding to a cytomegalovirus promoter transcription initiation site: effects on transcription.

    PubMed Central

    Macias, M P; Huang, L; Lashmit, P E; Stinski, M F

    1996-01-01

    We have previously shown that the IE2 protein of human cytomegalovirus (CMV) represses its own synthesis by binding to the major immediate-early promoter (M. P. Macias and M. F. Stinski, Proc. Natl. Acad. Sci. USA 90:707-711, 1993). The binding of a viral protein (IE2) and a cellular protein in the region of the transcription start site was investigated by site-specific mutational analysis and electrophoretic mobility shift assay. The viral protein and the cellular protein require different but adjacent core DNA sequence elements for binding. In situ chemical footprinting analysis of DNA-protein interactions with purified CMV IE2 protein or HeLa cell nuclear extracts demonstrated binding sites that overlap the transcription start site. The IE2 protein footprint was between bp -15 and +2, relative to the transcription start site, and the cellular protein was between bp -16 and +7. The ability of the unknown human cellular protein of approximately 150 kDa to bind the CMV major immediate-early promoter correlates with an increase in the level of transcription efficiency. Mutations in the core DNA sequence element for cellular protein binding significantly reduced the level of in vitro transcription efficiency. Mutations upstream and downstream of the core sequence moderately reduced the transcription efficiency level. Negative autoregulation of the CMV promoter by the viral IE2 protein may involve both binding to the DNA template and interference with the function of a cellular protein that binds to the transcription start site and enhances transcription efficiency. PMID:8648697

  4. 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

  5. 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.

  6. Implementation of Brownfield Initiative transforming a former steel manufacturing site into an industrial park

    SciTech Connect

    Salguero, J.P.; Quinlisk, J.M.

    1996-12-31

    The concept of Brownfield Redevelopment, or Land Recycling, alleviates the pressure to develop new sites while leaving former industrial sites dormant. This case history discusses the environmental methodology used to transform a former steel manufacturing facility into an industrial park. The investigation and remediation work was performed while the Pennsylvania`s Land Recycling and Environmental Remediation Standards Act (Act 2) was being developed. The Beaver County Corporation for Economic development (BCCED) purchased a former LTV manufacturing facility in Aliquippa, PA. Down gradient of the sites are the intakes of drinking water wells that supply the city of Akiquippa. Phase 1, 2 and 3 Environmental Site Assessments (ESAs) identified polychlorinated biphenyls (PCBs), petroleum hydrocarbons (TPHs), asbestos and heavy metal contamination. The groundwater assessment revealed traces of chlorinated compounds. An accelerated remedial investigation and focused feasibility study (RI/FFS) determined environmental and economic risks. Innovative and statistical techniques were used in the preparation of investigation workplans. Multiple media were samples. Complementary investigation phases were performed affording time for the regulatory agencies to interface with the engineer and owner. The use of a combined remedial actions derived from the proposed end-use proved to be most cost effective.

  7. 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.…

  8. Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex*

    PubMed Central

    Douglas, Max E.

    2016-01-01

    Mcm10 is required for the initiation of eukaryotic DNA replication and contributes in some unknown way to the activation of the Cdc45-MCM-GINS (CMG) helicase. How Mcm10 is localized to sites of replication initiation is unclear, as current models indicate that direct binding to minichromosome maintenance (MCM) plays a role, but the details and functional importance of this interaction have not been determined. Here, we show that purified Mcm10 can bind both DNA-bound double hexamers and soluble single hexamers of MCM. The binding of Mcm10 to MCM requires the Mcm10 C terminus. Moreover, the binding site for Mcm10 on MCM includes the Mcm2 and Mcm6 subunits and overlaps that for the loading factor Cdt1. Whether Mcm10 recruitment to replication origins depends on CMG helicase assembly has been unclear. We show that Mcm10 recruitment occurs via two modes: low affinity recruitment in the absence of CMG assembly (“G1-like”) and high affinity recruitment when CMG assembly takes place (“S-phase-like”). Mcm10 that cannot bind directly to MCM is defective in both modes of recruitment and is unable to support DNA replication. These findings indicate that Mcm10 is localized to replication initiation sites by directly binding MCM through the Mcm10 C terminus. PMID:26719337

  9. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27381287

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

    PubMed

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

    2016-01-01

    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. PMID:27381287

  12. Structural and biochemical studies on procaspase-8: new insights on initiator caspase activation.

    PubMed

    Keller, Nadine; Mares, Jirí; Zerbe, Oliver; Grütter, Markus G

    2009-03-11

    Caspases are proteases with an active-site cysteine and aspartate specificity in their substrates. They are involved in apoptotic cell death and inflammation, and dysfunction of these enzymes is directly linked to a variety of diseases. Caspase-8 initiates an apoptotic pathway triggered by external stimuli. It was previously characterized in its active inhibitor bound state by crystallography. Here we present the solution structure of the monomeric unprocessed catalytic domain of the caspase-8 zymogen, procaspase-8, showing for the first time the position of the linker and flexibility of the active site forming loops. Biophysical studies of carefully designed mutants allowed disentangling dimerization and processing, and we could demonstrate lack of activity of monomeric uncleaved procaspase-8 and of a processed but dimerization-incompetent mutant. The data provide experimental support in so-far unprecedented detail, and reveal why caspase-8 (and most likely other initiator caspases) needs the dimerization platform during activation.

  13. 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.

  14. Krakow clean fossil fuels and energy efficiency program. Final report: Initial site investigation

    SciTech Connect

    1995-11-15

    Original objective of this project was to retrofit the Balice Boilerhouse with a TCS Coal Micronization system and Amerex baghouses to achieve higher combustion efficiencies and lower air emission, including SO{sub 2}, NO{sub x}, CO and particulate matter. The Balice Boilerhouse is located adjacent to the Krakow Airport and provides heating steam for the Polish Military Unit No. 1616. In May 1995 the Polish Military announced it had decided to convert its boiler house to gas; thus cancelling the TCS Project. The balance of 1995 was spent considering alternative Project sites in Krakow for the application of the TCS coal Micronization technology.

  15. Development of soil chemical and biological properties in the initial stages of post-mining deposition sites.

    PubMed

    Monokrousos, Nikolaos; Boutsis, George; Diamantopoulos, John D

    2014-12-01

    The aim of this study was to assess the seasonal development of the physicochemical (pH, organic C, organic N, extractable P, Ca(2+), Mg(2+)) and biological soil properties (microbial biomass, activities of urease, dehydrogenase and alkaline phosphatase) of the topsoil of mine deposition sites that differed based on the material used exclusively for their creation: (a) marlstones, (b) red-grey formations (RGF), and (c) fly ash (FA), during the first year after their creation. Our hypothesis was that all deposition sites, regardless the material they consist of, present equal opportunities for the establishment of spontaneous vegetation. All macronutrients concentrations (P, Ca(2+), and Mg(2+)) remained constant with time and were found to be higher in the FA sites. Organic C, organic N, all enzyme activities, and microbial biomass were higher in the RGF and marl depositions, with marl sites presenting the highest values. All values of biological variables, with the exception of alkaline phosphatase, increased with time. The alkaline environment along with the slow improvement in soil biological properties of the FA sites seemed to present the most unfavorable conditions for spontaneous vegetation growth. On the contrary, the other two spoil materials presented significant improvement in the initial stages of soil formation in terms of soil functionality. PMID:25249044

  16. Density functional theory study of Fe, Co, and Ni adatoms and dimers adsorbed on graphene

    NASA Astrophysics Data System (ADS)

    Johll, Harman; Kang, Hway Chuan; Tok, Eng Soon

    2009-06-01

    Metal clusters have been investigated rather intensely for both fundamental and technological reasons. In this work we report the results of plane-wave density functional theory calculations of Fe, Co, and Ni adatoms and dimers adsorbed on graphene. We study both homonuclear and heteronuclear dimers, and the latter includes mixed dimers of Fe, Co, and Ni along with dimers of these elements with Pt. Our work is motivated by the fundamental interest in their configurational and magnetic properties. We calculated the adsorption site, the structure and relative stabilities of various adsorption configurations, the band structures, the atomic projected electronic density of states, and the magnetic moments of the adatoms and dimers. Contrary to previous work, our results show that adatoms bind weakly to graphene with binding energies ranging from 0.2 to 1.4 eV depending on the adsorption site and species. For both homonuclear and heteronuclear dimers the binding energies per atom are lower than the respective adatom cases, ranging from 0.1 to 0.5 eV per metal atom. The most strongly bound configurations for all the dimers studied are those with the dimer axis (nearly) perpendicular to the graphene plane and bound at the hole site. These configurations, which, to our knowledge, have not been considered in previous work, also turn out to have the largest enhancement of the magnetic moment at least for the atom farther from the graphene. The binding energies of these most strongly bound dimers are dependent on three factors, namely, the interconfigurational energy change in the dimer atom farther from graphene upon desorption, the charge transfer from the dimer to the graphene, and the adsorption site favored by the atom closer to the graphene sheet. The first factor is dominant for all the dimers studied here except for CoPt and NiPt. The relatively high electronegativity of Pt affects the character of the charge transfer from the dimer to graphene. In most of the dimers

  17. 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

  18. 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.

  19. 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.

  20. Versatile SPR aptasensor for detection of lysozyme dimer in oligomeric and aggregated mixtures.

    PubMed

    Vasilescu, Alina; Purcarea, Cristina; Popa, Elena; Zamfir, Medana; Mihai, Iuliana; Litescu, Simona; David, Sorin; Gaspar, Szilveszter; Gheorghiu, Mihaela; Jean-Louis Marty

    2016-09-15

    A Surface Plasmon Resonance (SPR) sensor for the quantitation of lysozyme dimer in monomer-dimer mixtures, reaching a detection limit of 1.4nM dimer, has been developed. The sensor is based on an aptamer which, although developed for the monomeric form, binds also the dimeric form but with a strikingly different kinetics. The aptasensor was calibrated using a dimer obtained by cross-linking. Sensorgrams acquired with the aptasensor in monomer-dimer mixtures were analysed using Principal Components Analysis and Multiple Regression to establish correlations with the dimer content in the mixtures. The method allows the detection of 0.1-1% dimer in monomer solutions without any separation. As an application, the aptasensor was used to qualitatively observe the initial stages of aggregation of lysozyme solutions at 60°C and pH 2, through the variations in lysozyme dimer amounts. Several other methods were used to characterize the lysozyme dimer obtained by cross-linking and confirm the SPR results. This work highlights the versatility of the aptasensor, which can be used, by simply tuning the experimental conditions, for the sensitive detection of either the monomer or the dimer and for the observation of the aggregation process of lysozyme.

  1. Tomato root: site of initial alteration in minor elements in cadmium treated plants

    SciTech Connect

    Clark, B.; Harkov, R.; Brennan, E.

    1982-01-01

    Tomato plants treated with 0.25 and 0.75 ppm Cd for one month exhibited no visual symptoms of toxicity, nor was the percent dry weight of leaf, stem, or root tissue altered. Cadmium concentrations in treated plants generally followed the trend: roots > leaves > stems. At such concentrations too low to cause phytotoxicity, cadmium altered the minor element concentration in tomato plants. The first changes occurred in the Mn and Fe concentrations of the roots. The reduction of these elements in the root may signal an early event in the mechanisms of Cd toxicity. To test the hypothesis that the root represents the primary site of injury from Cd, a kinetic study should be conducted in which root and shoot tissues are analyzed for Cd and the minor elements as the plants progress from an asymptomatic condition to one of severe Cd toxicity. (JMT)

  2. Lipid-directed vinculin dimerization.

    PubMed

    Chinthalapudi, Krishna; Patil, Dipak N; Rangarajan, Erumbi S; Rader, Christoph; Izard, Tina

    2015-05-01

    Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in cancer phenotypes, cardiovascular disease, and embryonic lethality. At the plasma cell membrane, the most abundant phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), binds the vinculin tail domain, Vt, and triggers homotypic and heterotypic interactions that amplify binding of vinculin to the actin network. Binding of PIP2 to Vt is necessary for maintaining optimal focal adhesions, for organizing stress fibers, for cell migration and spreading, and for the control of vinculin dynamics and turnover of focal adhesions. While the recently determined Vt/PIP2 crystal structure revealed the conformational changes occurring upon lipid binding and oligomerization, characterization of PIP2-induced vinculin oligomerization has been challenging in the adhesion biology field. Here, via a series of novel biochemical assays not performed in previous studies that relied on chemical cross-linking, we characterize the PIP2-induced vinculin oligomerization. Our results show that Vt/PIP2 forms a tight dimer with Vt or with the muscle-specific vinculin isoform, metavinculin, at sites of adhesion at the cell membrane. Insight into how PIP2 regulates clustering and into mechanisms that regulate cell adhesion allows the development for a more definite sensor for PIP2, and our developed techniques can be applied generally and thus open the door for the characterization of many other protein/PIP2 complexes under physiological conditions. PMID:25880222

  3. Lipid-directed vinculin dimerization.

    PubMed

    Chinthalapudi, Krishna; Patil, Dipak N; Rangarajan, Erumbi S; Rader, Christoph; Izard, Tina

    2015-05-01

    Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in cancer phenotypes, cardiovascular disease, and embryonic lethality. At the plasma cell membrane, the most abundant phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), binds the vinculin tail domain, Vt, and triggers homotypic and heterotypic interactions that amplify binding of vinculin to the actin network. Binding of PIP2 to Vt is necessary for maintaining optimal focal adhesions, for organizing stress fibers, for cell migration and spreading, and for the control of vinculin dynamics and turnover of focal adhesions. While the recently determined Vt/PIP2 crystal structure revealed the conformational changes occurring upon lipid binding and oligomerization, characterization of PIP2-induced vinculin oligomerization has been challenging in the adhesion biology field. Here, via a series of novel biochemical assays not performed in previous studies that relied on chemical cross-linking, we characterize the PIP2-induced vinculin oligomerization. Our results show that Vt/PIP2 forms a tight dimer with Vt or with the muscle-specific vinculin isoform, metavinculin, at sites of adhesion at the cell membrane. Insight into how PIP2 regulates clustering and into mechanisms that regulate cell adhesion allows the development for a more definite sensor for PIP2, and our developed techniques can be applied generally and thus open the door for the characterization of many other protein/PIP2 complexes under physiological conditions.

  4. Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome.

    PubMed Central

    Shieh, C K; Lee, H J; Yokomori, K; La Monica, N; Makino, S; Lai, M M

    1989-01-01

    We have previously shown that some strains of the murine coronavirus mouse hepatitis virus (MHV) synthesize an additional mRNA species (mRNA 2b, previously called mRNA 2a) with a size intermediate between that of mRNAs 2 and 3, suggesting the presence of an optional transcriptional initiation site. This transcriptional start is dependent on the leader sequence of the virus strains. To study the mechanism of coronavirus transcriptional regulation, we have cloned and sequenced the region of the viral genome corresponding to the 5' unique coding region of mRNA 2 of the JHM strain of MHV. In addition to the open reading frame (ORF) predicted to encode the viral nonstructural protein p30, a second complete ORF, with the potential to encode a 439-amino-acid polypeptide, was discovered. The transcriptional initiation sites of both mRNA 2a (formerly called mRNA 2) and mRNA 2b were determined by primer extension studies and RNA sequencing. The data indicated that transcription of mRNA 2a initiated at a site, UCUAUAC, that resembled the consensus intergenic sequence. In contrast, the start signal of the optional mRNA 2b, UAAUAAAC, deviated from the consensus sequence. mRNA 2b is a functional mRNA, as shown by in vitro translation studies of mRNA and ORF 2b and by the detection of an additional viral structural protein, gp65, in the JHM strain that synthesized this mRNA. Although the A59 strain of MHV was found to retain ORF 2b, it lacked the correct transcriptional and translational start signals for this gene. This study has therefore identified an optional gene product for murine coronaviruses and provided insights into the mechanism of regulation of MHV RNA transcription. Images PMID:2547994

  5. Impacts of alien invasive plants on soil nutrients are correlated with initial site conditions in NW Europe.

    PubMed

    Dassonville, Nicolas; Vanderhoeven, Sonia; Vanparys, Valérie; Hayez, Mathieu; Gruber, Wolf; Meerts, Pierre

    2008-08-01

    Alien invasive plants are capable of modifying ecosystem function. However, it is difficult to make generalisations because impacts often appear to be species- and site-specific. In this study, we examined the impacts of seven highly invasive plant species in NW Europe (Fallopia japonica, Heracleum mantegazzianum, Impatiens glandulifera, Prunus serotina, Rosa rugosa, Senecio inaequidens, Solidago gigantea) on nutrient pools in the topsoil and the standing biomass. We tested if the impacts follow predictable patterns, across species and sites or, alternatively, if they are entirely idiosyncratic. To that end, we compared invaded and adjacent uninvaded plots in a total of 36 sites with widely divergent soil chemistry and vegetation composition. For all species, invaded plots had increased aboveground biomass and nutrient stocks in standing biomass compared to uninvaded vegetation. This suggests that enhanced nutrient uptake may be a key trait of highly invasive plant species. The magnitude and direction of the impact on topsoil chemical properties were strongly site-specific. A striking finding is that the direction of change in soil properties followed a predictable pattern. Thus, strong positive impacts (higher topsoil nutrient concentrations in invaded plots compared to uninvaded ones) were most often found in sites with initially low nutrient concentrations in the topsoil, while negative impacts were generally found under the opposite conditions. This pattern was significant for potassium, magnesium, phosphorus, manganese and nitrogen. The particular site-specific pattern in the impacts that we observed provides the first evidence that alien invasive species may contribute to a homogenisation of soil conditions in invaded landscapes. PMID:18491146

  6. 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.

  7. 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.

  8. 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.

  9. Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth.

    PubMed

    Bloch, Daria; Pleskot, Roman; Pejchar, Přemysl; Potocký, Martin; Trpkošová, Pavlína; Cwiklik, Lukasz; Vukašinović, Nemanja; Sternberg, Hasana; Yalovsky, Shaul; Žárský, Viktor

    2016-10-01

    Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

  10. 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.

  11. Strength distribution of fatigue crack initiation sites in an Al-Li alloy

    NASA Astrophysics Data System (ADS)

    Zhai, T.

    2006-10-01

    The stress-number of cycles to failure (S-N) curves were measured along the short-transverse (S) and rolling (L) directions of a hot-cross-rolled AA 8090 Al-Li alloy plate (45-mm thick). The alloy was solution heat treated, quenched in water, strained by 6 pct, and peak aged. Fatigue tests were carried out in four-point bend at room temperature, 20 Hz, R=0.1, in air. It was found that the fatigue limits in the S and L directions were 147 and 197 MPa, respectively. The crack population on the surface of a sample at failure increased with the applied stress level and was found to be a Weibull function of the applied maximum stress in this alloy. The strength distribution of fatigue weakest links, where cracks were initiated, was derived from the Weibull function determined by the experimental data. The fatigue weakest-link density was defined as the crack population per unit area at a stress level close to the ultimate tensile stress and can be regarded as a materials property. The density and strength distribution of fatigue weakest links were found to be markedly different between the L and S directions, accounting for the difference in fatigue limit between the directions in this alloy. They were also found to be different between S-L and S-T samples, and between L-T and L-S samples of this alloy, which could not be revealed by the corresponding S-N curves measured. These differences were due to the anisotropy of the microstructures in different directions in this alloy.

  12. Basin constrained κ-dimer method for saddle point finding.

    PubMed

    Xiao, Penghao; Wu, Qiliang; Henkelman, Graeme

    2014-10-28

    Within the harmonic approximation to transition state theory, the rate of escape from a reactant is calculated from local information at saddle points on the boundary of the state. The dimer minimum-mode following method can be used to find such saddle points. But as we show, dimer searches that are initiated from a reactant state of interest can converge to saddles that are not on the boundary of the reactant state. These disconnected saddles are not directly useful for calculating the escape rate. Additionally, the ratio of disconnected saddles can be large, especially when the dimer searches are initiated far from the reactant minimum. The reason that the method finds disconnected saddles is a result of the fact that the dimer method tracks local ridges, defined as the set of points where the force is perpendicular to the negative curvature mode, and not the true ridge, defined as the boundary of the set of points which minimize to the reactant. The local ridges tend to deviate from the true ridge away from saddle points. Furthermore, the local ridge can be discontinuous and have holes which allow the dimer to cross the true ridge and escape the initial state. To solve this problem, we employ an alternative definition of a local ridge based upon the minimum directional curvature of the isopotential hyperplane, κ, which provides additional local information to tune the dimer dynamics. We find that hyperplanes of κ = 0 pass through all saddle points but rarely intersect with the true ridge elsewhere. By restraining the dimer within the κ < 0 region, the probability of converging to disconnected saddles is significantly reduced and the efficiency of finding connected saddles is increased.

  13. 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

  14. 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

  15. Specific dimerization of the light chains of human immunoglobulin.

    PubMed

    Stevenson, G T; Straus, D

    1968-07-01

    1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b). PMID:4174431

  16. 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.

  17. 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.

  18. Plasmon hybridization in nanoparticle dimers

    NASA Astrophysics Data System (ADS)

    Nordlander, Peter; Prodan, Emil; Oubre, Chris

    2004-03-01

    We apply the recently developed plasmon hybridization method (Science 302(2003)419-422) to solid sphere and nanoshell dimers. The results provide a simple and intuitive description of how the energy and excitation cross sections of the dimer plasmons depend on nanoparticle separation, D. We show that the dimer plasmons can be viewed as bonding and antibonding combinations, i.e. hybridization of the individual nanoparticle plasmons. For large D, the shifts of the dipolar dimer plasmons essentially follow the interaction energy between two classical dipoles (1/D^3). As D becomes smaller, the shifts of the dipolar dimer plasmons becomes much stronger and varies much faster with D due to the interaction of the dipolar plasmon of an individual particle with higher energy multipolar plasmons of the other particle. For the heterodimer, the hybridization between the individual nanoparticle plasmons on the different particles result in dimer plasmons whose energies as a function of D exhibit avoided crossings and other interesting effects. The results are compared with FDTD calculations. Work supported by ARO, TATP and the Robert A. Welch Foundation

  19. The VSV Polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation

    PubMed Central

    Barr, J.N.; Tang, Xiaoling; Hinzman, Edward; Shen, Ruizhong; Wertz, Gail W.

    2008-01-01

    Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependant on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependant RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bicistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bicistronic replicons were constructed containing a mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed. PMID:18241907

  20. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

    PubMed Central

    Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

    2016-01-01

    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. PMID:27364008

  1. Recognition of HIV TAR RNA by triazole linked neomycin dimers.

    PubMed

    Kumar, Sunil; Arya, Dev P

    2011-08-15

    A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5'-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (T(m)) of the HIV TAR RNA up to 10°C. Ethidium bromide displacement titrations revealed nanomolar IC(50) between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC(50) in the micromolar range is observed.

  2. 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.

  3. Recognition of HIV TAR RNA by triazole linked neomycin dimers

    PubMed Central

    Kumar, Sunil

    2013-01-01

    A series of neomycin dimers have been synthesized using “click chemistry” with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (Trans Activation Response) RNA region, a 59 base pair stem loop structure located at 5′-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement titrations revealed nanomolar IC50 between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC50 in the micromolar range is observed. PMID:21757341

  4. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    PubMed Central

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    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. PMID:21245530

  5. 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.

  6. Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica.

    PubMed

    Dhar, S K; Choudhury, N R; Mittal, V; Bhattacharya, A; Bhattacharya, S

    1996-05-01

    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

  7. 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.

  8. The Ammonia Dimer Revisited

    NASA Astrophysics Data System (ADS)

    Dawes, Richard; Van Der Avoird, Ad

    2012-06-01

    The conclusion from microwave spectra by Nelson, Fraser, and Klemperer that the ammonia dimer has a nearly cyclic structure led to much debate about the issue of whether (NH_3)_2 is hydrogen bonded. This structure was surprising because most {ab initio} calculations led to a classical, nearly linear, hydrogen-bonded structure. An obvious explanation of the discrepancy between the outcome of these calculations and the microwave data which led Nelson {et al.} to their ``surprising structure'' might be the effect of vibrational averaging: the electronic structure calculations focus on finding the minimum of the intermolecular potential, the experiment gives a vibrationally averaged structure. Isotope substitution studies seemed to indicate, however, that the complex is nearly rigid. Additional data became available from high-resolution molecular beam far-infrared spectroscopy in the Saykally group. These spectra, displaying large tunneling splittings, indicate that the complex is very floppy. The seemingly contradictory experimental data were explained when it became possible to calculate the vibration-rotation-tunneling (VRT) states of the complex on a six-dimensional intermolecular potential surface. The potential used was a simple model potential, with parameters fitted to the far-infrared data. Now, for the first time, a six-dimensional potential was computed by high level {ab initio} methods and this potential will be used in calculations of the VRT states of (NH_3)_2 and (ND_3)_2. So, we will finally be able to answer the question whether the conclusions from the model calculations are indeed a valid explanation of the experimental data. D. Nelson, G. T. Fraser, and W. Klemperer J. Chem. Phys. 83 6201 (1985) J. G. Loeser, C. A. Schmuttenmaer, R. C. Cohen, M. J. Elrod, D. W. Steyert, R. J. Saykally, R. E. Bumgarner, and G. A. Blake J. Chem. Phys. 97 4727 (1992) E. H. T. Olthof, A. van der Avoird, and P. E. S. Wormer J. Chem. Phys. 101 8430 (1994) E. H. T. Olthof

  9. 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

  10. 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.

  11. Mechanically Stabilized Tetrathiafulvalene Radical Dimers

    SciTech Connect

    Coskun, Ali; Spruell, Jason M.; Barin, Gokhan; Fahrenbach, Albert C.; Forgan, Ross S.; Colvin, Michael T.; Carmieli, Raanan; Benitez, Diego; Tkatchouk, Ekaterina; Friedman, Douglas C.; Sarjeant, Amy A.; Wasielewski, Michael R.; Goddard, William A.; Stoddart, J. Fraser

    2011-01-01

    Two donor-acceptor [3]catenanes—composed of a tetracationic molecular square, cyclobis(paraquat-4,4'-biphenylene), as the π-electron deficient ring and either two tetrathiafulvalene (TTF) and 1,5-dioxynaphthalene (DNP) containing macrocycles or two TTF-butadiyne-containing macrocycles as the π-electron rich components—have been investigated in order to study their ability to form TTF radical dimers. It has been proven that the mechanically interlocked nature of the [3]catenanes facilitates the formation of the TTF radical dimers under redox control, allowing an investigation to be performed on these intermolecular interactions in a so-called “molecular flask” under ambient conditions in considerable detail. In addition, it has also been shown that the stability of the TTF radical-cation dimers can be tuned by varying the secondary binding motifs in the [3]catenanes. By replacing the DNP station with a butadiyne group, the distribution of the TTF radical-cation dimer can be changed from 60% to 100%. These findings have been established by several techniques including cyclic voltammetry, spectroelectrochemistry and UV-vis-NIR and EPR spectroscopies, as well as with X-ray diffraction analysis which has provided a range of solid-state crystal structures. The experimental data are also supported by high-level DFT calculations. The results contribute significantly to our fundamental understanding of the interactions within the TTF radical dimers.

  12. Structural characterization of RNA polymerase II complexes arrested by a cyclobutane pyrimidine dimer in the transcribed strand of template DNA.

    PubMed

    Tornaletti, S; Reines, D; Hanawalt, P C

    1999-08-20

    We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase's 3'-->5' exonuclease, covers approximately 35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD.

  13. 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.

  14. 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.

  15. 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.

  16. Benchmarking of Optical Dimerizer Systems

    PubMed Central

    2015-01-01

    Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein–protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast. PMID:25350266

  17. Benchmarking of optical dimerizer systems.

    PubMed

    Pathak, Gopal P; Strickland, Devin; Vrana, Justin D; Tucker, Chandra L

    2014-11-21

    Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast. PMID:25350266

  18. Dimer-monomer model on the Towers of Hanoi graphs

    NASA Astrophysics Data System (ADS)

    Chen, Hanlin; Wu, Renfang; Huang, Guihua; Deng, Hanyuan

    2015-07-01

    The number of dimer-monomers (matchings) of a graph G is an important graph parameter in statistical physics. Following recent research, we study the asymptotic behavior of the number of dimer-monomers m(G) on the Towers of Hanoi graphs and another variation of the Sierpiński graphs which is similar to the Towers of Hanoi graphs, and derive the recursion relations for the numbers of dimer-monomers. Upper and lower bounds for the entropy per site, defined as μG = limv(G)→∞(lnm(G)/v(G)), where v(G) is the number of vertices in a graph G, on these Sierpiński graphs are derived in terms of the numbers at a certain stage. As the difference between these bounds converges quickly to zero as the calculated stage increases, the numerical value of the entropy can be evaluated with more than a hundred significant figures accuracy.

  19. 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/.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding.

    PubMed

    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; Fucini, Paola

    2016-04-19

    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

  5. 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

  6. 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. PMID:26618399

  7. 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. PMID:27043172

  8. 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.

  9. A Model for Dimerization of the SOX Group E Transcription Factor Family

    PubMed Central

    Ramsook, Sarah N.; Ni, Joyce; Shahangian, Shokofeh; Vakiloroayaei, Ana; Khan, Naveen; Kwan, Jamie J.

    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. PMID:27532129

  10. Photochemical dimerization of organic compounds

    SciTech Connect

    Crabtree, R.H.; Brown, S.H.; Muedas, C.A.; Ferguson, R.R.

    1992-04-14

    This patent describes improvement in a Group IIb photosensitized vapor phase dimerization of an organic compound in which a gaseous mixture of a Group IIB metal and the organic compound is irradiated in a reaction zone with a photosensitizing amount of radiant energy. The improvement comprises: a continuous stream of the gaseous mixture is passed as a vapor phase in a single pass through the reaction zone at a temperature at which the thus-produced dimer condenses immediately upon the formation thereof; the starting gaseous mixture comprises hydrogen and two ethylenically unsaturated compounds selected from the group consisting of alkenes of at least six carbon atoms, unsaturated nitriles, unsaturated epoxides, unsaturated silanes, unsaturated amines, unsaturated phosphines, and fluorinated alkenes; the gaseous mixture comprises nitrous oxide and the organic compound is a saturated compound with C-H bond strengths greater than 100 kcal/mol or a mixture of the saturated compound and an alkene; or the starting gaseous comprises an activating amount of hydrogen and the dimerization is a dehydrodimerization or cross-dimerization of a saturated hydrocarbon.

  11. Multiple initiators and C/EBP binding sites are involved in transcription from the TATA-less rat XDH/XO basal promoter.

    PubMed Central

    Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

    1995-01-01

    In the present study, we have explored further the organization of the TATA-less rat xanthine dehydrogenase/oxidase gene (XDH/XO). A DNase I hypersensitive site has been identified which it colocalizes with the basal promoter reported previously [Chow et al. (1994) Nucleic Acids Res., 22, 1846-1854]. Gel mobility shift assays indicate the presence of multiple binding factors located in the promoter. At least six footprints were detected of which two have been shown to be C/EBP binding sites. Members of the C/EBP-alpha and C/EBP-beta, but not C/EBP-delta, family are able to bind to these two sites. Deletional and mutational studies revealed that C/EBP binding is not essential for the basal level of transcription initiation of this promoter. Much of the transcriptional activity resides in the -102 to -7 DNA fragment, which contains all initiator activity which acts unidirectionally. Within this fragment, four putative initiator elements could be identified; interestingly, the linear integrity of these initiators is important for efficient transcription of the XDH/XO gene. Separation of the initiators leads to a complete loss of transcription activity; however, this loss could be partially restored by the introduction of an Sp1 binding site upstream of the separated initiators. Despite a difference in usage/frequency of initiation at the various initiators, primer extension analyses reveal similar positions for transcription initiations in both XDH/XO reporter constructs and in the endogenous XDH/XO gene. The differential usage of initiators may imply a possible post-transcriptional regulation for the XDH/XO gene. Images PMID:7667089

  12. 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

  13. 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.

  14. Properties of the Lennard-Jones dimeric fluid in two dimensions: An integral equation study

    PubMed Central

    Urbic, Tomaz; Dias, Cristiano L.

    2014-01-01

    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. PMID:24606372

  15. 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.

  16. Quantum dynamics of the dissociation of H2 on Cu(100): dependence of the site-reactivity on initial rovibrational state.

    PubMed

    McCormack, D A; Kroes, G J; Olsen, R A; Groeneveld, J A; van Stralen, J N; Baerends, E J; Mowrey, R C

    2000-01-01

    We perform six-dimensional (6D) quantum wavepacket calculations for H2 dissociatively adsorbing on Cu(100) from a variety of rovibrational initial states. The calculations are performed on a new potential energy surface (PES), the construction of which is also detailed. Reaction probabilities are in good agreement with experimental findings. Using a new flux analysis method, we calculate the reaction probability density as a function of surface site and collision energy, for a variety of initial states. This approach is used to study the effects of rotation and vibration on reaction at specific surface sites. The results are explained in terms of characteristics of the PES and intrinsically dynamic effects. An important observation is that, even at low collision energies, reaction does not necessarily proceed predominantly in the region of the minimum potential barrier, but can occur almost exclusively at a site with a higher barrier. This suggests that experimental control of initial conditions could be used to selectively induce reaction at particular surface sites. Our predictions for site-reactivity could be tested using contemporary experimental methods: The calculations predict that, for reacting molecules, there will be a dependence of the quadrupole alignment of j on the incident vibrational state, v. This is a direct result of PES topography in the vicinity of the preferred reaction sites of v = 0 and v = 1 molecules. Invoking detailed balance, evidence for this difference in preferred reaction site of v = 0 and 1 molecules could be obtained through associative desorption experiments.

  17. First Year Site Visits to Milwaukee Urban Systemic Initiative Schools. A Report on the Milwaukee Public Schools Milwaukee Urban Systemic Initiative.

    ERIC Educational Resources Information Center

    Huinker, DeAnn; Pearson, Gretchen; Posnanski, Tracy; Coan, Cheryl; Porter, Corrie

    The Urban Systemic Initiatives (USI) program is an effort sponsored by the National Science Foundation (NSF) that targets large urban school systems with the goal of sustainable implementation of high-quality, standards-based teaching for the purpose of attaining system-wide increases in students' learning of challenging mathematics and science.…

  18. 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.

  19. Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers.

    PubMed

    Watkins, Derrick; Kumar, Sunil; Green, Keith D; Arya, Dev P; Garneau-Tsodikova, Sylvie

    2015-07-01

    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.

  20. Rubidium dimers in paraffin-coated cells

    NASA Astrophysics Data System (ADS)

    Acosta, V. M.; Jarmola, A.; Windes, D.; Corsini, E.; Ledbetter, M. P.; Karaulanov, T.; Auzinsh, M.; Rangwala, S. A.; Kimball, D. F. Jackson; Budker, D.

    2010-08-01

    Measurements were made to determine the density of rubidium dimer vapor in paraffin-coated cells. The number density of dimers and atoms in similar paraffin-coated and uncoated cells was measured by optical spectroscopy. Due to the relatively low melting point of paraffin, a limited temperature range of 43-80 °C was explored, with the lower end corresponding to a dimer density of less than 107 cm- 3. With 1 min integration time, a sensitivity to dimer number density of better than 106 cm- 3 was achieved. No significant difference in dimer density between the cells was observed.

  1. Dimerization-driven degradation of C. elegans and human E proteins

    PubMed Central

    Sallee, Maria D.; Greenwald, Iva

    2015-01-01

    E proteins are conserved regulators of growth and development. We show that the Caenorhabditis elegans E-protein helix–loop–helix-2 (HLH-2) functions as a homodimer in directing development and function of the anchor cell (AC) of the gonad, the critical organizer of uterine and vulval development. Our structure–function analysis of HLH-2 indicates that dimerization drives its degradation in other uterine cells (ventral uterine precursor cells [VUs]) that initially have potential to be the AC. We also provide evidence that this mode of dimerization-driven down-regulation can target other basic HLH (bHLH) dimers as well. Remarkably, human E proteins can functionally substitute for C. elegans HLH-2 in regulating AC development and also display dimerization-dependent degradation in VUs. Our results suggest that dimerization-driven regulation of bHLH protein stability may be a conserved mechanism for differential regulation in specific cell contexts. PMID:26159995

  2. Edge Magnon Excitation in Spin Dimer Systems

    NASA Astrophysics Data System (ADS)

    Sakaguchi, Ryo; Matsumoto, Masashige

    2016-10-01

    Magnetic excitation in a spin dimer system on a bilayer honeycomb lattice is investigated in the presence of a zigzag edge, where disordered and ordered phases can be controlled by a quantum phase transition. In analogy with the case of graphene with a zigzag edge, a flat edge magnon mode appears in the disordered phase. In an ordered phase, a finite magnetic moment generates a mean-field potential to the magnon. Since the potential is nonuniform on the edge and bulk sites, it affects the excitation, and the dispersion of the edge mode deviates from the flat shape. We investigate how the edge magnon mode evolves when the phase changes through the quantum phase transition and discuss the similarities to ordered spin systems on a monolayer honeycomb lattice.

  3. Dimerization transitions in spin-1 chains

    NASA Astrophysics Data System (ADS)

    Chepiga, Natalia; Affleck, Ian; Mila, Frédéric

    2016-06-01

    We study spontaneous dimerization transitions in a Heisenberg spin-1 chain with additional next-nearest-neighbor and three-site interactions using extensive numerical simulations and a conformal field-theory analysis. We show that the transition can be second order in the Wess-Zumino-Witten (WZW) SU (2) 2 or Ising universality class, or first order. We argue that these features are generic because of a marginal operator in the WZW SU (2) 2 model and because of two topologically distinct nondimerized phases with or without edge states. We also provide explicit numerical evidence of conformal towers of singlets inside the spin gap at the Ising transition. Implications for other models are briefly discussed.

  4. Mechanism of FGF receptor dimerization and activation

    PubMed Central

    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. PMID:26725515

  5. Dimerization, trimerization and quantum pumping

    NASA Astrophysics Data System (ADS)

    Guo, Huaiming

    2014-03-01

    We study one-dimensional topological models with dimerization and trimerization and show that these models can be generated using interaction or optical superlattice. The topological properties of these models are demonstrated by the appearance of edge states and the mechanism of dimerization and trimerization is analyzed. Then we show that a quantum pumping process can be constructed based on each one-dimensional topological model. The quantum pumping process is explicitly demonstrated by the instantaneous energy spectrum and local current. The result shows that the pumping is assisted by the gapless states connecting the bands and one charge is pumped during a cycle, which also defines a nonzero Chern number. Our study systematically shows the connection of one-dimensional topological models and quantum pumping, and is useful for the experimental studies on topological phases in optical lattices and photonic quasicrystals.

  6. Dimeric guaianolides from Artemisia absinthium.

    PubMed

    Turak, Ablajan; Shi, She-Po; Jiang, Yong; Tu, Peng-Fei

    2014-09-01

    Five dimeric guaianolides, absinthins A-E, and seven known dimeric guaianolides were isolated from Artemisia absinthium. Their structures were elucidated based on 1D- and 2D-NMR experiments, including (1)H NMR, (13)C NMR, DEPT, (1)H-(1)H COSY, HSQC, HMBC, and NOESY, and through HRESIMS data analysis. The absolute configuration of the known compound, anabsinthin, was determined by X-ray crystallographic analysis. The isolated compounds were tested to assess their inhibitory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 cells; absinthin C and isoanabsinthin exhibited significant inhibitory effects with IC50 values of 1.52 and 1.98μM, respectively.

  7. Photocyclizable resorcin[4]arene dimers.

    PubMed

    Schäfer, Christian; Strübe, Frank; Bringmann, Sebastian; Mattay, Jochen

    2008-12-01

    The synthesis of covalently linked dimers, containing two resorcin[4]arene moieties connected over two 9,10-functionalized anthracene units, is reported. Besides the synthetic route, which involves for example the formation of anthracene-9,10-dialkanols ( and ), and characterization of the compounds, the photochemical properties, introduced through the anthracene groups, were investigated by means of UV/VIS spectroscopy. Both resorcin[4]arene dimers ( and ) were able to undergo an intramolecular [4+4] cycloaddition, therefore changing the size of the inner cavity. Unfortunately, the back reaction, which was expected to take place on irradiation below 300 nm or upon heating, was not observed yet and will be the focus of our future work.

  8. 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.

  9. 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.

  10. Dimerize ethylene to butene-1

    SciTech Connect

    Commereuc, D.; Andrews, J.; Chauvin, Y.; Gillard, J.; Leonard, J.

    1984-11-01

    Institut Francais du Petrole (IFP) has developed a new process, Alphabutol, to dimerize selectively ethylene to butene-1. The Alphabutol process uses a homogeneous catalyst which means that reactants, products and catalyst are all soluble in the same liquid phase. The new catalytic system used in the Alphabutol process avoids isomerization of butene-1 to butene-2. Therefore, there is no need for product superfractionation.

  11. 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.

  12. Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

    PubMed

    Danielsson, O; Shafqat, J; Estonius, M; el-Ahmad, M; Jörnvall, H

    1996-11-19

    The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a

  13. DNA replication during amplification of the C3 puff of Rhynchosciara americana initiates at multiple sites in a 6 kb region.

    PubMed

    Yokosawa, J; Soares, M A; Dijkwel, P A; Stocker, A J; Hamlin, J L; Lara, F J

    1999-09-01

    Two independent two-dimensional agarose gel electrophoresis methods have been used to map the origin of replication that directs amplification of the C3 DNA puff of Rhynchosciara americana. The results of neutral/neutral two-dimensional gel electrophoresis show that DNA replication initiates at multiple sites in a zone of at least 6 kb situated immediately upstream from the promoter of the main transcription unit of this puff. The complementary neutral/alkaline two-dimensional gel electrophoresis technique shows that, within the initiation zone, forks move in both directions. In contrast, unidirectional fork movement away from the initiation zone is observed at the ends of the region, implying that it is the only place in the amplified region of the C3 puff where initiations occur. Since the initiation zone coincides with the region that is most highly amplified, amplification of the C3 puff probably occurs by an onion skin-type mechanism. PMID:10525965

  14. 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

  15. 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.

  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. PMID:25900042

  17. Antifreeze protein dimer: when two ice-binding faces are better than one.

    PubMed

    Baardsnes, Jason; Kuiper, Michael J; Davies, Peter L

    2003-10-01

    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.

  18. Transfer and decay of an exciton coupled to vibrations in a dimer

    SciTech Connect

    Schanz, H.; Barvik, I.; Esser, B.

    1997-05-01

    Transfer and decay dynamics of an exciton coupled to a polarization vibration in a dimer is investigated in a mixed quantum-classical picture with the exciton decay incorporated by a sink site. Using a separation of time scales, it is possible to explain analytically the most important characteristics of the model. If the vibronic subsystem is fast, these are the enhancement of nonlinear self-trapping due to the sink and the slowing down of the exciton decay for large coupling or sink strength. Numerical results obtained recently for the discrete self-trapping (DST) approximation to the model are quantitatively explained and dynamic effects beyond this approximation are found. If the vibronic subsystem is slow, the behavior of the system follows closely the predictions of the adiabatic approximation. In this regime, the exciton decay crucially depends on the initial conditions of the vibronic subsystem. In the transition regime between the adiabatic and DST approximation, complex dynamics is observed by numerical computation. We discuss the correspondence to the chaotic behavior of the excitonic-vibronic coupled dimer without trap. {copyright} {ital 1997} {ital The American Physical Society}

  19. Probing the low temperature initiation sites in Fe-, Mn-promoted sulfated zirconia via CO and H{sub 2} adsorption

    SciTech Connect

    Sayari, A.; Yang, Y.

    1999-10-01

    Exposure of freshly activated Fe-, Mn-promoted sulfated zirconia (SFMZ) to carbon monoxide at temperatures up to 50 C induced permanent loss of activity, while the addition of CO after the beginning of the reaction had a reversible effect, regardless of whether the butane flow has been interrupted or not. Similar experiments using dissociated hydrogen instead of CO led to irreversible poisoning in all cases. These findings were interpreted based on (1) the occurrence of initiation sites that are consumed stoichiometrically and very rapidly upon exposure to butane, (2) such initiation sites which are also consumed by CO or dissociated hydrogen, (3) CO which competes effectively for adsorption sites without affecting accumulated reaction intermediates, and (4) such intermediates that are removed in the presence of dissociated hydrogen.

  20. Quantum chemical interaction energy surfaces of ethylene and propene dimers.

    PubMed

    Jalkanen, Jukka-Pekka; Pulkkinen, Sallaraisa; Pakkanen, Tapani A; Rowley, Richard L

    2005-03-31

    Ab initio studies of nonbonding interactions for ethylene and propene dimers were conducted at the MP2/6-311+G(2df,2pd) level. The dimers were attractive in all of the orientations studied; however, the attraction was <0.1 kcal/mol for ethylene D2h and C2h dimers, for which the pi-electron clouds or H atoms interact closely. A previously introduced transferable potential model, NIPE [Jalkanen, J.-P.; Pakkanen, T. A.; Yang, Y.; Rowley, R. L. J. Chem. Phys. 2003, 118, 5474], which is based on quantum chemical calculations of small alkane molecules, was tested against the propene and ethylene dimer data. Comparisons of results showed that interaction energies for orientations dominated by interactions between the propene methyl groups or two hydrogens were accurately predicted with the NIPE model. Interactions involving the double bond were not predicted as well, because the original NIPE regression data set did not contain any information about pi-electron systems. An extension of the NIPE model to include pi-electron interactions is proposed. Additional interaction sites are used with the same energy function as atomic interactions. This addition provides a more accurate description of the interaction energies of both ethylene and propene and extends the transferability of the NIPE model to alkenes.

  1. 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.

  2. Technical approaches to characterizing and cleaning up iron and steel mill sites under the brownfields initiative. Final report

    SciTech Connect

    1998-12-01

    EPA has developed this guide to provide decision-makers, such as city planners, private sector developers, and other involved in redeveloping brownfields, with a better understanding of the technical issues involved in assessing and cleaning up iron and steel mill sites so they can make the most informed decisions possible. This overview of the technical process involved in assessing and cleaning up brownfields sites can assist planners in making decisions at various stages of the project. An understanding of land use and industrial processes conducted in the past at a site can help the planner to conceptualize the site and identify likely areas of contamination that may require cleanup. Numerous resources are suggested to facilitate characterization of the site and consideration of cleanup technologies.

  3. The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation.

    PubMed

    Ross, Wilma; Vrentas, Catherine E; Sanchez-Vazquez, Patricia; Gaal, Tamas; Gourse, Richard L

    2013-05-01

    The global regulatory nucleotide ppGpp ("magic spot") regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp's target site on RNAP, and therefore its mechanism of action, has remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β', and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

  4. Structural basis for RNA recognition by a dimeric PPR-protein complex.

    PubMed

    Ke, Jiyuan; Chen, Run-Ze; Ban, Ting; Zhou, X Edward; Gu, Xin; Tan, M H Eileen; Chen, Chen; Kang, Yanyong; Brunzelle, Joseph S; Zhu, Jian-Kang; Melcher, Karsten; Xu, H Eric

    2013-12-01

    Thylakoid assembly 8 (THA8) is a pentatricopeptide repeat (PPR) RNA-binding protein required for the splicing of the transcript of ycf3, a gene involved in chloroplast thylakoid-membrane biogenesis. Here we report the identification of multiple THA8-binding sites in the ycf3 intron and present crystal structures of Brachypodium distachyon THA8 either free of RNA or bound to two of the identified RNA sites. The apostructure reveals a THA8 monomer with five tandem PPR repeats arranged in a planar fold. The complexes of THA8 bound to the two short RNA fragments surprisingly reveal asymmetric THA8 dimers with the bound RNAs at the dimeric interface. RNA binding induces THA8 dimerization, with a conserved G nucleotide of the bound RNAs making extensive contacts with both monomers. Together, these results establish a new model of RNA recognition by RNA-induced formation of an asymmetric dimer of a PPR protein.

  5. 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

  6. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Functional Significance of Serotonin Receptor Dimerization

    PubMed Central

    Herrick-Davis, Katharine

    2013-01-01

    The original model of G protein activation by a single G-protein-coupled receptor (GPCR) is giving way to a new model wherein two protomers of a GPCR dimer interact with a single G protein. This article will review the evidence suggesting that 5-HT receptors form dimers/oligomers and will compare the findings with results obtained from studies with other biogenic amine receptors. Topics to be covered include the origin or biogenesis of dimer formation, potential dimer interface(s), and oligomer size (dimer versus tetramer or higher order). The functional significance will be discussed in terms of G-protein activation following ligand binding to one or two protomers in a dimeric structure, the formation of heterodimers and the development of bivalent ligands. PMID:23811735

  8. 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.

  9. Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

    PubMed Central

    Chesterton, C. James; Coupar, Barbara E. H.; Butterworth, Peter H. W.

    1974-01-01

    The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA. PMID:4464858

  10. Enhanced Chiral Recognition by Cyclodextrin Dimers

    PubMed Central

    Voskuhl, Jens; Schaepe, Kira; Ravoo, Bart Jan

    2011-01-01

    In this article we investigate the effect of multivalency in chiral recognition. To this end, we measured the host-guest interaction of a β-cyclodextrin dimer with divalent chiral guests. We report the synthesis of carbohydrate-based water soluble chiral guests functionalized with two borneol, menthol, or isopinocampheol units in either (+) or (−) configuration. We determined the interaction of these divalent guests with a β-cyclodextrin dimer using isothermal titration calorimetry. It was found that—in spite of a highly unfavorable conformation—the cyclodextrin dimer binds to guest dimers with an increased enantioselectivity, which clearly reflects the effect of multivalency. PMID:21845101

  11. ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response.

    PubMed

    Ross, Wilma; Sanchez-Vazquez, Patricia; Chen, Albert Y; Lee, Jeong-Hyun; Burgos, Hector L; Gourse, Richard L

    2016-06-16

    Throughout the bacterial domain, the alarmone ppGpp dramatically reprograms transcription following nutrient limitation. This "stringent response" is critical for survival and antibiotic tolerance and is a model for transcriptional regulation by small ligands. We report that ppGpp binds to two distinct sites 60 Å apart on E. coli RNA polymerase (RNAP), one characterized previously (site 1) and a second identified here at an interface of RNAP and the transcription factor DksA (site 2). The location and unusual tripartite nature of site 2 account for the DksA-ppGpp synergism and suggest mechanisms for ppGpp enhancement of DksA's effects on RNAP. Site 2 binding results in the majority of ppGpp's effects on transcription initiation in vitro and in vivo, and strains lacking site 2 are severely impaired for growth following nutritional shifts. Filling of the two sites at different ppGpp concentrations would expand the dynamic range of cellular responses to changes in ppGpp levels. PMID:27237053

  12. ppGpp Binding to a Site at the RNAP-DksA Interface Accounts for Its Dramatic Effects on Transcription Initiation during the Stringent Response.

    PubMed

    Ross, Wilma; Sanchez-Vazquez, Patricia; Chen, Albert Y; Lee, Jeong-Hyun; Burgos, Hector L; Gourse, Richard L

    2016-06-16

    Throughout the bacterial domain, the alarmone ppGpp dramatically reprograms transcription following nutrient limitation. This "stringent response" is critical for survival and antibiotic tolerance and is a model for transcriptional regulation by small ligands. We report that ppGpp binds to two distinct sites 60 Å apart on E. coli RNA polymerase (RNAP), one characterized previously (site 1) and a second identified here at an interface of RNAP and the transcription factor DksA (site 2). The location and unusual tripartite nature of site 2 account for the DksA-ppGpp synergism and suggest mechanisms for ppGpp enhancement of DksA's effects on RNAP. Site 2 binding results in the majority of ppGpp's effects on transcription initiation in vitro and in vivo, and strains lacking site 2 are severely impaired for growth following nutritional shifts. Filling of the two sites at different ppGpp concentrations would expand the dynamic range of cellular responses to changes in ppGpp levels.

  13. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    PubMed

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  14. 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.

  15. Definition of the transcription initiation site of human plasminogen gene in liver and non hepatic cell lines.

    PubMed

    Malgaretti, N; Bruno, L; Pontoglio, M; Candiani, G; Meroni, G; Ottolenghi, S; Taramelli, R

    1990-12-31

    We have mapped the cap site of the human plasminogen mRNA by primer extension and PCR techniques and found that it is located at position -161 relative to the first ATG, 97 bases upstream to the 5' end of the previously isolated cDNA clone. Seven human hepatic and non hepatic cell lines and fresh liver cells were tested for human plasminogen mRNA expression: the liver and the liver derived HepG2 cell line represent the major site of plasminogen RNA synthesis while the other cell lines (Hep3B, HeLa, IMR, 293 CaCo and SW626) show much lower levels.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Dimerization effect of sucrose octasulfate on rat FGF1

    PubMed Central

    Kulahin, N.; Kiselyov, V.; Kochoyan, A.; Kristensen, O.; Kastrup, Jette S.; Berezin, V.; Bock, E.; Gajhede, M.

    2008-01-01

    Fibroblast growth factors (FGFs) constitute a family of at least 23 structurally related heparin-binding proteins that are involved in regulation of cell growth, survival, differentiation and migration. Sucrose octasulfate (SOS), a chemical analogue of heparin, has been demonstrated to activate FGF signalling pathways. The structure of rat FGF1 crystallized in the presence of SOS has been determined at 2.2 Å resolution. SOS-mediated dimerization of FGF1 was observed, which was further supported by gel-filtration experiments. The major contributors to the sulfate-binding sites in rat FGF1 are Lys113, Lys118, Arg122 and Lys128. An arginine at position 116 is a consensus residue in mammalian FGF molecules; however, it is a serine in rat FGF1. This difference may be important for SOS-mediated FGF1 dimerization in rat. PMID:18540049

  1. The water dimer I: Experimental characterization

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Anamika; Cole, William T. S.; Saykally, Richard J.

    2015-07-01

    As the archetype of water hydrogen bonding, the water dimer has been studied extensively by both theory and experiment for nearly seven decades. In this article, we present a detailed chronological review of the experimental dimer studies and the insights into the complex nature of water and hydrogen bonding gained from them. A subsequent letter will review the corresponding theoretical advances.

  2. Site-directed subsurface environmental initiative: Five year summary and plan for fundamental research in subsoils and in groundwater, FY 1989-FY 1993

    SciTech Connect

    Not Available

    1987-10-01

    The overall goal of this research initiative is to develop the necessary scientific basis for resolution of key technical obstacles to defining and remediating contamination at DOE and other waste sites. To accomplish this goal, the resouces of the national laboratories, universities, and DOE sites will be fully utilized to develop and demonstrate improved, cost-effective, and environmentally acceptable techniques for predicting the behavior of contaminants and reducing their concentrations in ground water. This document is a preliminary plan to set general research directions for a program extending into the 1990s. The needs and milestones identified in this plan may change with additional guidance from DOE sites. Promising research opportunities will be identified as part of national laboratory submissions of preliminary proposals.

  3. Free energy calculations of gramicidin dimer dissociation.

    PubMed

    Wanasundara, Surajith N; Krishnamurthy, Vikram; Chung, Shin-Ho

    2011-11-24

    Molecular dynamics simulations, combined with umbrella sampling, is used to study how gramicidin A (gA) dimers dissociate in the lipid bilayer. The potential of mean force and intermolecular potential energy are computed as functions of the distance between center of masses of the two gA monomers in two directions of separation: parallel to the bilayer surface and parallel to the membrane normal. Results from this study show that the dissociation of gA dimers occurs via lateral displacement of gA monomers followed by tilting of dimers with respect to the lipid bilayer normal. It is found that the dissociation energy of gA dimers in the dimyristoylphosphatidylcholine bilayer is 14 kcal mol(-1) (~22 kT), which is approximately equal to the energy of breaking six intermolecular hydrogen bonds that stabilize the gA channel dimer.

  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. Inhibition of a viral enzyme by a small-molecule dimer disruptor.

    PubMed

    Shahian, Tina; Lee, Gregory M; Lazic, Ana; Arnold, Leggy A; Velusamy, Priya; Roels, Christina M; Guy, R Kiplin; Craik, Charles S

    2009-09-01

    We identified small-molecule dimer disruptors that inhibit an essential dimeric protease of human Kaposi's sarcoma-associated herpesvirus (KSHV) by screening an alpha-helical mimetic library. Next, we synthesized a second generation of low-micromolar inhibitors with improved potency and solubility. Complementary methods including size exclusion chromatography and 1H-13C HSQC titration using selectively labeled 13C-Met samples revealed that monomeric protease is enriched in the presence of inhibitor. 1H-15N HSQC titration studies mapped the inhibitor binding site to the dimer interface, and mutagenesis studies targeting this region were consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of a dynamic intermediate. These results validate the interface of herpesvirus proteases and other similar oligomeric interactions as suitable targets for the development of small-molecule inhibitors.

  6. Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation.

    PubMed

    Jambrina, Pablo G; Rauch, Nora; Pilkington, Ruth; Rybakova, Katja; Nguyen, Lan K; Kholodenko, Boris N; Buchete, Nicolae-Viorel; Kolch, Walter; Rosta, Edina

    2016-01-18

    RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N-terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC-helix and charged residues upstream of the NtA motif. Additionally, we show that the R-spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure-based mechanism for the auto-transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization-related drug resistance.

  7. 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}.

  8. 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

  9. 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.

  10. Ratchet rotation of a 3D dimer on a vibrating plate.

    PubMed

    Wang, Jiao; Liu, Caishan; Jia, Yan-Bin; Ma, Daolin

    2014-01-01

    This work studies the dynamics of a 3D dimer bouncing upon a horizontal plate undergoing a vertical harmonic vibration. Despite complex interactions within the system due to impacts and friction, numerical simulation shows that, under certain conditions prescribed for the dynamics, the center of mass of the dimer, when projected onto a horizontal plane, will follow a circular orbit. The phenomenon is like a particle under Coulomb friction performing a ratchet motion that rotates around. Investigations further reveal that the dimer dynamics bear some typical characteristics of a nonlinear system, including sensitivity to the initial conditions and bifurcation behaviors related to the physical parameters of the dynamics. Our results indicate that the coefficient of restitution and the plate's vibration intensity play critical roles in exciting the circular orbit, while the dimer's geometry and the vibration frequency mainly influence the trajectory characteristics. These findings may help understand transport mechanisms underlying systems of granular matter with anisotropic particles. PMID:24458553

  11. An Inter-Comparison of Two Independent Site Test Interferometers Located in Goldstone, California: Initial Study Results

    NASA Technical Reports Server (NTRS)

    Morabito, David; D'Addario, Larry; Acosta, Roberto J.; Nessel, James A.

    2012-01-01

    Site Test Interferometers (STIs) have been deployed at two different locations at the NASA Deep Space Network (DSN) tracking complex in Goldstone, California. An STI measures the difference in path length between a geostationary satellite and two antennas on the Earth, producing a measure of atmospheric phase fluctuations over spatial distances comparable to those between elements of possible microwave phased arrays. The purposes of the Goldstone STIs are to assess the suitability of Goldstone as an array site and to statistically characterize atmospheric induced delay fluctuations for application to future array scenarios.The two STI's are separated by 13 km across the Goldstone complex. Each instrument is composed of two small-diameter antennas and associated electronics separated by approx. 200 meters in a principally east-west configuration. The antennas continuously observe signals emitted by geo-stationary satellites and produce data that contain information on the phase difference between signals received by both antennas. The fluctuation in delay (or path length difference) statistics derived from these data sets can be used to infer power loss for particular array configurations.We report on a comparison of the root mean square (RMS) phase delay statistics estimated over 10-minute intervals between the two Goldstone STIs. We have achieved good statistical agreement between the data acquired from the two STIs, given that each instrument is observing different satellites, at different frequencies, over different baseline lengths, with very different implementations, and are located 13 km apart in widely separated terrain at different geodetic altitudes.

  12. Airways abnormalities and rheumatoid arthritis-related autoantibodies in subjects without arthritis: early injury or initiating site of autoimmunity?

    PubMed Central

    Demoruelle, M. Kristen; Weisman, Michael H.; Simonian, Philip L.; Lynch, David A.; Sachs, Peter B.; Pedraza, Isabel F.; Harrington, Annie R.; Kolfenbach, Jason R.; Striebich, Christopher C.; Pham, Quyen N.; Strickland, Colin D.; Petersen, Brian D.; Parish, Mark C.; Derber, Lezlie A.; Norris, Jill M.; Holers, V. Michael; Deane, Kevin D.

    2011-01-01

    Objective To evaluate the presence of pulmonary abnormalities in subjects with rheumatoid arthritis (RA)-related autoantibody (Ab) positivity without inflammatory arthritis (IA). Methods 42 subjects without IA but with elevations of anti-cyclic citrullinated peptide antibodies and/or 2 or more rheumatoid factor isotypes (a profile that is 96% specific for RA), 15 Ab(−) controls and 12 patients with early established seropositive RA (<1 year duration) underwent spirometry and high-resolution computed tomographic (HRCT) lung imaging. Results The median age of Ab(+) subjects was 54 years-old, 52% were female and 38% were smokers (not significantly different than Ab(−) controls). No Ab(+) subject had IA on joint examination. On HRCT, 76% of Ab(+) subjects had airways abnormalities including bronchial wall thickening, bronchiectasis, centrilobular opacities and air trapping, compared to 33% of Ab(−) controls (p=0.005). The Ab(+) subjects had similar prevalence and type of lung abnormalities compared to patients with early RA. Two Ab(+) subjects with airways disease developed IA classifiable as articular RA ~13 months after lung evaluation. Conclusion Airways abnormalities that are consistent with inflammation are common in Ab(+) subjects without IA, and similar to airways abnormalities seen in early RA. These findings suggest that the lung may be an early site of autoimmune-related injury, and potentially a site of generation of RA-related autoimmunity. Further studies are needed to define the mechanistic role of lung inflammation in the development of RA. PMID:22183986

  13. Shared Skies Partnership: A Dual-Site All-Sky Live Remote Observing Initiative for Research and Education

    NASA Astrophysics Data System (ADS)

    Kielkopf, John F.; Hart, R.; Carter, B.; Collins, K. A.; Brown, C.; Hay, J.; Hons, A.; Marsden, S.

    2014-01-01

    The University of Southern Queensland's Mt. Kent Observatory in Queensland, Australia, and the University of Louisville's Moore Observatory in Kentucky, USA, are collaborating in the development of live remote observing for research, student training, and education. With a focus on flexible operation assisted by semi-autonomous controllers, rather than completely robotic data acquisition, the partnership provides interactive hands-on experience to students at all levels, optimized performance based on real-time observations, and flexible scheduling for transient events and targets of opportunity. Two sites on opposites sides of the globe cover the entire sky, and for equatorial regions allow nearly continuous coverage. The facilites include 0.5-m corrected Dall-Kirkham (CDK) telescopes at both sites, a 0.6 m Ritchie-Chretien telescope at Moore, and a new Nasmyth design 0.7-meter CDK at Mt. Kent instrumented for milli-magnitude precision photometry and wide field imaging, with spectrographs under development. We will describe the operational and data acquisition software, recent research results, and how remote access is being made available to students and observers.

  14. 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 ecosystems in the United States. These impacts arise primarily from the improper disposal of large volumes of saline water produced with oil and gas, from accidental hydrocarbon and produced water releases, and from abandoned oil wells that were not correctly sealed. It is important to understand the long-term and short-term effects of produced water and hydrocarbon releases from these sites in order to develop risk-based remediation plans. Remediation is particularly needed in aging and depleted fields where land use is changing from petroleum production to residential, agricultural or recreational uses. About 20 scientists from the USGS and other governmental agencies and academia are involved in a multidisciplinary investigation to study the transport, fate, and natural attenuation of inorganic salts, trace metals, organic compounds and radionuclides present in produced water, and their impacts at the Osage-Skiatook Petroleum Environmental Research (OSPER) 'A' and 'B' sites, located on the Osage Reservation in Osage County, Oklahoma. Stakeholders in the project include the Osage Nation, which holds the mineral rights, the Bureau of Indian Affairs with trust responsibility, and the Army Corps of Engineers, which owns the surface rights at these sites and manages adjacent Skiatook Lake. The 4250-hectare Skiatook Lake provides drinking water to local Tulsa suburban communities and a rural water district, and offers recreational fishing and boating opportunities to tens of thousands of visitors each year. Approximately 1.5 and 1.0 hectare of land at the OSPER 'A' (depleted Lester lease) and 'B' (active Branstetter lease) sites, respectively, are affected by salt scarring, tree kills, soil salinization and brine and petroleum contamination due to the leakage of produced water and associated hydrocarbons from brine pits and accidental

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Vibrational coupling in carboxylic acid dimers

    NASA Astrophysics Data System (ADS)

    Nandi, Chayan K.; Hazra, Montu K.; Chakraborty, Tapas

    2005-09-01

    The vibrational level splitting in the ground electronic state of carboxylic acid dimers mediated by the doubly hydrogen-bonded networks are investigated using pure and mixed dimers of benzoic acid with formic acid as molecular prototypes. Within the 0-2000-cm-1 range, the frequencies for the fundamental and combination vibrations of the two dimers are experimentally measured by using dispersed fluorescence spectroscopy in a supersonic jet expansion. Density-functional-theory calculations predict that most of the dimer vibrations are essentially in-phase and out-of-phase combinations of the monomer modes, and many of such combinations show significantly large splitting in vibrational frequencies. The infrared spectrum of the jet-cooled benzoic acid dimer, reported recently by Bakker et al. [J. Chem. Phys. 119, 11180 (2003)], has been used along with the dispersed fluorescence spectra to analyze the coupled g-u vibrational levels. Assignments of the dispersed fluorescence spectra of the mixed dimer are suggested by comparing the vibronic features with those in the homodimer spectrum and the predictions of density-functional-theory calculation. The fluorescence spectra measured by excitations of the low-lying single vibronic levels of the mixed dimer reveal that the hydrogen-bond vibrations are extensively mixed with the ring modes in the S1 surface.

  20. Vibrational coupling in carboxylic acid dimers.

    PubMed

    Nandi, Chayan K; Hazra, Montu K; Chakraborty, Tapas

    2005-09-22

    The vibrational level splitting in the ground electronic state of carboxylic acid dimers mediated by the doubly hydrogen-bonded networks are investigated using pure and mixed dimers of benzoic acid with formic acid as molecular prototypes. Within the 0-2000-cm(-1) range, the frequencies for the fundamental and combination vibrations of the two dimers are experimentally measured by using dispersed fluorescence spectroscopy in a supersonic jet expansion. Density-functional-theory calculations predict that most of the dimer vibrations are essentially in-phase and out-of-phase combinations of the monomer modes, and many of such combinations show significantly large splitting in vibrational frequencies. The infrared spectrum of the jet-cooled benzoic acid dimer, reported recently by Bakker et al. [J. Chem. Phys. 119, 11180 (2003)], has been used along with the dispersed fluorescence spectra to analyze the coupled g-u vibrational levels. Assignments of the dispersed fluorescence spectra of the mixed dimer are suggested by comparing the vibronic features with those in the homodimer spectrum and the predictions of density-functional-theory calculation. The fluorescence spectra measured by excitations of the low-lying single vibronic levels of the mixed dimer reveal that the hydrogen-bond vibrations are extensively mixed with the ring modes in the S1 surface. PMID:16392485

  1. Energetic Coupling between Ligand Binding and Dimerization in Escherichia coli Phosphoglycerate Mutase.

    PubMed

    Gardner, Nathan W; Monroe, Lyman K; Kihara, Daisuke; Park, Chiwook

    2016-03-29

    Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding of an exceptional quality. The homodimeric Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. We found that, in the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization.

  2. Energetic Coupling between Ligand Binding and Dimerization in E. coli Phosphoglycerate Mutase

    PubMed Central

    Gardner, Nathan W.; Monroe, Lyman K.; Kihara, Daisuke; Park, Chiwook

    2016-01-01

    Energetic coupling of two molecular events in a protein molecule is ubiquitous in biochemical reactions mediated by proteins, such as catalysis and signal transduction. Here, we investigate energetic coupling between ligand binding and folding of a dimer using a model system that shows three-state equilibrium unfolding in an exceptional quality. The homodimeric E. coli cofactor-dependent phosphoglycerate mutase (dPGM) was found to be stabilized by ATP in a proteome-wide screen, although dPGM does not require or utilize ATP for enzymatic function. We investigated the effect of ATP on the thermodynamic stability of dPGM using equilibrium unfolding. In the absence of ATP, dPGM populates a partially unfolded, monomeric intermediate during equilibrium unfolding. However, addition of 1.0 mM ATP drastically reduces the population of the intermediate by selectively stabilizing the native dimer. Using a computational ligand docking method, we predicted ATP binds to the active site of the enzyme using the triphosphate group. By performing equilibrium unfolding and isothermal titration calorimetry with active-site variants of dPGM, we confirmed that active-site residues are involved in ATP binding. Our findings show that ATP promotes dimerization of the protein by binding to the active site, which is distal from the dimer interface. This cooperativity suggests an energetic coupling between the active-site and the dimer interface. We also propose a structural link to explain how ligand binding to the active site is energetically coupled with dimerization. PMID:26919584

  3. Glutamate Racemase Dimerization Inhibits Dynamic Conformational Flexibility and Reduces Catalytic Rates

    SciTech Connect

    Mehboob, Shahila; Guo, Liang; Fu, Wentao; Mittal, Anuradha; Yau, Tiffany; Truong, Kent; Johlfs, Mary; Long, Fei; Fung, Leslie W.-M.; Johnson, Michael E.

    2009-09-15

    Glutamate racemase (RacE) is a bacterial enzyme that converts L-glutamate to D-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with D-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the D-Glu to L-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.

  4. CLEC-2 activates Syk through dimerization.

    PubMed

    Hughes, Craig E; Pollitt, Alice Y; Mori, Jun; Eble, Johannes A; Tomlinson, Michael G; Hartwig, John H; O'Callaghan, Christopher A; Fütterer, Klaus; Watson, Steve P

    2010-04-01

    The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor. PMID:20154219

  5. RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts.

    PubMed

    Sanuki, Yosuke; Kubota, Yumiko; Kanemaki, Masato T; Takahashi, Tatsuro S; Mimura, Satoru; Takisawa, Haruhiko

    2015-01-01

    Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.

  6. 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.

  7. Initial excavation and dating of Ngalue Cave: a Middle Stone Age site along the Niassa Rift, Mozambique.

    PubMed

    Mercader, Julio; Asmerom, Yemane; Bennett, Tim; Raja, Mussa; Skinner, Anne

    2009-07-01

    Direct evidence for a systematic occupation of the African tropics during the early late Pleistocene is lacking. Here, we report a record of human occupation between 105-42ka, based on results from a radiometrically-dated cave section from the Mozambican segment of the Niassa (Malawi/Nyasa) Rift called Ngalue. The sedimentary sequence from bottom to top has five units. We concentrate on the so-called "Middle Beds," which contain a Middle Stone Age industry characterized by the use of the discoidal reduction technique. A significant typological feature is the presence of formal types such as points, scrapers, awls, and microliths. Special objects consist of grinders/core-axes covered by ochre. Ngalue is one of the few directly-dated Pleistocene sites located along the biogeographical corridor for modern human dispersals that links east, central, and southern Africa, and, with further study, may shed new light on hominin cave habitats during the late Pleistocene.

  8. Spin 3/2 dimer model

    NASA Astrophysics Data System (ADS)

    Rachel, S.

    2009-05-01

    We present a parent Hamiltonian for weakly dimerized valence bond solid states for arbitrary half-integral S. While the model reduces for S=1/2 to the Majumdar-Ghosh Hamiltonian, we discuss this model and its properties for S=3/2. Its degenerate ground state is the most popular toy model state for discussing dimerization in spin 3/2 chains. In particular, it describes the impurity-induced dimer phase in Cr8Ni as proposed recently. We point out that the explicit construction of the Hamiltonian and its main features apply to arbitrary half-integral spin S.

  9. Quantum dimer model for the pseudogap metal

    PubMed Central

    Punk, Matthias; Allais, Andrea; Sachdev, Subir

    2015-01-01

    We propose a quantum dimer model for the metallic state of the hole-doped cuprates at low hole density, p. The Hilbert space is spanned by spinless, neutral, bosonic dimers and spin S=1/2, charge +e fermionic dimers. The model realizes a “fractionalized Fermi liquid” with no symmetry breaking and small hole pocket Fermi surfaces enclosing a total area determined by p. Exact diagonalization, on lattices of sizes up to 8×8, shows anisotropic quasiparticle residue around the pocket Fermi surfaces. We discuss the relationship to experiments. PMID:26195771

  10. Biomimetic synthesis of active isorhapontigenin dimers.

    PubMed

    Wang, Xian-Fen; Zhang, Yuan; Lin, Ming-Bao; Hou, Qi; Yao, Chun-Suo; Shi, Jian-Gong

    2014-01-01

    Synthetic isorhapontigenin was treated with several kinds of inorganic reagents and peroxidase so as to prepare active stilbene dimers. Among them, silver acetate in methanol gave two new isorhapontigenin dimers 4 and 5, together with four known natural stilbene dimers 2, 3, 6, and 7. Their structures and relative configurations were determined on the basis of spectral analysis, and their possible formation mechanisms were discussed, respectively. Compounds 2, 6, and 7 were artificially synthesized for the first time. All the products were evaluated for anti-inflammatory activities.

  11. Multiple-charge transfer and trapping in DNA dimers

    NASA Astrophysics Data System (ADS)

    Tornow, Sabine; Bulla, Ralf; Anders, Frithjof B.; Zwicknagl, Gertrud

    2010-11-01

    We investigate the charge transfer characteristics of one and two excess charges in a DNA base-pair dimer using a model Hamiltonian approach. The electron part comprises diagonal and off-diagonal Coulomb matrix elements such a correlated hopping and the bond-bond interaction, which were recently calculated by Starikov [E. B. Starikov, Philos. Mag. Lett. 83, 699 (2003)10.1080/0950083031000151374] for different DNA dimers. The electronic degrees of freedom are coupled to an ohmic or a superohmic bath serving as dissipative environment. We employ the numerical renormalization group method in the nuclear tunneling regime and compare the results to Marcus theory for the thermal activation regime. For realistic parameters, the rate that at least one charge is transferred from the donor to the acceptor in the subspace of two excess electrons significantly exceeds the rate in the single charge sector. Moreover, the dynamics is strongly influenced by the Coulomb matrix elements. We find sequential and pair transfer as well as a regime where both charges remain self-trapped. The transfer rate reaches its maximum when the difference of the on-site and intersite Coulomb matrix element is equal to the reorganization energy which is the case in a guanine/cytosine (GC)-dimer. Charge transfer is completely suppressed for two excess electrons in adenine/thymine (AT)-dimer in an ohmic bath and replaced by damped coherent electron-pair oscillations in a superohmic bath. A finite bond-bond interaction W alters the transfer rate: it increases as function of W when the effective Coulomb repulsion exceeds the reorganization energy (inverted regime) and decreases for smaller Coulomb repulsion.

  12. dimerization and DNA binding alter phosphorylation of Fos and Jun

    SciTech Connect

    Abate, C.; Baker, S.J.; Curran, T. ); Lees-Miller, S.P.; Anderson, C.W. ); Marshak, D.R. )

    1993-07-15

    Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcription factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here the authors show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34[sup cdc2] (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation. 44 refs., 4 figs.

  13. An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

    PubMed Central

    Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

    2016-01-01

    The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

  14. Bicompartmental phase transfer vehicles based on colloidal dimers.

    PubMed

    Wang, Sijia; Wu, Ning

    2014-11-26

    Colloidal particles have been used extensively for stabilizing oil-water interfaces in petroleum, food, and cosmetics industries. They have also demonstrated promising potential in the encapsulation and delivery of drugs. Our work is motivated by challenging applications that require protecting and transporting active agents across the water-oil interfaces, such as delivering catalysts to underground oil phase through water flooding for in situ cracking of crude oil. In this Research Article, we successfully design, synthesize, and test a unique type of bicompartmental targeting vehicle that encapsulates catalytic molecules, finds and accumulates at oil-water interface, releases the catalysts toward the oil phase, and performs hydrogenation reaction of unsaturated oil. This vehicle is based on colloidal dimers that possess structural anisotropy between two compartments. We encapsulate active species, such as fluorescent dye and catalytic molecules in one lobe which consists of un-cross-linked polymers, while the other polymeric lobe is highly cross-linked. Although dimers are dispersible in water initially, the un-cross-linked lobe swells significantly upon contact with a trace amount of oil in aqueous phase. The dimers then become amphiphilic, migrate toward, and accumulate at the oil-water interface. As the un-cross-linked lobe swells and eventually dissolves in oil, the encapsulated catalysts are fully released. We also show that hydrogenation of unsaturated oil can be performed subsequently with high conversion efficiency. By further creating the interfacial anisotropy on the dimers, we can reduce the catalyst release time from hundred hours to 30 min. Our work demonstrates a new concept in making colloidal emulsifiers and phase-transfer vehicles that are important for encapsulation and sequential release of small molecules across two different phases.

  15. Quantitative analysis of cyclic dimer fatty acid content in the dimerization product by proton NMR spectroscopy.

    PubMed

    Park, Kyun Joo; Kim, Minyoung; Seok, Seunghwan; Kim, Young-Wun; Kim, Do Hyun

    2015-01-01

    In this work, (1)H NMR is utilized for the quantitative analysis of a specific cyclic dimer fatty acid in a dimer acid mixture using the pseudo-standard material of mesitylene on the basis of its structural similarity. Mesitylene and cyclic dimer acid levels were determined using the signal of the proton on the cyclic ring (δ=6.8) referenced to the signal of maleic acid (δ=6.2). The content of the cyclic dimer fatty acid was successfully determined through the standard curve of mesitylene and the reported equation. Using the linearity of the mesitylene curve, the cyclic dimer fatty acid in the oil mixture was quantified. The results suggest that the proposed method can be used to quantify cyclic compounds in mixtures to optimize the dimerization process.

  16. Initial field testing definition of subsurface sealing and backfilling tests in unsaturated tuff; Yucca Mountain Site Characterization Project

    SciTech Connect

    Fernandez, J.A.; Case, J.B.; Tyburski, J.R.

    1993-05-01

    This report contains an initial definition of the field tests proposed for the Yucca Mountain Project repository sealing program. The tests are intended to resolve various performance and emplacement concerns. Examples of concerns to be addressed include achieving selected hydrologic and structural requirements for seals, removing portions of the shaft liner, excavating keyways, emplacing cementitious and earthen seals, reducing the impact of fines on the hydraulic conductivity of fractures, efficient grouting of fracture zones, sealing of exploratory boreholes, and controlling the flow of water by using engineered designs. Ten discrete tests are proposed to address these and other concerns. These tests are divided into two groups: Seal component tests and performance confirmation tests. The seal component tests are thorough small-scale in situ tests, the intermediate-scale borehole seal tests, the fracture grouting tests, the surface backfill tests, and the grouted rock mass tests. The seal system tests are the seepage control tests, the backfill tests, the bulkhead test in the Calico Hills unit, the large-scale shaft seal and shaft fill tests, and the remote borehole sealing tests. The tests are proposed to be performed in six discrete areas, including welded and non-welded environments, primarily located outside the potential repository area. The final selection of sealing tests will depend on the nature of the geologic and hydrologic conditions encountered during the development of the Exploratory Studies Facility and detailed numerical analyses. Tests are likely to be performed both before and after License Application.

  17. Theoretical investigation of structures and energetics of sodium adatom and its dimer on graphene: DFT study

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

    Extensive ab initio calculations have been performed to study the energetics of a sodium (Na) atom and its dimer adsorbed on graphene using the SIESTA package Soler et al. (2002) [1] which works within a DFT(density functional theory)-GGA (generalized gradient approximation) pseudopotential framework. The adsorption energy, geometry, charge transfer, ionization potential and density of states (DOS), partial density states (PDOS) of adatom/dimer-graphene system have been calculated. After considering various sites for adsorption of Na on graphene, the center of a hexagonal ring of carbon atoms is found to be the preferred site of adsorption while the Na2 dimer prefers to rest parallel to the graphene sheet. We find insignificant energy differences among adsorption configurations involving different possible sites in parallel orientation, which implies high mobility of the dimer on the graphene sheet. We also notice only a slight distortion of the graphene sheet perpendicular to its plane upon adatom adsorption. However, some lateral displacements seen are more perceptible. Summary The adsorption energy, geometry, charge transfer, ionization potential and density of states (DOS) and PDOS of adatom/dimer-graphene system have been calculated using SIESTA package Soler et al. (2002) [1] which works within a DFT(density functional theory)-GGA (generalized gradient approximation) pseudopotential framework. Preferred site for adsorption of a sodium atom on graphene is the hollow site. For the Na dimer adsorption, we found that horizontal orientation is favored over the vertical one. From DOS plots, it is clear that graphene's states are nearly unaffected by the adsorption of Na adatom and Interaction between sodium and graphene is predominantly ionic

  18. The influence of the ``hot''-dimer adsorption mechanism on the kinetics of a monomer-dimer surface reaction

    NASA Astrophysics Data System (ADS)

    Pereyra, V. D.; Albano, E. V.

    1993-09-01

    “Hot” dimers are molecules which after adsorption dissociate and each of the remaining “hot” monomers fly apart up to a maximum distance R from the original adsorption site. The influence of the “hot”-dimer adsorption mechanism on relevant aspects of the bimolecular catalyzed reaction of the type A - (1/2) B 2(“hot”) → AB is studied by means of the Monte-Carlo simulation technique. The temporal evolution of both the reactant's coverages as well as the rate of AB-production is evaluated and discussed. Due to the enhanced probability of “hot” species for encounters with other adsorbed particles, the rate of AB-production becomes faster when increasing R. This behavior may be relevant in the dynamic of some catalyzed reactions such as for example the oxidation of carbon monoxide on transition metal surfaces, i.e. A≡CO, B 2≡O2, and AB≡CO2. Also the sticking coefficient of “hot” dimers and the average distance traveled by the “hot” monomers are evaluated and discussed.

  19. Role of Human DNA Polymerase kappa in Extension Opposite from a cis-syn Thymine Dimer

    SciTech Connect

    R Vasquez-Del Carpio; T Silverstein; S Lone; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase {Kappa} (Pol{Kappa}), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Pol{Kappa} in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Pol{Kappa} in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.

  20. Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity*

    PubMed Central

    Lay, Fung T.; Mills, Grant D.; Poon, Ivan K. H.; Cowieson, Nathan P.; Kirby, Nigel; Baxter, Amy A.; van der Weerden, Nicole L.; Dogovski, Con; Perugini, Matthew A.; Anderson, Marilyn A.; Kvansakul, Marc; Hulett, Mark D.

    2012-01-01

    The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens. PMID:22511788

  1. Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth1[OPEN

    PubMed Central

    Bloch, Daria; Pleskot, Roman; Vukašinović, Nemanja

    2016-01-01

    Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. PMID:27516531

  2. Best Practices for Siting Solar Photovoltaics on Municipal Solid Waste Landfills. 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

    Kiatreungwattana, K.; Mosey, G.; Jones-Johnson, S.; Dufficy, C.; Bourg, J.; Conroy, A.; Keenan, M.; Michaud, W.; Brown, K.

    2013-04-01

    The Environmental Protection Agency and the National Renewable Energy Laboratory developed this best practices document to address common technical challenges for siting solar photovoltaics (PV) on municipal solid waste (MSW) landfills. The purpose of this document is to promote the use of MSW landfills for solar energy systems. Closed landfills and portions of active landfills with closed cells represent thousands of acres of property that may be suitable for siting solar photovoltaics (PV). These closed landfills may be suitable for near-term construction, making these sites strong candidate to take advantage of the 30% Federal Business Energy Investment Tax Credit. It was prepared in response to the increasing interest in siting renewable energy on landfills from solar developers; landfill owners; and federal, state, and local governments. It contains examples of solar PV projects on landfills and technical considerations and best practices that were gathered from examining the implementation of several of these projects.

  3. 21 CFR 176.120 - Alkyl ketene dimers.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Alkyl ketene dimers. 176.120 Section 176.120 Food... Use Only as Components of Paper and Paperboard § 176.120 Alkyl ketene dimers. Alkyl ketene dimers may... section. (a) The alkyl ketene dimers are manufactured by the dehydrohalogenation of the acyl...

  4. 21 CFR 176.120 - Alkyl ketene dimers.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Alkyl ketene dimers. 176.120 Section 176.120 Food... Paperboard § 176.120 Alkyl ketene dimers. Alkyl ketene dimers may be safely used as a component of articles..., transporting, or holding food, subject to the provisions of this section. (a) The alkyl ketene dimers...

  5. 21 CFR 176.120 - Alkyl ketene dimers.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Alkyl ketene dimers. 176.120 Section 176.120 Food... Use Only as Components of Paper and Paperboard § 176.120 Alkyl ketene dimers. Alkyl ketene dimers may... section. (a) The alkyl ketene dimers are manufactured by the dehydrohalogenation of the acyl...

  6. 21 CFR 176.120 - Alkyl ketene dimers.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Alkyl ketene dimers. 176.120 Section 176.120 Food... Use Only as Components of Paper and Paperboard § 176.120 Alkyl ketene dimers. Alkyl ketene dimers may... section. (a) The alkyl ketene dimers are manufactured by the dehydrohalogenation of the acyl...

  7. 21 CFR 176.120 - Alkyl ketene dimers.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Alkyl ketene dimers. 176.120 Section 176.120 Food... Use Only as Components of Paper and Paperboard § 176.120 Alkyl ketene dimers. Alkyl ketene dimers may... section. (a) The alkyl ketene dimers are manufactured by the dehydrohalogenation of the acyl...

  8. Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer*

    PubMed Central

    Poluri, Krishna Mohan; Joseph, Prem Raj B.; Sawant, Kirti V.; Rajarathnam, Krishna

    2013-01-01

    Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (Kd = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue. PMID:23864653

  9. Mechanism of Inducible Nitric-oxide Synthase Dimerization Inhibition by Novel Pyrimidine Imidazoles*

    PubMed Central

    Nagpal, Latika; Haque, Mohammad M.; Saha, Amit; Mukherjee, Nirmalya; Ghosh, Arnab; Ranu, Brindaban C.; Stuehr, Dennis J.; Panda, Koustubh

    2013-01-01

    Overproduction of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) has been etiologically linked to several inflammatory, immunological, and neurodegenerative diseases. As dimerization of NOS is required for its activity, several dimerization inhibitors, including pyrimidine imidazoles, are being evaluated for therapeutic inhibition of iNOS. However, the precise mechanism of their action is still unclear. Here, we examined the mechanism of iNOS inhibition by a pyrimidine imidazole core compound and its derivative (PID), having low cellular toxicity and high affinity for iNOS, using rapid stopped-flow kinetic, gel filtration, and spectrophotometric analysis. PID bound to iNOS heme to generate an irreversible PID-iNOS monomer complex that could not be converted to active dimers by tetrahydrobiopterin (H4B) and l-arginine (Arg). We utilized the iNOS oxygenase domain (iNOSoxy) and two monomeric mutants whose dimerization could be induced (K82AiNOSoxy) or not induced (D92AiNOSoxy) with H4B to elucidate the kinetics of PID binding to the iNOS monomer and dimer. We observed that the apparent PID affinity for the monomer was 11 times higher than the dimer. PID binding rate was also sensitive to H4B and Arg site occupancy. PID could also interact with nascent iNOS monomers in iNOS-synthesizing RAW cells, to prevent their post-translational dimerization, and it also caused irreversible monomerization of active iNOS dimers thereby accomplishing complete physiological inhibition of iNOS. Thus, our study establishes PID as a versatile iNOS inhibitor and therefore a potential in vivo tool for examining the causal role of iNOS in diseases associated with its overexpression as well as therapeutic control of such diseases. PMID:23696643

  10. An Initial Evaluation Of Characterization And Closure Options For Underground Pipelines Within A Hanford Site Single-Shell Tank Farm

    SciTech Connect

    Badden, Janet W.; Connelly, Michael P.; Seeley, Paul N.; Hendrickson, Michelle L.

    2013-01-10

    The Hanford Site includes 149 single-shell tanks, organized in 12 'tank farms,' with contents managed as high-level mixed waste. The Hanford Federal Facility Agreement and Consent Order requires that one tank farm, the Waste Management Area C, be closed by June 30, 2019. A challenge to this project is the disposition and closure of Waste Management Area C underground pipelines. Waste Management Area C contains nearly seven miles of pipelines and 200 separate pipe segments. The pipelines were taken out of service decades ago and contain unknown volumes and concentrations of tank waste residuals from past operations. To understand the scope of activities that may be required for these pipelines, an evaluation was performed. The purpose of the evaluation was to identify what, if any, characterization methods and/or closure actions may be implemented at Waste Management Area C for closure of Waste Management Area C by 2019. Physical and analytical data do not exist for Waste Management Area C pipeline waste residuals. To develop estimates of residual volumes and inventories of contamination, an extensive search of available information on pipelines was conducted. The search included evaluating historical operation and occurrence records, physical attributes, schematics and drawings, and contaminant inventories associated with the process history of plutonium separations facilities and waste separations and stabilization operations. Scoping analyses of impacts to human health and the environment using three separate methodologies were then developed based on the waste residual estimates. All analyses resulted in preliminary assessments, indicating that pipeline waste residuals presented a comparably low long-term impact to groundwater with respect to soil, tank and other ancillary equipment residuals, but exceeded Washington State cleanup requirement values. In addition to performing the impact analyses, the assessment evaluated available sampling technologies and

  11. Dimerization of a Viral SET Protein Endows its Function

    SciTech Connect

    H Wei; M Zhou

    2011-12-31

    Histone modifications are regarded as the most indispensible phenomena in epigenetics. Of these modifications, lysine methylation is of the greatest complexity and importance as site- and state-specific lysine methylation exerts a plethora of effects on chromatin structure and gene transcription. Notably, paramecium bursaria chlorella viruses encode a conserved SET domain methyltransferase, termed vSET, that functions to suppress host transcription by methylating histone H3 at lysine 27 (H3K27), a mark for eukaryotic gene silencing. Unlike mammalian lysine methyltransferases (KMTs), vSET functions only as a dimer, but the underlying mechanism has remained elusive. In this study, we demonstrate that dimeric vSET operates with negative cooperativity between the two active sites and engages in H3K27 methylation one site at a time. New atomic structures of vSET in the free form and a ternary complex with S-adenosyl homocysteine and a histone H3 peptide and biochemical analyses reveal the molecular origin for the negative cooperativity and explain the substrate specificity of H3K27 methyltransferases. Our study suggests a 'walking' mechanism, by which vSET acts all by itself to globally methylate host H3K27, which is accomplished by the mammalian EZH2 KMT only in the context of the Polycomb repressive complex.

  12. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Brisbane Baylands Brownfield Site in Brisbane, California. 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.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Brisbane Baylands site in Brisbane, California, 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.

  13. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Standard Chlorine of Delaware Superfund Site in Delaware City, Delaware. 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.

    2013-06-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Standard Chlorine of Delaware site in Delaware City, Delaware, 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.

  14. Feasibility Study of Economics and Performance of Solar Photovoltaics at the TechCity East Campus Resource Conservation and Recovery Act Site in Kingston, New York. 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. W.; Mosey, G.; Healey, V.

    2014-01-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the TechCity East Campus site in Kingston, New York, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) provided technical assistance for this project. The purpose of this study 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.

  15. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Snohomish County Cathcart Landfill Site in Snohomish County, Washington. 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

    Olis, D.; Salasovich, J.; Mosey, G.; Healey, V.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Snohomish County Cathcart Landfill Site in Snohomish County, Washington, 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.

  16. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Sky Park Landfill Site in Eau Claire, Wisconsin. 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

    Simon, J.; Mosey, G.

    2013-01-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Sky Park Landfill site in Eau Claire, Wisconsin, 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.

  17. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Former Bethlehem Steel Plant Brownfield Site in Lackawanna, New York. 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.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Former Bethlehem Steel Plant site in Lackawanna, New York, 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.

  18. Feasibility Study of Economics and Performance of Solar Photovoltaics at the VAG Mine Site in Eden and Lowell, Vermont. 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

    Simon, J.; Mosey, G.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Vermont Asbestos Group (VAG) Mine site in Eden, Vermont, and Lowell, Vermont, 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.

  19. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Former Fort Ord Army Base Site in Marina, California. 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

    Stoltenberg, B.; Konz, C.; Mosey, G.

    2013-05-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Former Fort Ord Army Base (FOAB) site in Marina, California, 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.

  20. Feasibility Study of Economics and Performance of Geothermal Power Generation at the Lakeview Uranium Mill Site in Lakeview, Oregon. 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

    Hillesheim, M.; Mosey, G.

    2013-11-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Lakeview Uranium Mill site in Lakeview, Oregon, for a feasibility study of renewable energy production. The EPA contracted with the National Renewable Energy Laboratory (NREL) to provide technical assistance for the project. The purpose of this report is to describe an assessment of the site for possible development of a geothermal power generation facility and to estimate the cost, performance, and site impacts for the facility. In addition, the report recommends development pathways that could assist in the implementation of a geothermal power system at the site.

  1. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Crazy Horse Landfill Site in Salinas, California. 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

    Stoltenberg, B.; Konz, C.; Mosey, G.

    2013-03-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Crazy Horse Landfill site in Salinas, California, for a feasibility study of renewable energy production. The National Renewable Energy Laboratory (NREL) was contacted to provide 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, operation and maintenance requirements, 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.

  2. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Kerr McGee Site in Columbus, Mississippi. 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

    Simon, J.; Mosey, G.

    2013-01-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Kerr McGee site in Columbus, Mississippi, 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.

  3. Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites

    SciTech Connect

    Rossi, M.; Anderson, C.; Demidov, O. N.; Appella, E.; Mazur, S. J.

    2008-12-01

    PPM1D (Wip1), a type PP2C phosphatase, is expressed at low levels in most normal tissues but is overexpressed in several types of cancers. In cells containing wild-type p53, the levels of PPM1D mRNA and protein increase following exposure to genotoxic stress, but the mechanism of regulation by p53 was unknown. PPM1D also has been identified as a CREB-regulated gene due to the presence of a cyclic AMP response element (CRE) in the promoter. Transient transfection and chromatin immunoprecipitation experiments in HCT116 cells were used to characterize a conserved p53 response element located in the 5' untranslated region (UTR) of the PPM1D gene that is required for the p53-dependent induction of transcription from the human PPM1D promoter. CREB binding to the CRE contributes to the regulation of basal expression of PPM1D and directs transcription initiation at upstream sites. Following exposure to ultraviolet (UV) or ionizing radiation, the abundance of transcripts with short 5' UTRs increased in cells containing wild-type p53, indicating increased utilization of downstream transcription initiation sites. In cells containing wild-type p53, exposure to UV resulted in increased PPM1D protein levels even when PPM1D mRNA levels remained constant, indicating post-transcriptional regulation of PPM1D protein levels.

  4. The nucleotide mapping of DNA double-strand breaks at the CYS3 initiation site of meiotic recombination in Saccharomyces cerevisiae.

    PubMed Central

    de Massy, B; Rocco, V; Nicolas, A

    1995-01-01

    Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae occurs by localized DNA double-strand breaks (DSBs) at several locations in the genome, corresponding to hot spots for meiotic gene conversion and crossing over. The meiotic DSBs occur in regions of chromatin that are hypersensitive to nucleases. To gain insight into the molecular mechanism involved in the formation of these DSBs, we have determined their positions at the nucleotide level at the CYS3 hot spot of gene conversion on chromosome I. We found four major new features of these DSBs: (i) sites of DSBs are multiple with varying intensities and spacing within the promoter region of the CYS3 gene; (ii) no consensus sequence can be found at these sites, indicating that the activity involved in DSB formation has little or no sequence specificity; (iii) the breaks are generated by blunt cleavages; and (iv) the 5' ends are modified in rad50S mutant strains, where the processing of these ends is known to be prevented. We present a model for the initiation of meiotic recombination taking into account the implications of these results. Images PMID:7556102

  5. RecFOR is not required for pneumococcal transformation but together with XerS for resolution of chromosome dimers frequently formed in the process.

    PubMed

    Johnston, Calum; Mortier-Barrière, Isabelle; Granadel, Chantal; Polard, Patrice; Martin, Bernard; Claverys, Jean-Pierre

    2015-01-01

    Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR

  6. RecFOR Is Not Required for Pneumococcal Transformation but Together with XerS for Resolution of Chromosome Dimers Frequently Formed in the Process

    PubMed Central

    Johnston, Calum; Mortier-Barrière, Isabelle; Granadel, Chantal; Polard, Patrice; Martin, Bernard; Claverys, Jean-Pierre

    2015-01-01

    Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA - cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR

  7. Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

    PubMed Central

    2010-01-01

    Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration. PMID:20687923

  8. Structure of a rabbit muscle fructose-1, 6-bisphosphate aldolase A dimer variant

    SciTech Connect

    Sherawat, Manashi; Tolan, Dean R.; Allen, Karen N.

    2008-05-01

    The X-ray crystallographic structure of a dimer variant of fructose-1, 6-bisphosphate aldolase demonstrates a stable oligomer that mirrors half of the native tetramer. The presence of product demonstrates that this is an active form. 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 Å 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.

  9. Effects of rotation on the nonlinear friction of a damped dimer sliding on a periodic substrate.

    PubMed

    Neide, I G; Kenkre, V M; Gonçalves, S

    2010-10-01

    Rotational effects on the nonlinear sliding friction of a damped dimer moving over a substrate are studied within a largely one-dimensional model. The model consists of two masses connected rigidly, internally damped, and sliding over a sinusoidal (substrate) potential while being free to rotate in the plane containing the masses and the direction of sliding. Numerical simulations of the dynamics performed by throwing the dimer with an initial center of mass velocity along the substrate direction show a richness of phenomena including the appearance of three separate regimes of motion. The orientation of the dimer performs tiny oscillations around values that are essentially constant in each regime. The constant orientations form an intricate pattern determined by the ratio of the dimer length to the substrate wavelength as well as by the initial orientations chosen. Corresponding evolution of the center of mass velocity consists, respectively, of regular oscillations in the first and the third regimes, but a power law decay in the second regime; the center of mass motion is effectively damped in this regime because of the coupling to the rotation. Depending on the initial orientation of the dimer, there is considerable variation in the overall behavior. For small initial angles to the vertical, an interesting formal connection can be established to earlier results known in the literature for a vibrating, rather than rotating, dimer. But for large angles, on which we focus in the present paper, quite different evolution occurs. Some of the numerical observations are explained successfully on the basis of approximate analytical arguments but others pose puzzling problems.

  10. Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

    PubMed Central

    Ruff, Karen M.

    2014-01-01

    The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform. PMID:25246650

  11. Dimerization and DNA recognition rules of mithramycin and its analogues.

    PubMed

    Weidenbach, Stevi; Hou, Caixia; Chen, Jhong-Min; Tsodikov, Oleg V; Rohr, Jürgen

    2016-03-01

    The antineoplastic and antibiotic natural product mithramycin (MTM) is used against cancer-related hypercalcemia and, experimentally, against Ewing sarcoma and lung cancers. MTM exerts its cytotoxic effect by binding DNA as a divalent metal ion (Me(2+))-coordinated dimer and disrupting the function of transcription factors. A precise molecular mechanism of action of MTM, needed to develop MTM analogues selective against desired transcription factors, is lacking. Although it is known that MTM binds G/C-rich DNA, the exact DNA recognition rules that would allow one to map MTM binding sites remain incompletely understood. Towards this goal, we quantitatively investigated dimerization of MTM and several of its analogues, MTM SDK (for Short side chain, DiKeto), MTM SA-Trp (for Short side chain and Acid), MTM SA-Ala, and a biosynthetic precursor premithramycin B (PreMTM B), and measured the binding affinities of these molecules to DNA oligomers of different sequences and structural forms at physiological salt concentrations. We show that MTM and its analogues form stable dimers even in the absence of DNA. All molecules, except for PreMTM B, can bind DNA with the following rank order of affinities (strong to weak): MTM=MTM SDK>MTM SA-Trp>MTM SA-Ala. An X(G/C)(G/C)X motif, where X is any base, is necessary and sufficient for MTM binding to DNA, without a strong dependence on DNA conformation. These recognition rules will aid in mapping MTM sites across different promoters towards development of MTM analogues as useful anticancer agents.

  12. Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: Insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family

    SciTech Connect

    Nath, Seema; Banerjee, Ramanuj; Sen, Udayaditya

    2014-07-18

    Highlights: • VcLMWPTP-1 forms dimer in solution. • The dimer is catalytically active unlike other reported dimeric LMWPTPs. • The formation of extended dimeric surface excludes the active site pocket. • The surface bears closer resemblance to eukaryotic LMWPTPs. - Abstract: Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization in a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45 Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.

  13. UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells

    SciTech Connect

    Protic-Sabljic, M.; Tuteja, N.; Munson, P.J.; Hauser, J.; Kraemer, K.H.; Dixon, K.

    1986-10-01

    We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.

  14. Effects of Cd{sup 2+} on cis-dimer structure of E-cadherin in living cells

    SciTech Connect

    Takeda, Hiroshi

    2014-02-21

    Highlights: • The effects of Cd on the dimer of cadherin in living cells was analyzed. • Cd induced cadherin dimer formation was not detected in living cell with low Ca. • Ca mediated structural cooperativity and allostery in the native cadherin. • Ca concentration-dependent competitive displacement of Cd from cadherin is proposed. - Abstract: E-cadherin, a calcium (Ca{sup 2+})-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca{sup 2+} plays an important role in the cis-dimer formation of cadherin. However, the molecular mechanisms by which Ca{sup 2+} interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated. As expected for a Ca{sup 2+} antagonist, cadmium (Cd{sup 2+}) disrupts cadherin function by displacing Ca{sup 2+} from its binding sites on the cadherin molecules. We used Cd{sup 2+} as a probe for investigating the role of Ca{sup 2+} in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd{sup 2+}, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd{sup 2+}-switch” experiments, we did not find that Cd{sup 2+}-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca{sup 2+} medium. In the present study, we demonstrated for the first time the effects of Cd{sup 2+} on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.

  15. Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts.

    PubMed

    Roza, L; Vermeulen, W; Bergen Henegouwen, J B; Eker, A P; Jaspers, N G; Lohman, P H; Hoeijmakers, J H

    1990-03-15

    UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, e.g., (6-4) photoproducts, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

  16. The Diamagnetic Susceptibility of the Tubulin Dimer

    PubMed Central

    Torbet, James; Diakun, Gregory P.; Rikken, Geert L. J. A.; Diaz, J. Fernando

    2014-01-01

    An approximate value of the diamagnetic anisotropy of the tubulin dimer, Δχdimer, has been determined assuming axial symmetry and that only the α-helices and β-sheets contribute to the anisotropy. Two approaches have been utilized: (a) using the value for the Δχα for an α-helical peptide bond given by Pauling (1979) and (b) using the previously determined anisotropy of fibrinogen as a calibration standard. The Δχdimer ≈ 4 × 10−27 JT−2 obtained from these measurements are similar to within 20%. Although Cotton-Mouton measurements alone cannot be used to estimate Δχ directly, the value we measured, CMdimer = (1.41 ± 0.03) × 10−8 T−2cm2mg−1, is consistent with the above estimate for Δχdimer. The method utilized for the determination of the tubulin dimer diamagnetic susceptibility is applicable to other proteins and macromolecular assemblies as well. PMID:24701206

  17. Linking in domain-swapped protein dimers

    PubMed Central

    Baiesi, Marco; Orlandini, Enzo; Trovato, Antonio; Seno, Flavio

    2016-01-01

    The presence of knots has been observed in a small fraction of single-domain proteins and related to their thermodynamic and kinetic properties. The exchanging of identical structural elements, typical of domain-swapped proteins, makes such dimers suitable candidates to validate the possibility that mutual entanglement between chains may play a similar role for protein complexes. We suggest that such entanglement is captured by the linking number. This represents, for two closed curves, the number of times that each curve winds around the other. We show that closing the curves is not necessary, as a novel parameter G′, termed Gaussian entanglement, is strongly correlated with the linking number. Based on 110 non redundant domain-swapped dimers, our analysis evidences a high fraction of chains with a significant intertwining, that is with |G′| > 1. We report that Nature promotes configurations with negative mutual entanglement and surprisingly, it seems to suppress intertwining in long protein dimers. Supported by numerical simulations of dimer dissociation, our results provide a novel topology-based classification of protein-swapped dimers together with some preliminary evidence of its impact on their physical and biological properties. PMID:27659606

  18. Isolation of novel single-chain Cro proteins targeted for binding to the bcl-2 transcription initiation site by repertoire selection and subunit combinatorics.

    PubMed

    Jonas, Kristina; Van Der Vries, Erhard; Nilsson, Mikael T I; Widersten, Mikael

    2005-11-01

    New designed DNA-binding proteins may be recruited to act as transcriptional regulators and could provide new therapeutic agents in the treatment of genetic disorders such as cancer. We have isolated tailored DNA-binding proteins selected for affinity to a region spanning the transcription initiation site of the human bcl-2 gene. The proteins were derived from a single-chain derivative of the lambda Cro protein (scCro), randomly mutated in its recognition helices to construct libraries of protein variants of distinct DNA-binding properties. By phage display-afforded affinity selections combined with recombination of shuffled subunits, protein variants were isolated, which displayed high affinity for the target bcl-2 sequence, as determined by electrophoretic mobility shift and biosensor assays. The proteins analyzed were moderately sequence-specific but provide a starting point for further maturation of desired function.

  19. Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct.

    PubMed

    Hunter, R H F; Gadea, J

    2014-08-01

    This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca(2+) ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm-sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.

  20. Exotic Protonated Species Produced by UV-Induced Photofragmentation of a Protonated Dimer: Metastable Protonated Cinchonidine.

    PubMed

    Alata, Ivan; Scuderi, Debora; Lepere, Valeria; Steinmetz, Vincent; Gobert, Fabrice; Thiao-Layel, Loïc; Le Barbu-Debus, Katia; Zehnacker-Rentien, Anne

    2015-10-01

    A metastable protonated cinchona alkaloid was produced in the gas phase by UV-induced photodissociation (UVPD) of its protonated dimer in a Paul ion trap. The infrared multiple photon dissociation (IRMPD) spectrum of the molecular ion formed by UVPD was obtained and compared to DFT calculations to characterize its structure. The protonation site obtained thereby is not accessible by classical protonation ways. The protonated monomer directly formed in the ESI source or by collision-induced dissociation (CID) of the dimer undergoes protonation at the most basic alkaloid nitrogen. In contrast, protonation occurs at the quinoline aromatic ring nitrogen in the UVPD-formed monomer. PMID:26347997

  1. Exotic Protonated Species Produced by UV-Induced Photofragmentation of a Protonated Dimer: Metastable Protonated Cinchonidine.

    PubMed

    Alata, Ivan; Scuderi, Debora; Lepere, Valeria; Steinmetz, Vincent; Gobert, Fabrice; Thiao-Layel, Loïc; Le Barbu-Debus, Katia; Zehnacker-Rentien, Anne

    2015-10-01

    A metastable protonated cinchona alkaloid was produced in the gas phase by UV-induced photodissociation (UVPD) of its protonated dimer in a Paul ion trap. The infrared multiple photon dissociation (IRMPD) spectrum of the molecular ion formed by UVPD was obtained and compared to DFT calculations to characterize its structure. The protonation site obtained thereby is not accessible by classical protonation ways. The protonated monomer directly formed in the ESI source or by collision-induced dissociation (CID) of the dimer undergoes protonation at the most basic alkaloid nitrogen. In contrast, protonation occurs at the quinoline aromatic ring nitrogen in the UVPD-formed monomer.

  2. Electromers of the benzene dimer radical cation.

    PubMed

    Błoch-Mechkour, Anna; Bally, Thomas

    2015-04-28

    The well-studied benzene dimer radical cation, which is prototypical for this class of species, has been reinvestigated computationally. Thereby it turned out that both the σ-hemibonded and the half-shifted sandwich structures of the benzene dimer cation, which had been independently proposed, represent stationary points on the B2PLYP-D potential energy surfaces. However, these structures belong to distinct electronic states, both of which are associated with potential surfaces that are very flat with regard to rotation of the two benzene rings in an opposite sense relative to each other. The surfaces of these two "electromers" of the benzene dimer cation are separated by only 3-4 kcal mol(-1) and do not intersect along the rotation coordinate, which represents a rather unique electronic structure situation. When moving on either of the two surfaces the title complex is an extremely fluxional species, in spite of its being bound by over 20 kcal mol(-1).

  3. Slab photonic crystals with dimer colloid bases

    SciTech Connect

    Riley, Erin K.; Liddell Watson, Chekesha M.

    2014-06-14

    The photonic band gap properties for centered rectangular monolayers of asymmetric dimers are reported. Colloids in suspension have been organized into the phase under confinement. The theoretical model is inspired by the range of asymmetric dimers synthesized via seeded emulsion polymerization and explores, in particular, the band structures as a function of degree of lobe symmetry and degree of lobe fusion. These parameters are varied incrementally from spheres to lobe-tangent dimers over morphologies yielding physically realizable particles. The work addresses the relative scarcity of theoretical studies on photonic crystal slabs with vertical variation that is consistent with colloidal self-assembly. Odd, even and polarization independent gaps in the guided modes are determined for direct slab structures. A wide range of lobe symmetry and degree of lobe fusion combinations having Brillouin zones with moderate to high isotropy support gaps between odd mode band indices 3-4 and even mode band indices 1-2 and 2-3.

  4. Theory of absorption bands in molecular dimers: Interpolating between optical asymmetries

    SciTech Connect

    Wagenknecht, Hans; Esser, Bernd

    2003-02-01

    Absorption band shapes of an asymmetric dimer system constituted by monomers with different excitation energies and optical transition matrix elements are considered in the semiclassical parameter region. Optical transition matrix elements originating from arbitrary initial vibrational states are analyzed on the basis of a spin representation of the eigenstates of an associated symmetry broken spin-boson Hamiltonian. Correlations between the spin-down and spin-up coefficients of these eigenstates are shown to exist and investigated in detail. Using these correlations, an asymmetry interpolation of the intensity of absorption lines between dimer configurations with different optical monomer transition matrix elements is proposed.

  5. Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface.

    PubMed

    Calgaro, Marcos R; Neto, Mario de Oliveira; Figueira, Ana Carolina M; Santos, Maria A M; Portugal, Rodrigo V; Guzzi, Carolina A; Saidemberg, Daniel M; Bleicher, Lucas; Vernal, Javier; Fernandez, Pablo; Terenzi, Hernán; Palma, Mario Sergio; Polikarpov, Igor

    2007-08-01

    The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.

  6. Structural plasticity in Ig superfamily domain 4 of ICAM-1 mediates cell surface dimerization

    PubMed Central

    Chen, Xuehui; Kim, Thomas Doohun; Carman, Christopher V.; Mi, Li-Zhi; Song, Gang; Springer, Timothy A.

    2007-01-01

    The Ig superfamily (IgSF) intercellular adhesion molecule-1 (ICAM-1) equilibrates between monomeric and dimeric forms on the cell surface, and dimerization enhances cell adhesion. A crystal structure of ICAM-1 IgSF domains (D) 3–5 revealed a unique dimerization interface in which D4s of two protomers fuse through edge β-strands to form a single super β-sandwich domain. Here, we describe a crystal structure at 2.7-Å resolution of monomeric ICAM-1 D3–D5, stabilized by the monomer-specific Fab CA7. CA7 binds to D5 in a region that is buried in the dimeric interface and is distal from the dimerization site in D4. In monomeric ICAM-1 D3–D5, a 16-residue loop in D4 that is disordered in the dimeric structure could clearly be traced as a BC loop, a short C strand, and a CE meander with a cis-Pro followed by a solvent-exposed, flexible four-residue region. Deletions of 6 or 10 residues showed that the C-strand is essential for monomer stability, whereas a distinct six-residue deletion showed little contribution of the CE meander. Mutation of two inward-pointing Leu residues in edge β-strand E to Lys increased monomer stability, confirming the hypothesis that inward-pointing charged side chains on edge β-strands are an important design feature to prevent β-supersheet formation. Overall, the studies reveal that monomer–dimer transition is associated with a surprisingly large, physiologically relevant, IgSF domain rearrangement. PMID:17881562

  7. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    SciTech Connect

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  8. A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11

    PubMed Central

    Shi, Yuejun; Li, Youwen; Lei, Yingying; Ye, Gang; Shen, Zhou; Sun, Limeng; Luo, Rui; Wang, Dang; Fu, Zhen F.; Xiao, Shaobo

    2016-01-01

    ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the “active site loop” (His129 to His144) and “supporting loop” (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales. PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and

  9. Gene V protein dimerization and cooperativity of binding of poly(dA).

    PubMed

    Terwilliger, T C

    1996-12-24

    Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence, C = [d log(Kint omega)]/[d log(NaCl)], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976) J. Mol. Biol. 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C. This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.

  10. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    PubMed

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology. PMID:24524625

  11. Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches.

    PubMed

    Canales, Angeles; Nieto, Lidia; Rodríguez-Salarichs, Javier; Sánchez-Murcia, Pedro A; Coderch, Claire; Cortés-Cabrera, Alvaro; Paterson, Ian; Carlomagno, Teresa; Gago, Federico; Andreu, José M; Altmann, Karl-Heinz; Jiménez-Barbero, Jesús; Díaz, J Fernando

    2014-04-18

    The binding of epothilones to dimeric tubulin and to microtubules has been studied by means of biochemical and NMR techniques. We have determined the binding constants of epothilone A (EpoA) and B (EpoB) to dimeric tubulin, which are 4 orders of magnitude lower than those for microtubules, and we have elucidated the conformation and binding epitopes of EpoA and EpoB when bound to tubulin dimers and microtubules in solution. The determined conformation of epothilones when bound to dimeric tubulin is similar to that found by X-ray crystallographic techniques for the binding of EpoA to the Tubulin/RB3/TTL complex; it is markedly different from that reported for EpoA bound to zinc-induced sheets obtained by electron crystallography. Likewise, only the X-ray structure of EpoA bound to the Tubulin/RB3/TTL complex at the luminal site, but not the electron crystallography structure, is compatible with the results obtained by STD on the binding epitope of EpoA bound to dimeric tubulin, thus confirming that the allosteric change (structuring of the M-loop) is the biochemical mechanism of induction of tubulin assembly by epothilones. TR-NOESY signals of EpoA bound to microtubules have been obtained, supporting the interaction with a transient binding site with a fast exchange rate (pore site), consistent with the notion that epothilones access the luminal site through the pore site, as has also been observed for taxanes. Finally, the differences in the tubulin binding affinities of a series of epothilone analogues has been quantitatively explained using the newly determined binding pose and the COMBINE methodology.

  12. Evaluation of the boron tolerant grass, Puccinellia distans, as an initial vegetative cover for the Phytorestoration of a boron-contaminated mining site in Southern California.

    PubMed

    Stiles, Amanda R; Liu, Chunguang; Kayama, Yuriko; Wong, Josephine; Doner, Harvey; Funston, Roger; Terry, Norman

    2011-10-15

    Land damaged by boron (B) mining should be restored to its natural state with a zero net impact on biodiversity. In an earlier study (Environ. Sci. Technol.2010,44, 7089-7095), we characterized a Turkish ecotype of the grass, Puccinellia distans, which exhibited extreme tolerance to B. Here we evaluated the use of a US ecotype of P. distans as an initial vegetative cover for the phytorestoration of a B mine in southern California. Hydroponic studies revealed that this P. distans ecotype tolerated B concentrations >100 mg B/L and could be germinated and grown in B-contaminated soils taken from the sites to be restored. P. distans grew well in moderately B-contaminated soil (∼88 mg B/L saturated extract) amended with added organic matter (peat moss); other soil treatments such as gypsum addition or pH correction were not needed. P. distans also grew in severely B-contaminated soil (∼1506 mg B/L) provided that toxic levels of soil B were diluted by the addition of sand and/or organic matter. Our results provide evidence in support of the concept of using the US ecotype of P. distans as an initial vegetative cover for the phytorestoration of B-contaminated soil. PMID:21882844

  13. Redefining the Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter and transcription initiation site in group I Burkitt lymphoma cell lines.

    PubMed Central

    Schaefer, B C; Strominger, J L; Speck, S H

    1995-01-01

    The Epstein-Barr virus-encoded nuclear antigen EBNA-1 gene promoter for the restricted Epstein-Barr virus (EBV) latency program operating in group I Burkitt lymphoma (BL) cell lines was previously identified incorrectly. Here we present evidence from RACE (rapid amplification of cDNA ends) cloning, reverse transcription-PCR, and S1 nuclease analyses, which demonstrates that the EBNA-1 gene promoter in group I BL cell lines is located in the viral BamHI Q fragment, immediately upstream of two low-affinity EBNA-1 binding sites. Transcripts initiated from this promoter, referred to as Qp, have the previously reported Q/U/K exon splicing pattern. Qp is active in group I BL cell lines but not in group III BL cell lines or in EBV immortalized B-lymphoblastoid cell lines. In addition, transient transfection of Qp-driven reporter constructs into both an EBV-negative BL cell line and a group I BL cell line gave rise to correctly initiated transcripts. Inspection of Qp revealed that it is a TATA-less promoter whose architecture is similar to the promoters of housekeeping genes, suggesting that Qp may be a default promoter which ensures EBNA-1 expression in cells that cannot run the full viral latency program. Elucidation of the genetic mechanism responsible for the EBNA-1-restricted program of EBV latency is an essential step in understanding control of viral latency in EBV-associated tumors. Images Fig. 1 Fig. 3 Fig. 4 PMID:7479841

  14. A Novel Allosteric Mechanism of NF-κB Dimerization and DNA Binding Targeted by an Anti-Inflammatory Drug

    PubMed Central

    Ashkenazi, Shaked; Plotnikov, Alexander; Bahat, Anat; Ben-Zeev, Efrat; Warszawski, Shira

    2016-01-01

    The NF-κB family plays key roles in immune and stress responses, and its deregulation contributes to several diseases. Therefore its modulation has become an important therapeutic target. Here, we used a high-throughput screen for small molecules that directly inhibit dimerization of the NF-κB protein p65. One of the identified inhibitors is withaferin A (WFA), a documented anticancer and anti-inflammatory compound. Computational modeling suggests that WFA contacts the dimerization interface on one subunit and surface residues E285 and Q287 on the other. Despite their locations far from the dimerization site, E285 and Q287 substitutions diminished both dimerization and the WFA effect. Further investigation revealed that their effects on dimerization are associated with their proximity to a conserved hydrophobic core domain (HCD) that is crucial for dimerization and DNA binding. Our findings established NF-κB dimerization as a drug target and uncovered an allosteric domain as a target of WFA action. PMID:26830231

  15. The dimeric transmembrane domain of prolyl dipeptidase DPP-IV contributes to its quaternary structure and enzymatic activities.

    PubMed

    Chung, Kuei-Min; Cheng, Jai-Hong; Suen, Ching-Shu; Huang, Chih-Hsiang; Tsai, Cheng-Han; Huang, Li-Hao; Chen, Yi-Rong; Wang, Andrew H-J; Jiaang, Weir-Torn; Hwang, Ming-Jing; Chen, Xin

    2010-09-01

    Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the extracellular active site. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM.

  16. Regulation of UVR8 photoreceptor dimer/monomer photo-equilibrium in Arabidopsis plants grown under photoperiodic conditions.

    PubMed

    Findlay, Kirsten M W; Jenkins, Gareth I

    2016-08-01

    The UV RESISTANCE LOCUS 8 (UVR8) photoreceptor specifically mediates photomorphogenic responses to UV-B. Photoreception induces dissociation of dimeric UVR8 into monomers to initiate responses. However, the regulation of dimer/monomer status in plants growing under photoperiodic conditions has not been examined. Here we show that UVR8 establishes a dimer/monomer photo-equilibrium in plants growing in diurnal photoperiods in both controlled environments and natural daylight. The photo-equilibrium is determined by the relative rates of photoreception and dark-reversion to the dimer. Experiments with mutants in REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2 show that these proteins are crucial in regulating the photo-equilibrium because they promote reversion to the dimer. In plants growing in daylight, the UVR8 photo-equilibrium is most strongly correlated with low ambient fluence rates of UV-B (up to 1.5 μmol m(-2) s(-1) ), rather than higher fluence rates or the amount of photosynthetically active radiation. In addition, the rate of reversion of monomer to dimer is reduced at lower temperatures, promoting an increase in the relative level of monomer at approximately 8-10 °C. Thus, UVR8 does not behave like a simple UV-B switch under photoperiodic growth conditions but establishes a dimer/monomer photo-equilibrium that is regulated by UV-B and also influenced by temperature.

  17. Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

    PubMed Central

    Ross, T K; Achberger, E C; Braymer, H D

    1989-01-01

    The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine. Images PMID:2649480

  18. Evaluation of D-dimer and lactate dehydrogenase plasma levels in patients with relapsed acute leukemia

    PubMed Central

    HU, WANGQIANG; WANG, XIAOXIA; YANG, RONGRONG

    2016-01-01

    Despite the outstanding advances made over the past decade regarding our knowledge of acute leukemia (AL), relapsed AL remains to be associated with a dismal prognosis. A better understanding of AL relapse and monitoring of the D-dimer and lactate dehydrogenase (LDH) plasma levels following chemotherapy may aid clinicians in determining whether relapse may occur in the subsequent phases of the disease. The present study evaluated D-dimer and LDH levels in 204 patients with relapsed AL. Data were collected at the initial onset of AL, at complete remission (CR) and in patients with relapsed AL. D-dimer plasma levels were significantly increased in patients with initial AL and in patients with relapsed AL (P=0.005 and P=0.007, respectively) but not in those with CR. LDH levels were significantly increased in AL patients at the initial onset of disease and at relapse compared with patients achieving CR, irrespective of cell type. Plasma prothrombin time, activated partial thromboplastin time and fibrinogen levels were not significantly different across patients (with the exception of acute promyelocytic leukemia patients) at the initial onset, relapsed AL or CR. Routine hematological parameters (white blood cell count, hemoglobin, platelet count) were significantly different at the initial onset of AL (P=0.002, P<0.001 and P=0.001, respectively) and during relapsed AL (P=0.009, P=0.003 and P<0.001, respectively) compared with patients achieving CR, suggesting an association between D-dimer, LDH and relapsed AL. These results also indicate that determination of D-dimer and LDH levels may be useful for predicting the probability of relapse during chemotherapy, but should also be combined with routine hematological parameters. PMID:27347185

  19. P1 plasmid replication: measurement of initiator protein concentration in vivo.

    PubMed Central

    Swack, J A; Pal, S K; Mason, R J; Abeles, A L; Chattoraj, D K

    1987-01-01

    To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable. Images PMID:3611028

  20. Dimer liquid state in the quantum dimer-pentamer model on the square lattice

    NASA Astrophysics Data System (ADS)

    Myers, Owen; Herdman, C. M.

    2015-03-01

    We study the ground state of the quantum dimer-pentamer model (QDPM) on the square lattice. This model is a generalization of the square lattice quantum dimer model (QDM) as its configuration space comprises fully-packed hard-core dimer coverings as well as configurations containing pentamers, where four dimers touch a vertex. Thus in the QDPM, the fully-packed, hard-core constraint of the QDM is relaxed such that the local dimer number at each vertex is fixed modulo 3; correspondingly, the local U (1) gauge symmetry of the QDM Hilbert space is reduced to a local Z3 gauge symmetry in the QDPM. We construct a local Hamiltonian for which the Rokhsar-Kivelson (RK) state (the equal superposition of all configurations in a topological sector) is the exact ground state and has a 9-fold topological degeneracy on the torus. Using Monte Carlo calculations, we find no spontaneous symmetry breaking in the RK wavefunction and that its dimer-dimer correlation function decays exponentially. Additionally, we discuss the possibility of Z3 topological order in the ground state of the QDPM.

  1. Rubidium dimer destruction by a diode laser

    SciTech Connect

    Ban, T.; Aumiler, D.; Pichler, G.

    2005-02-01

    We observed rubidium dimer destruction by excitation of rubidium vapor with diode laser light tuned across the Rb D{sub 2} resonance line in a 2400 GHz tuning interval. The destruction was measured for rubidium atom concentrations in the (1-9)x10{sup 16} cm{sup -3} range, pump beam power up to 43 mW, and with a 5 Torr of the helium buffer gas. We discuss the physical mechanisms involved and specify the molecular pathways which may effectively lead to the observed dimer destruction.

  2. Selective inhibition of c-Myc/Max dimerization and DNA binding by small molecules.

    PubMed

    Kiessling, Anke; Sperl, Bianca; Hollis, Angela; Eick, Dirk; Berg, Thorsten

    2006-07-01

    bZip and bHLHZip protein family members comprise a large fraction of eukaryotic transcription factors and need to bind DNA in order to exert most of their fundamental biological roles. Their binding to DNA requires homo- or heterodimerization via alpha-helical domains, which generally do not contain obvious binding sites for small molecules. We have identified two small molecules, dubbed Mycro1 and Mycro2, which inhibit the protein-protein interactions between the bHLHZip proteins c-Myc and Max. Mycros are the first inhibitors of c-Myc/Max dimerization, which have been demonstrated to inhibit DNA binding of c-Myc with preference over other dimeric transcription factors in vitro. Mycros inhibit c-Myc-dependent proliferation, gene transcription, and oncogenic transformation in the low micromolar concentration range. Our data support the idea that dimeric transcription factors can be druggable even in the absence of obvious small-molecule binding pockets.

  3. The Nature of Binding in the Phenalenyl Dimer and its Derivatives

    NASA Astrophysics Data System (ADS)

    Kolb, Brian; Thonhauser, Timo; Kertesz, Miklos

    2012-02-01

    The biradical phenalenyl (π)-dimer and its derivatives have attracted interest recently because of their potentially useful electrical, optical, and magnetic properties. These properties can be tuned by adjusting the binding characteristics between the monomers within the dimer. Typically, this is done by substituting electron withdrawing or donating groups onto the (α) or (β)-site carbons. An understanding of this binding lies at the heart of useful application of these materials. In this work, the binding characteristics of phenalenyl dimers were investigated using density functional theory. In particular, the vdW-DF functional was used to explore the role of van der Waals interactions in the binding within this system. A comparison of the binding curves with those of the closed shell derivatives wherein the central carbons have been replaced by either nitrogen or boron sheds light into the nature of the interactions between the monomers.

  4. Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum

    SciTech Connect

    Clark, J.M.; Deering, R.A.

    1987-02-01

    A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostellium discoideum. Analysis of the frequency of T4 endonuclease V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (rad/sup +/) removed >98% of the dimers induced by irradiation at 40 J/m/sup 2/ (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8% were removed during the 10- to 15- min period immediately after irradiation, whereas NC4 excised 64% during this interval. HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic acid at the sites of pyrimidine dimers.

  5. Molecular Design Principles Underlying beta-strand Swapping in the Adhesive Dimerization of Cadherins

    SciTech Connect

    J Vendome; S Posy; X Jin; F Bahna; G Ahlsen; L Shapiro; B Honig

    2011-12-31

    Cell adhesion by classical cadherins is mediated by dimerization of their EC1 domains through the 'swapping' of N-terminal {beta}-strands. We use molecular simulations, measurements of binding affinities and X-ray crystallography to provide a detailed picture of the structural and energetic factors that control the adhesive dimerization of cadherins. We show that strand swapping in EC1 is driven by conformational strain in cadherin monomers that arises from the anchoring of their short N-terminal strand at one end by the conserved Trp2 and at the other by ligation to Ca{sup 2+} ions. We also demonstrate that a conserved proline-proline motif functions to avoid the formation of an overly tight interface where affinity differences between different cadherins, crucial at the cellular level, are lost. We use these findings to design site-directed mutations that transform a monomeric EC2-EC3 domain cadherin construct into a strand-swapped dimer.

  6. HIV-1 Protease Dimerization Dynamics Reveals a Transient Druggable Binding Pocket at the Interface

    PubMed Central

    Pietrucci, Fabio; Vargiu, Attilio Vittorio; Kranjc, Agata

    2015-01-01

    The binding mechanism of HIV-1 protease monomers leading to the catalytically competent dimeric enzyme has been investigated by means of state-of-the-art atomistic simulations. The emerging picture allows a deeper understanding of experimental observations and reveals that water molecules trapped at the interface have an important role in slowing down the kinetics of the association process. Unexpectedly, a cryptic binding pocket is identified at the interface of the complex, corresponding to a partially bound dimer that lacks enzymatic function. The pocket has a transient nature with a lifetime longer than 1 μs, and it displays very favorable druggability features. Docking as well as MM-GBSA free-energy calculations further support the possibility to target the new binding site by means of inhibitors able to prevent the complete dimerization by capturing the inactive conformation. This discovery could open the way to the rational design of a new class of anti-HIV drugs. PMID:26692118

  7. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    SciTech Connect

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J.

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  8. Transition metal dimer on Au(111) surface: A first principle study

    SciTech Connect

    Sahoo, Suman Kalyan; Nigam, Sandeep; Sarkar, Pranab; Majumder, Chiranjib

    2012-06-05

    The adsorption behaviour of transition metal dimers M{sub 2} (M= Cu, Ag, Au) on the Au(111) surface have been studied using the density functional theory formalism. The projector augmented wave method under the spin polarized version of generalized gradient approximation scheme was employed to calculate the total energy. The results suggest that all dimers prefer to orient in parallel to the surface plane, where two M atoms are adsorbed on two nearby threefold fcc sites. We have investigated the chemical interaction between M atoms and Au surface through electronic density of state analysis. It is found that on deposition, the electronic density of states (EDOS) of M{sub 2} dimer becomes broader in comparison to their gas phase spectrum.

  9. HIV-1 Protease Dimerization Dynamics Reveals a Transient Druggable Binding Pocket at the Interface.

    PubMed

    Pietrucci, Fabio; Vargiu, Attilio Vittorio; Kranjc, Agata

    2015-01-01

    The binding mechanism of HIV-1 protease monomers leading to the catalytically competent dimeric enzyme has been investigated by means of state-of-the-art atomistic simulations. The emerging picture allows a deeper understanding of experimental observations and reveals that water molecules trapped at the interface have an important role in slowing down the kinetics of the association process. Unexpectedly, a cryptic binding pocket is identified at the interface of the complex, corresponding to a partially bound dimer that lacks enzymatic function. The pocket has a transient nature with a lifetime longer than 1 μs, and it displays very favorable druggability features. Docking as well as MM-GBSA free-energy calculations further support the possibility to target the new binding site by means of inhibitors able to prevent the complete dimerization by capturing the inactive conformation. This discovery could open the way to the rational design of a new class of anti-HIV drugs. PMID:26692118

  10. Dimerization Induced Deprotonation of Water on RuO2(110)

    SciTech Connect

    Mu, Rentao; Cantu Cantu, David; Lin, Xiao; Glezakou, Vassiliki Alexandra; Wang, Zhitao; Lyubinetsky, Igor; Rousseau, Roger J.; Dohnalek, Zdenek

    2014-10-02

    RuO2 has proven to be indispensable as a co-catalyst in numerous systems designed for photocatalytic water splitting. In this study we have carried out a detailed mechanistic study of water behavior on the most stable RuO2 face, RuO2(110), by employing variable temperature scanning tunneling microscopy and density functional theory calculations. We show that water monomers adsorb molecularly on Ru sites, become mobile above 238 K, diffuse along the Ru rows and form water dimers. The onset for dimer diffusion is observed at ~277 K indicating significantly higher diffusion barrier than that for monomers. More importantly, we find that water dimers deprotonate readily to form Ru-bound H3O2 and bridging OH species. The observed behavior is compared and contrasted with that observed for water on isostructural rutile TiO2(110).

  11. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  12. Transition metal dimer on Au(111) surface: A first principle study

    NASA Astrophysics Data System (ADS)

    Sahoo, Suman Kalyan; Nigam, Sandeep; Sarkar, Pranab; Majumder, Chiranjib

    2012-06-01

    The adsorption behaviour of transition metal dimers M2 (M= Cu, Ag, Au) on the Au(111) surface have been studied using the density functional theory formalism. The projector augmented wave method under the spin polarized version of generalized gradient approximation scheme was employed to calculate the total energy. The results suggest that all dimers prefer to orient in parallel to the surface plane, where two M atoms are adsorbed on two nearby threefold fcc sites. We have investigated the chemical interaction between M atoms and Au surface through electronic density of state analysis. It is found that on deposition, the electronic density of states (EDOS) of M2 dimer becomes broader in comparison to their gas phase spectrum.

  13. An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway.

    PubMed

    Garner, Thomas P; Reyna, Denis E; Priyadarshi, Amit; Chen, Hui-Chen; Li, Sheng; Wu, Yang; Ganesan, Yogesh Tengarai; Malashkevich, Vladimir N; Almo, Steve S; Cheng, Emily H; Gavathiotis, Evripidis

    2016-08-01

    Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition. PMID:27425408

  14. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

    PubMed Central

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

    2016-01-01

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

  15. Structural basis for the PufX-mediated dimerization of bacterial photosynthetic core complexes.

    PubMed

    Busselez, Johan; Cottevieille, Magali; Cuniasse, Philippe; Gubellini, Francesca; Boisset, Nicolas; Lévy, Daniel

    2007-12-01

    In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 A by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of approximately 300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center (RC). The LH1 consists of 15 resolved alpha/beta heterodimers and is interrupted. Within the opening, PufX polypeptide is assigned at a position facing the Q(B) site of the RC. This core complex is different from a dissociated dimer of the core complex of Rba. sphaeroides revealing that PufX in Rba. veldkampii is unable to dimerize. The absence in PufX of Rba. veldkampii of a G(31)XXXG(35) dimerization motif highlights the transmembrane interactions between PufX subunits involved in the dimerization of the core complexes of Rhodobacter species.

  16. Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

    PubMed Central

    Kabe, Yasuaki; Nakane, Takanori; Koike, Ikko; Yamamoto, Tatsuya; Sugiura, Yuki; Harada, Erisa; Sugase, Kenji; Shimamura, Tatsuro; Ohmura, Mitsuyo; Muraoka, Kazumi; Yamamoto, Ayumi; Uchida, Takeshi; Iwata, So; Yamaguchi, Yuki; Krayukhina, Elena; Noda, Masanori; Handa, Hiroshi; Ishimori, Koichiro; Uchiyama, Susumu; Kobayashi, Takuya; Suematsu, Makoto

    2016-01-01

    Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. PMID:26988023

  17. Distinct roles of Jun : Fos and Jun : ATF dimers in oncogenesis.

    PubMed

    van Dam, H; Castellazzi, M

    2001-04-30

    Jun : Fos and Jun : ATF complexes represent two classes of AP-1 dimers that (1) preferentially bind to either heptameric or octameric AP-1 binding sites, and (2) are differently regulated by cellular signaling pathways and oncogene products. To discriminate between the functions of Jun : Fos, Jun : ATF and Jun : Jun, mutants were developed that restrict the ability of Jun to dimerize either to itself, or to Fos(-like) or ATF(-like) partners. Introduction of these mutants in chicken embryo fibroblasts shows that Jun : Fra2 and Jun : ATF2 dimers play distinct, complementary roles in in vitro oncogenesis by inducing either anchorage independence or growth factor independence, respectively. v-Jun : ATF2 rather than v-Jun : Fra2 triggers the development of primary fibrosarcomas in the chicken wing. Genes encoding extracellular matrix components seem to constitute an important subset of v-Jun : ATF2-target genes. Repression of the matrix component SPARC by Jun is essential for the induction of fibrosarcomas. Avian primary cells transformed by either Jun : Fra2 or Jun : ATF2 thus provide powerful tools for the investigation of the downstream pathways involved in oncogenesis. Further genetic studies with Jun dimerization mutants will be required to be precise and extend the specific roles of the Jun : Fos and Jun : ATF dimers during cancer progression in avian and mammalian systems.

  18. Dimerization kinetics and products of. alpha. -substituted o-quinodimethanes derived from benzene and furan

    SciTech Connect

    Leung, Man-kit.

    1992-07-20

    Effects of the {alpha}-substitutions on the termini of the reactive diene unit of o-quinodimethanes revealed a non-concerted mechanism for furan-based and benzene-based o-quinodimethane (o-QDM) dimerizations. In section one, the coexistence of the cisoid and transoid transition states in the diradical formation step is evidenced by the stereochemistry of the dimers. In view of the results of the furan-based o-QDM dimerizauons, it is believed that the regioselectivity in the diradical cyclization step is controlled mainly by the interaction between the active sites on the furan moieties in the diradical ring closure step, not by the intemal bond rotations of the carbon chain of the diradical intermediate. In section two, it was found that the trend of the regioselectivity. along the size of the {alpha}-substituents, of benzene-based o-QDM dimerizations is opposite to that of the Diels-Alder reactions. On the basis of the trends, it is suggested that the Diels-Alder reaction mechanism of benzene-based o-QDM's is concerted while the dimerization mechanism of benzene-based o-QDM's is stepwise. Because of their similar activation parameters, it is proposed that the parent o-xylylene and other o-xylylenes dimerize via a similar two step, diradical mechanism.

  19. Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

    PubMed Central

    Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  20. Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

    PubMed

    Flausino, O A; Dufau, L; Regasini, L O; Petrônio, M S; Silva, D H S; Rose, T; Bolzani, V S; Reboud-Ravaux, M

    2012-01-01

    The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the β-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular β-sheets involved in protein-protein interactions.

  1. Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization.

    PubMed

    Charbel Issa, Peter; Barnard, Alun R; Herrmann, Philipp; Washington, Ilyas; MacLaren, Robert E

    2015-07-01

    Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.

  2. Single-Molecule Rotational Switch on a Dangling Bond Dimer Bearing.

    PubMed

    Godlewski, Szymon; Kawai, Hiroyo; Kolmer, Marek; Zuzak, Rafał; Echavarren, Antonio M; Joachim, Christian; Szymonski, Marek; Saeys, Mark

    2016-09-27

    One of the key challenges in the construction of atomic-scale circuits and molecular machines is to design molecular rotors and switches by controlling the linear or rotational movement of a molecule while preserving its intrinsic electronic properties. Here, we demonstrate both the continuous rotational switching and the controlled step-by-step single switching of a trinaphthylene molecule adsorbed on a dangling bond dimer created on a hydrogen-passivated Ge(001):H surface. The molecular switch is on-surface assembled when the covalent bonds between the molecule and the dangling bond dimer are controllably broken, and the molecule is attached to the dimer by long-range van der Waals interactions. In this configuration, the molecule retains its intrinsic electronic properties, as confirmed by combined scanning tunneling microscopy/spectroscopy (STM/STS) measurements, density functional theory calculations, and advanced STM image calculations. Continuous switching of the molecule is initiated by vibronic excitations when the electrons are tunneling through the lowest unoccupied molecular orbital state of the molecule. The switching path is a combination of a sliding and rotation motion over the dangling bond dimer pivot. By carefully selecting the STM conditions, control over discrete single switching events is also achieved. Combined with the ability to create dangling bond dimers with atomic precision, the controlled rotational molecular switch is expected to be a crucial building block for more complex surface atomic-scale devices. PMID:27504525

  3. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly.

    PubMed

    Bowden, Thomas A; Crispin, Max; Harvey, David J; Jones, E Yvonne; Stuart, David I

    2010-06-01

    Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

  4. Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

    PubMed Central

    Ghodke, Harshad; Wang, Hong; Hsieh, Ching L.; Woldemeskel, Selamawit; Watkins, Simon C.; Rapić-Otrin, Vesna; Van Houten, Bennett

    2014-01-01

    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. PMID:24760829

  5. Gold atom and dimer adsorbed on perfect and defective graphene and boron nitride monolayer: A first-principles study

    NASA Astrophysics Data System (ADS)

    Li, Guihua; Li, Feng; Wang, Xiaopeng; Zhao, Mingwen; Liu, Xiangdong

    2014-05-01

    Energetic and structural properties of gold atom (Au) and gold dimer (Au dimer) adsorbed on pristine and defective graphene (Gra) and boron nitride monolayer (BN) are investigated using density functional theory. Substitutional doping models in the neutral charge state are considered by replacing the C site in graphene with B or N atom impurities (Gra-CB and Gra-CN) or by doping the B or N sites in the BN sheet by a C atom (BN-BC and BN-NC). It is shown that while the binding of Au/Au-dimer to a pristine support is weak, stronger binding could be achieved by introducing a defect in the surface indicating that defects can trap metal atoms. It is found that Gra-CB and BN-NC support Au/Au-dimer well and BN-NC is more preferable from aspect of adsorption energy. Interaction between Au/Au-dimer and the BN-NC substrates is explained by assigning appropriate partial charge densities of the valence band maximum (VBM) and conduction band minimum (CBM) at the Г point and projected densities of states (PDOS). The results demonstrate that both pristine and defective BN surfaces can no longer be treated as inert supports for Au/Au-dimer.

  6. Ligand regulation of a constitutively dimeric EGF receptor

    NASA Astrophysics Data System (ADS)

    Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

    2015-06-01

    Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

  7. A p-quinodimethane-bridged porphyrin dimer.

    PubMed

    Zeng, Wangdong; Ishida, Masatoshi; Lee, Sangsu; Sung, Young Mo; Zeng, Zebing; Ni, Yong; Chi, Chunyan; Kim, Dongho; Wu, Jishan

    2013-12-01

    A p-quinodimethane (p-QDM)-bridged porphyrin dimer 1 has been prepared for the first time. An unexpected Michael addition reaction took place when we attempted to synthesize compound 1 by reaction of the cross-conjugated keto-linked porphyrin dimers 8a and 8b with alkynyl/aryl Grignard reagents. Alternatively, compound 1 could be successfully prepared by intramolecular Friedel-Crafts alkylation of the diol-linked porphyrin dimer 14 with concomitant oxidation in air. Compound 1 shows intense one-photon absorption (OPA, λ(max)=955 nm, ε=45400 M(-1) cm(-1)) and a large two-photon absorption (TPA) cross-section (σ((2))(max)=2080 GM at 1800 nm) in the near-infrared (NIR) region due to its extended π-conjugation and quinoidal character. It also exhibits a short singlet excited-state lifetime of 25 ps. The cyclic voltammogram of 1 displays multiple redox waves with a small electrochemical energy gap of 0.86 eV. The ground-state geometry, electronic structure, and optical properties of 1 have been further studied by density functional theory (DFT) calculations and compared with those of the keto-linked dimer 8b. This research has revealed that incorporation of a p-QDM unit into the porphyrin framework had a significant impact on its optical and electronic properties, leading to a novel NIR OPA and TPA chromophore.

  8. Adsorption of dimeric surfactants in lamellar silicates

    NASA Astrophysics Data System (ADS)

    Balcerzak, Mateusz; Pietralik, Zuzanna; Domka, Ludwik; Skrzypczak, Andrzej; Kozak, Maciej

    2015-12-01

    The adsorption of different types of cationic surfactants in lamellar silicates changes their surface character from hydrophilic to hydrophobic. This study was undertaken to obtain lamellar silicates modified by a series of novel dimeric (gemini) surfactants of different length alkyl chains and to characterise these organophilised materials. Synthetic sodium montmorillonite SOMASIF® ME 100 (M) and enriched bentonite of natural origin (Nanoclay - hydrophilic bentonite®) were organophilised with dimeric (gemini) surfactants (1,1‧-(1,4-butanediyl)bis(alkoxymethyl)imidazolium dichlorides). As a result of surfactant molecule adsorption in interlamellar space, the d-spacing (d001) increased from 0.97 nm (for the anhydrous structure) to 2.04 nm. A Fourier transform infrared spectroscopy (FTIR) analysis of the modified systems reveals bands assigned to the stretching vibrations of the CH2 and CH3 groups and the scissoring vibrations of the NH group from the structure of the dimeric surfactants. Thermogravimetric (TG) and derivative thermogravimetric (DTG) studies imply a four-stage process of surfactant decomposition. Scanning electron microscopy (SEM) images provide information on the influence of dimeric surfactant intercalation into the silicate structures. Particles of the modified systems show a tendency toward the formation of irregularly shaped agglomerates.

  9. Nonrandom behavior of amphiphilic dimers in solution

    SciTech Connect

    Wu, D.-W.; Aranovich, G. L.; Donohue, M. D.

    2000-08-22

    A simple lattice theory is developed for amphiphilic dimers. An analytical solution is derived by taking into account the most important configurations of nearest neighbors. Numerical calculations and Monte Carlo simulations were performed to compare with analytical solutions. (c) 2000 American Institute of Physics.

  10. Stability and Formation of Isobutylene Dimers.

    ERIC Educational Resources Information Center

    Goldsmith, Robert H.

    1983-01-01

    Isobutylene is an important bulk chemical for the petroleum industry. Dimerization and hydrogenation reactions produce the standard fuel octane rating comparison. This classic chemistry is often misrepresented in modern texts, however, and this paper attempts to correlate the physical organic principles that apply. (Author)

  11. Dimers on the 33 .42 lattice

    NASA Astrophysics Data System (ADS)

    Li, Shuli; Yan, Weigen

    2016-06-01

    In this work, we obtain explicit expression of the number of close-packed dimers (perfect matchings) of the 33 .42 lattice with cylindrical boundary condition. Particularly, we show that the entropy of 33 .42 lattice is the same for cylindrical and toroidal boundary conditions.

  12. Dimerization and the effectiveness of ICAM-1 in mediating LFA-1-dependent adhesion

    PubMed Central

    Jun, Chang-Duk; Shimaoka, Motomu; Carman, Christopher V.; Takagi, Junichi; Springer, Timothy A.

    2001-01-01

    Dimeric intercellular adhesion molecule-1 (ICAM-1) binds more efficiently to lymphocyte function-associated antigen-1 (LFA-1) than monomeric ICAM-1. However, it is unknown whether dimerization enhances binding simply by providing two ligand-binding sites and thereby increasing avidity, or whether it serves to generate a single “fully competent” LFA-1-binding surface. Domain 1 of ICAM-1 contains both the binding site for LFA-1, centered on residue E34, and a homodimerization interface. Whether the LFA-1-binding site extends across the homodimerization interface has not been tested. To address this question, we constructed four different heterodimeric soluble forms of ICAM-1 joined at the C terminus via an α-helical coiled coil (ACID-BASE). These heterodimeric ICAM-1 constructs include, (i) E34/E34 (two intact LFA-1-binding sites), (ii) E34/K34 (one disrupted LFA-1-binding site), (iii) E34/ΔD1–2 (one deleted LFA-1-binding site), and (iv) K34/K34 (two disrupted LFA-1-binding sites). Cells bearing activated LFA-1 bound similarly to surfaces coated with either E34/K34 or E34/ΔD1–2 and with an ≈2-fold reduction in efficiency compared with E34/E34, suggesting that D1 dimerization, which is precluded in E34/ΔD1-D2, is not necessary for optimal LFA-1 binding. Furthermore, BIAcore (BIAcore, Piscataway, NJ) affinity measurements revealed that soluble open LFA-1 I domain bound to immobilized soluble ICAM-1, E34/E34, E34/K34, and E34/ΔD1-D2 with nearly identical affinities. These studies demonstrate that a single ICAM-1 monomer, not dimeric ICAM-1, represents the complete, “fully competent” LFA-1-binding surface. PMID:11391003

  13. Pyrimidine dimers block simian virus 40 replication forks

    SciTech Connect

    Berger, C.A.; Edenberg, H.J.

    1986-10-01

    UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion. We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

  14. Dimers of mitochondrial ATP synthase form the permeability transition pore

    PubMed Central

    Giorgio, Valentina; von Stockum, Sophia; Antoniel, Manuela; Fabbro, Astrid; Fogolari, Federico; Forte, Michael; Glick, Gary D.; Petronilli, Valeria; Zoratti, Mario; Szabó, Ildikó; Lippe, Giovanna; Bernardi, Paolo

    2013-01-01

    Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the FOF1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca2+. Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca2+, addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg2+/ADP. These results indicate that the PTP forms from dimers of the ATP synthase. PMID:23530243

  15. Dimerization of visual pigments in vivo.

    PubMed

    Zhang, Tao; Cao, Li-Hui; Kumar, Sandeep; Enemchukwu, Nduka O; Zhang, Ning; Lambert, Alyssia; Zhao, Xuchen; Jones, Alex; Wang, Shixian; Dennis, Emily M; Fnu, Amrita; Ham, Sam; Rainier, Jon; Yau, King-Wai; Fu, Yingbin

    2016-08-01

    It is a deeply engrained notion that the visual pigment rhodopsin signals light as a monomer, even though many G protein-coupled receptors are now known to exist and function as dimers. Nonetheless, recent studies (albeit all in vitro) have suggested that rhodopsin and its chromophore-free apoprotein, R-opsin, may indeed exist as a homodimer in rod disk membranes. Given the overwhelmingly strong historical context, the crucial remaining question, therefore, is whether pigment dimerization truly exists naturally and what function this dimerization may serve. We addressed this question in vivo with a unique mouse line (S-opsin(+)Lrat(-/-)) expressing, transgenically, short-wavelength-sensitive cone opsin (S-opsin) in rods and also lacking chromophore to exploit the fact that cone opsins, but not R-opsin, require chromophore for proper folding and trafficking to the photoreceptor's outer segment. In R-opsin's absence, S-opsin in these transgenic rods without chromophore was mislocalized; in R-opsin's presence, however, S-opsin trafficked normally to the rod outer segment and produced functional S-pigment upon subsequent chromophore restoration. Introducing a competing R-opsin transmembrane helix H1 or helix H8 peptide, but not helix H4 or helix H5 peptide, into these transgenic rods caused mislocalization of R-opsin and S-opsin to the perinuclear endoplasmic reticulum. Importantly, a similar peptide-competition effect was observed even in WT rods. Our work provides convincing evidence for visual pigment dimerization in vivo under physiological conditions and for its role in pigment maturation and targeting. Our work raises new questions regarding a potential mechanistic role of dimerization in rhodopsin signaling. PMID:27462111

  16. Ultraviolet Spectrum And Chemical Reactivity Of CIO Dimer

    NASA Technical Reports Server (NTRS)

    Demore, William B.; Tschuikow-Roux, E.

    1992-01-01

    Report describes experimental study of ultraviolet spectrum and chemical reactivity of dimer of chlorine monoxide (CIO). Objectives are to measure absorption cross sections of dimer at near-ultraviolet wavelengths; determine whether asymmetrical isomer (CIOCIO) exists at temperatures relevant to Antarctic stratosphere; and test for certain chemical reactions of dimer. Important in photochemistry of Antarctic stratosphere.

  17. Structure analysis of archaeal AMP phosphorylase reveals two unique modes of dimerization.

    PubMed

    Nishitani, Yuichi; Aono, Riku; Nakamura, Akira; Sato, Takaaki; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2013-08-01

    AMP phosphorylase (AMPpase) catalyzes the initial reaction in a novel AMP metabolic pathway recently found in archaea, converting AMP and phosphate into adenine and ribose 1,5-bisphosphate. Gel-filtration chromatography revealed that AMPpase from Thermococcus kodakarensis (Tk-AMPpase) forms an exceptionally large macromolecular structure (>40-mers) in solution. To investigate its unique multimerization feature, we determined the first crystal structures of Tk-AMPpase, in the apo-form and in complex with substrates. Structures of two truncated forms of Tk-AMPpase (Tk-AMPpaseΔN84 and Tk-AMPpaseΔC10) clarified that this multimerization is achieved by two dimer interfaces within a single molecule: one by the central domain and the other by the C-terminal domain, which consists of an unexpected domain-swapping interaction. The N-terminal domain, characteristic of archaeal enzymes, is essential for enzymatic activity, participating in multimerization as well as domain closure of the active site upon substrate binding. Moreover, biochemical analysis demonstrated that the macromolecular assembly of Tk-AMPpase contributes to its high thermostability, essential for an enzyme from a hyperthermophile. Our findings unveil a unique archaeal nucleotide phosphorylase that is distinct in both function and structure from previously known members of the nucleoside phosphorylase II family.

  18. Theoretical study of defect formation during the initial stages of native-oxide growth on GaSb (001)

    SciTech Connect

    Bermudez, V. M.

    2014-04-07

    The formation of defects during the initial stages of native-oxide growth on the GaSb (001)-α(4 × 3) surface has been studied computationally using spin-unrestricted density functional theory. It is found that insertion into a Ga-Sb adatom dimer to form a peroxo Ga-O-O-Sb bridge is the most energetically favorable process with insertion into Ga-Sb back-bonds being somewhat less so. A Ga-O-O-Ga bridge between dimers is also favorable, but Sb-O-O-Sb bridges show little if any stability. In the course of analyzing molecular adsorption, a particularly reactive site has been identified that leads to O{sub 2} dissociation with little or no barrier. This process is initiated in the vicinity of an Sb-Sb dimer in the terminating layer and leads to sub-surface Ga and Sb defect sites (i.e., coordinatively unsaturated atoms) and to strained Ga-Sb bonds that may be susceptible to further O{sub 2} attack. However, the defects formed in these reactions do not produce states in the gap.

  19. Theoretical study of defect formation during the initial stages of native-oxide growth on GaSb (001)

    NASA Astrophysics Data System (ADS)

    Bermudez, V. M.

    2014-04-01

    The formation of defects during the initial stages of native-oxide growth on the GaSb (001)-α(4 × 3) surface has been studied computationally using spin-unrestricted density functional theory. It is found that insertion into a Ga-Sb adatom dimer to form a peroxo Ga-O-O-Sb bridge is the most energetically favorable process with insertion into Ga-Sb back-bonds being somewhat less so. A Ga-O-O-Ga bridge between dimers is also favorable, but Sb-O-O-Sb bridges show little if any stability. In the course of analyzing molecular adsorption, a particularly reactive site has been identified that leads to O2 dissociation with little or no barrier. This process is initiated in the vicinity of an Sb-Sb dimer in the terminating layer and leads to sub-surface Ga and Sb defect sites (i.e., coordinatively unsaturated atoms) and to strained Ga-Sb bonds that may be susceptible to further O2 attack. However, the defects formed in these reactions do not produce states in the gap.

  20. Suprafacial Orientation of the SCF[superscript Cdc4] Dimer Accommodates Multiple Geometries for Substrate Ubiquitination

    SciTech Connect

    Tang, Xiaojing; Orlicky, Stephen; Lin, Zhenyuan; Willems, Andrew; Neculai, Dante; Ceccarelli, Derek; Mercurio, Frank; Shilton, Brian H.; Sicheri, Frank; Tyers, Mike

    2008-07-15

    SCF ubiquitin ligases recruit substrates for degradation via F box protein adaptor subunits. WD40 repeat F box proteins, such as Cdc4 and {beta}-TrCP, contain a conserved dimerization motif called the D domain. Here, we report that the D domain protomers of yeast Cdc4 and human {beta}-TrCP form a superhelical homotypic dimer. Disruption of the D domain compromises the activity of yeast SCF{sup Cdc4} toward the CDK inhibitor Sic1 and other substrates. SCF{sup Cdc4} dimerization has little effect on the affinity for Sic1 but markedly stimulates ubiquitin conjugation. A model of the dimeric holo-SCF{sup Cdc4} complex based on small-angle X-ray scatter measurements reveals a suprafacial configuration, in which substrate-binding sites and E2 catalytic sites lie in the same plane with a separation of 64 {angstrom} within and 102 {angstrom} between each SCF monomer. This spatial variability may accommodate diverse acceptor lysine geometries in both substrates and the elongating ubiquitin chain and thereby increase catalytic efficiency.

  1. IL-4 and IL-13 induce SOCS-1 gene expression in A549 cells by three functional STAT6-binding motifs located upstream of the transcription initiation site.

    PubMed

    Hebenstreit, Daniel; Luft, Petra; Schmiedlechner, Angela; Regl, Gerhard; Frischauf, Anna-Maria; Aberger, Fritz; Duschl, Albert; Horejs-Hoeck, Jutta

    2003-12-01

    Proteins of the suppressors of cytokine signaling (SOCS) family have important functions as negative regulators of cytokine signaling. We show here that SOCS-1 expression can be induced in the human epithelial lung cell line A549 by IL-4 and IL-13. Analysis of reporter gene constructs under control of the SOCS-1 promoter provides evidence that IL-4- and IL-13-induced up-regulation is dependent on three IFN-gamma-activated sequence motifs of the sequence TTC(N)(4)GAA, which is known for binding STAT6. The three motifs are situated close to each other approximately 600 bp upstream of the transcriptional initiation site. When mutations were inserted into all three IFN-gamma-activated sequence motifs at the same time, IL-4-IL-13-induced luciferase activity was abrogated. With single and double mutants, promoter activity was diminished in comparison with the wild-type promoter. STAT6 is therefore required for IL-4-IL-13-dependent SOCS-1 expression in A549 cells, and the three identified binding motifs cooperate to induce maximal transcription. EMSAs conducted with nuclear extracts of IL-4- and IL-13-stimulated A549 cells showed that STAT6 was able to bind to each of the three binding motifs. Finally, cotransfection of a SOCS-1 expression vector inhibited activation of SOCS-1 promoter luciferase constructs. Thus, SOCS-1 is able to autoregulate its expression via a negative feedback loop.

  2. The blood-brain barrier in the cerebrum is the initial site for the Japanese encephalitis virus entering the central nervous system.

    PubMed

    Liu, Tsan-Hsiun; Liang, Li-Ching; Wang, Chien-Chih; Liu, Huei-Chung; Chen, Wei-June

    2008-11-01

    Japanese encephalitis (JE) virus is a member of the encephalitic flaviviruses and frequently causes neurological sequelae in a proportion of patients who survive the acute phase of the infection. In the present study, we molecularly identified viral infection in the brain of mice with rigidity of hindlimbs and/or abnormal gait, in which JE virus particles appeared within membrane-bound vacuoles of neurons throughout the central nervous system. Deformation of tight junctions (TJs) shown as dissociation of endothelial cells in capillaries, implying that the integrity of the blood-brain barrier (BBB) has been compromised by JE virus infection. BBB permeability evidently increased in the cerebrum, but not in the cerebellum, of JE virus-infected mice intravenously injected with the tracer of Evans blue dye. This suggests that the permeability of the BBB differentially changed in response to viral infection, leading to the entry of JE virions and/or putatively infected leukocytes from the periphery to the cerebrum as the initial site of infection in the central nervous system (CNS). Theoretically, the virus spread to the cerebellum soon after the cerebrum became infected.

  3. Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading.

    PubMed

    Lucon, Janice; Qazi, Shefah; Uchida, Masaki; Bedwell, Gregory J; LaFrance, Ben; Prevelige, Peter E; Douglas, Trevor

    2012-10-01

    Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

  4. A geographic analysis of chronically homeless adults before and after enrollment in a multi-site supported housing initiative: community characteristics and migration.

    PubMed

    Tsai, Jack; Mares, Alvin S; Rosenheck, Robert A

    2011-12-01

    The current study examined the community characteristics and migration of chronically homeless adults before and after entry into a multi-site supported housing initiative. A total of 394 participants were geocoded at baseline and 12-month follow up. Data from geographic information systems indicate that the median distance participants traveled from their last residence to their residence 1 year after program entry was 4.6 miles and 12% of participants traveled more than 100 miles. Participants moved into communities with higher population densities, larger proportions of Whites, and smaller proportions of Blacks following their entry into supported housing, but continued to live in communities with higher crime rates, lower education levels, and lower income levels then the state average. At 12 months, Black participants residing in communities with higher population densities and larger Black populations reported higher social support and lower subjective distress. This underscores the importance of considering client preferences in housing. Together, these findings suggest that supported housing programs may be successful in finding housing for homeless clients, but are not placing them in improved communities. Special attention may also be needed for some clients who travel long distances between residences.

  5. D-Dimer Levels Predict Myocardial Injury in ST-Segment Elevation Myocardial Infarction: A Cardiac Magnetic Resonance Imaging Study

    PubMed Central

    Song, Young Bin; Lima, Joao A. C.; Guallar, Eliseo; Choe, Yeon Hyeon; Hwang, Jin Kyung; Kim, Eun Kyoung; Yang, Jeong Hoon; Hahn, Joo-Yong; Choi, Seung-Hyuk; Lee, Sang-Chol; Lee, Sang Hoon; Gwon, Hyeon-Cheol

    2016-01-01

    Objectives Elevated D-dimer levels on admission predict prognosis in patients undergoing primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI), but the association of D-dimer levels with structural markers of myocardial injury in these patients is unknown. Methods We performed cardiac magnetic resonance (CMR) imaging in 208 patients treated with primary PCI for STEMI. CMR was performed a median of 3 days after the index procedure. Of the 208 patients studied, 75 patients had D-dimer levels above the normal range on admission (>0.5 μg/mL; high D-dimer group) while 133 had normal levels (≤0.5 μg/mL; low D-dimer group). The primary outcome was myocardial infarct size assessed by CMR. Secondary outcomes included area at risk (AAR), microvascular obstruction (MVO) area, and myocardial salvage index (MSI). Results In CMR analysis, myocardial infarct size was larger in the high D-dimer group than in the low D-dimer group (22.3% [16.2–30.5] versus 18.8% [10.7–26.7]; p = 0.02). Compared to the low D-dimer group, the high D-dimer group also had a larger AAR (38.1% [31.7–46.9] versus 35.8% [24.2–45.3]; p = 0.04) and a smaller MSI (37.7 [28.2–46.9] versus 47.1 [33.2–57.0]; p = 0.01). In multivariate analysis, high D-dimer levels were significantly associated with larger myocardial infarct (OR 2.59; 95% CI 1.37–4.87; p<0.01) and lower MSI (OR 2.62; 95% CI 1.44–4.78; p<0.01). Conclusions In STEMI patients undergoing primary PCI, high D-dimer levels on admission were associated with a larger myocardial infarct size, a greater extent of AAR, and lower MSI, as assessed by CMR data. Elevated initial D-dimer level may be a marker of advanced myocardial injury in patients treated with primary PCI for STEMI. PMID:27513758

  6. Biopolymer from microbial assisted in situ hydrolysis of triglycerides and dimerization of fatty acids.

    PubMed

    Kavitha, V; Radhakrishnan, N; Madhavacharyulu, E; Sailakshmi, G; Sekaran, G; Reddy, B S R; Rajkumar, G Suseela; Gnanamani, Arumugam

    2010-01-01

    The present study demonstrates biopolymer production by in situ bio-based dimerization of fatty acids by microorganism isolated from marine sediments. Microbial isolate grown in Zobell medium in the presence of triglycerides for the period of 24-240 h at 37 degrees C, hydrolyze the applied triglycerides and sequentially dimerized the hydrolyzed products and subsequently polymerized and transformed to a biopolymer having appreciable adhesive properties. Physical (nature, odour, stickyness and tensile strength), chemical (instrumentation) and biochemical (cell free broth) methods of analyses carried out provided the hypotheses involved in the formation of the product as well as the nature of the product formed. Results revealed, lipolytic enzymes released during initial period of growth and the biosurfactant production during later period, respectively, hydrolyze the applied triglycerides and initiate the dimerization and further accelerated when the incubation period extended. The existence and the non-existence of in situ hydrolysis of various triglycerides followed by dimerization and polymerization and the mechanism of transformation of triglycerides to biopolymer are discussed in detail.

  7. Differential lipid binding of vinculin isoforms promotes quasi-equivalent dimerization.

    PubMed

    Chinthalapudi, Krishna; Rangarajan, Erumbi S; Brown, David T; Izard, Tina

    2016-08-23

    The main cause of death globally remains debilitating heart conditions, such as dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), which are often due to mutations of specific components of adhesion complexes. Vinculin regulates these complexes and plays essential roles in intercalated discs that are necessary for muscle cell function and coordinated movement and in the development and function of the heart. Humans bearing familial or sporadic mutations in vinculin suffer from chronic, progressively debilitating DCM that ultimately leads to cardiac failure and death, whereas autosomal dominant mutations in vinculin can also provoke HCM, causing acute cardiac failure. The DCM/HCM-associated mutants of vinculin occur in the 68-residue insert unique to the muscle-specific, alternatively spliced isoform of vinculin, termed metavinculin (MV). Contrary to studies that suggested that phosphoinositol-4,5-bisphosphate (PIP2) only induces vinculin homodimers, which are asymmetric, we show that phospholipid binding results in a domain-swapped symmetric MV dimer via a quasi-equivalent interface compared with vinculin involving R975. Although one of the two PIP2 binding sites is preserved, the symmetric MV dimer that bridges two PIP2 molecules differs from the asymmetric vinculin dimer that bridges only one PIP2 Unlike vinculin, wild-type MV and the DCM/HCM-associated R975W mutant bind PIP2 in their inactive conformations, and R975W MV fails to dimerize. Mutating selective vinculin residues to their corresponding MV residues, or vice versa, switches the isoform's dimeric constellation and lipid binding site. Collectively, our data suggest that MV homodimerization modulates microfilament attachment at muscular adhesion sites and furthers our understanding of MV-mediated cardiac remodeling. PMID:27503891

  8. Characterizing the importance of the biotin carboxylase domain dimer for S. aureus pyruvate carboxylase catalysis

    PubMed Central

    Yu, Linda P. C.; Chou, Chi-Yuan; Choi, Philip H.; Tong, Liang

    2013-01-01

    Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO2 donor. Studies with E. coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations in the BC domain dimer interface of S. aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive. PMID:23286247

  9. Spectroscopically determined force field for water dimer: physically enhanced treatment of hydrogen bonding in molecular mechanics energy functions.

    PubMed

    Mannfors, Berit; Palmo, Kim; Krimm, Samuel

    2008-12-11

    Our ab initio transformed spectroscopically determined force field (SDFF) methodology emphasizes, in addition to accurate structure and energy performance, comparable prediction of vibrational properties in order to improve reproduction of interaction forces. It is now applied to the determination of a molecular mechanics (MM) force field for the water monomer and dimer as an initial step in developing a more physically based treatment of the hydrogen bonding that not only underlies condensed-phase water but also must be important in molecular-level protein-water interactions. Essential electrical components of the SDFF for monomer water are found to be the following: an off-plane charge distribution, this distribution consisting of four off-atom charge sites in traditional lone pair (LP) but also in inverted lone pair (ILP) positions; allowance for a diffuse size to these off-atom sites; and the incorporation of charge fluxes (i.e., the change in charge with change in internal coordinate). Parametrization of such an LP/ILP model together with the SDFF analytically transformed valence force field results in essentially exact agreement with ab initio (in this case MP2/6-31++G(d,p)) structure, electrical, and vibrational properties. Although we demonstrate that the properties of this monomer electrical model together with its van der Waals and polarization interactions are transferable to the dimer, this is not sufficient in reproducing comparable dimer properties, most notably the huge increase in infrared intensity of a donor OH stretch mode. This deficiency, which can be eliminated by a large dipole-derivative-determined change in the effective charge flux of the donor hydrogen-bonded OH bond, is not accounted for by the charge flux change in this bond due to the induction effects of the acceptor electric field alone, and can only be fully removed by an added bond flux associated with the extent of overlap of the wave functions of the two molecules. We show that

  10. Characterisation of the single copy trefoil peptides intestinal trefoil factor and pS2 and their ability to form covalent dimers.

    PubMed

    Chinery, R; Bates, P A; De, A; Freemont, P S

    1995-01-01

    A bacterial recombinant expression system was established to produce biologically active rat Intestinal Trefoil Factor (rITF). Characterisation of purified rITF shows that both monomers and dimers can be observed under reducing and non-reducing conditions, respectively. Site-directed mutagenesis studies show that Cys57 is necessary for rITF dimer formation. Samples of human gastrointestinal tissue following biopsy also demonstrated the presence of reducible human pS2 and ITF covalent dimers. Three-dimensional models for pS2 and ITF support the hypothesis that both pS2 and ITF can exist as disulphide-linked dimers in vivo and that any proposed function for these peptides must take dimer formation into account.

  11. Terminal Interface Conformations Modulate Dimer Stability Prior to Amino Terminal Autoprocessing of HIV-1 Protease

    SciTech Connect

    Agniswamy, Johnson; Sayer, Jane M.; Weber, Irene T.; Louis, John M.

    2012-04-17

    The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel {beta}-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S{sup -4}FNF{sup -1}) and interface residues (P{sup 1}QIT{sup 4}). A 180{sup o} rotation of the T{sup 4}-L{sup 5} peptide bond is accompanied by a new Q{sup 2}-L{sup 5} hydrogen bond and complete disengagement of PQIT from the {beta}-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14-20 C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 x 10{sup 4})-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal {beta}-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site-PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.

  12. Topology, Dimerization, and Stability of the Single-Span Membrane Protein CadC

    PubMed Central

    Lindner, Eric; White, Stephen H.

    2014-01-01

    Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel Signal Peptidase I (SPase I) cleavage assay, we show that CadC is a Type II single-span membrane protein (MP) with a cytoplasmic DNA binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ—a Type II MP involved in the maintenance of the rod shape of E. coli—replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the TM domain of the chimera with the glycophorin-A (GpA) TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM-helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the GpA domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal-peptidase assay, we inserted a SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occured with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations. PMID:24946151

  13. Antistaphylococcal activity of DNA-interactive pyrrolobenzodiazepine (PBD) dimers and PBD-biaryl conjugates

    PubMed Central

    Rahman, Khondaker M.; Rosado, Helena; Moreira, Joao B.; Feuerbaum, Eva-Anne; Fox, Keith R.; Stecher, Eva; Howard, Philip W.; Gregson, Stephen J.; James, Colin H.; de la Fuente, Maria; Waldron, Denise E.; Thurston, David E.; Taylor, Peter W.

    2012-01-01

    Objectives Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. Methods DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. Results Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. Conclusions PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates. PMID:22547662

  14. Potential Impacts of Leakage from Black Rock Reservoir on the Hanford Site Unconfined Aquifer: Initial Hypothetical Simulations of Flow and Contaminant Transport

    SciTech Connect

    Freedman, Vicky L.

    2007-03-09

    Initial scoping calculations of the unconfined aquifer at the Hanford Site were carried out for the U.S. Bureau of Reclamation (USBR) to investi¬gate the potential impacts on the Hanford unconfined aquifer that would result from leakage from the proposed Black Rock Reservoir to the west. Although impacts on groundwater flow and contaminant transport were quantified based on numerical simulation results, the investigation represented a quali¬tative assessment of the potential lateral recharge that could result in adverse effects on the aquifer. Because the magnitude of the potential leakage is unknown, hypothetical bounding calculations were performed. When a quantitative analysis of the magnitude of the potential recharge from Black Rock Reservoir is obtained, the hydrologic impacts analysis will be revisited. The analysis presented in this report represent initial bounding calculations. A maximum lateral recharge (i.e., upland flux) was determined in the first part of this study by executing steady-state flow simulations that raised the water table no higher than the elevation attained in the Central Plateau during the Hanford operational period. This metric was selected because it assumed a maximum remobilization of contaminants that existed under previous fully saturated conditions. Three steady-state flow fields were then used to analyze impacts to transient contaminant transport: a maximum recharge (27,000 acre-ft/yr), a no additional flux (365 acre-ft/yr), and an intermediate recharge case (16,000 acre-ft/yr). The transport behavior of four radionuclides was assessed for a 300 year simula¬tion period with the three flow fields. The four radionuclides are current contaminants of concern (COCs) in the Central Plateau and include tritium, iodine-129, technetium-99, and uranium-238. Transient flow and transport simulations were used to establish hypothetical concentration distributions in the subsurface. Using the simulated concentration distributions in 2005

  15. Initiation and growth of multiple-site damage in the riveted lap joint of a curved stiffened fuselage panel: An experimental and analytical study

    NASA Astrophysics Data System (ADS)

    Ahmed, Abubaker Ali

    As part of the structural integrity research of the National Aging Aircraft Research Program, a comprehensive study on multiple-site damage (MSD) initiation and growth in a pristine lap-joint fuselage panel has been conducted. The curved stiffened fuselage panel was tested at the Full-Scale Aircraft Structural Test Evaluation and Research (FASTER) facility located at the Federal Aviation Administration William J. Hughes Technical Center. A strain survey test was conducted to verify proper load application. The panel was then subjected to a fatigue test with constant-amplitude cyclic loading. The applied loading spectrum included underload marker cycles so that crack growth history could be reconstructed from post-test fractographic examinations. Crack formation and growth were monitored via nondestructive and high-magnification visual inspections. Strain gage measurements recorded during the strain survey tests indicated that the inner surface of the skin along the upper rivet row of the lap joint experienced high tensile stresses due to local bending. During the fatigue loading, cracks were detected by eddy-current inspections at multiple rivet holes along the upper rivet row. Through-thickness cracks were detected visually after about 80% of the fatigue life. Once MSD cracks from two adjacent rivet holes linked up, there was a quick deterioration in the structural integrity of the lap joint. The linkup resulted in a 2.87" (72.9-mm) lead fatigue crack that rapidly propagated across 12 rivet holes and crossed over into the next skin bay, at which stage the fatigue test was terminated. A post-fatigue residual strength test was then conducted by loading the panel quasi-statically up to final failure. The panel failed catastrophically when the crack extended instantaneously across three additional bays. Post-test fractographic examinations of the fracture surfaces in the lap joint of the fuselage panel were conducted to characterize subsurface crack initiation and

  16. Potential Impacts of Leakage from Black Rock Reservoir on the Hanford Site Unconfined Aquifer: Initial Hypothetical Simulations of Flow and Contaminant Transport

    SciTech Connect

    Freedman, Vicky L.

    2008-01-30

    Initial scoping calculations of the unconfined aquifer at the Hanford Site were carried out for the U.S. Bureau of Reclamation (USBR) to investigate the potential impacts on the Hanford unconfined aquifer that would result from leakage from the proposed Black Rock Reservoir to the west. Although impacts on groundwater flow and contaminant transport were quantified based on numerical simulation results, the investigation represented a qualitative assessment of the potential lateral recharge that could result in adverse effects on the aquifer. Because the magnitude of the potential leakage is unknown, hypothetical bounding calculations were performed. When a quantitative analysis of the magnitude of the potential recharge from Black Rock Reservoir is obtained, the hydrologic impacts analysis will be revisited. The analysis presented in this report represents initial bounding calculations. A maximum lateral recharge (i.e., upland flux) was determined in the first part of this study by executing steady-state flow simulations that raised the water table no higher than the elevation attained in the Central Plateau during the Hanford operational period. This metric was selected because it assumed a maximum remobilization of contaminants that existed under previous fully saturated conditions. Three steady-state flow fields were then used to analyze impacts to transient contaminant transport: a maximum recharge (27,000 acre-ft/yr), a no additional flux (365 acre-ft/yr), and an intermediate recharge case (16,000 acre-ft/yr). The transport behavior of four radionuclides was assessed for a 300 year simulation period with the three flow fields. The four radionuclides are tritium, iodine-129, technetium-99, and uranium-238. Transient flow and transport simulations were used to establish hypothetical concentration distributions in the subsurface. Using the simulated concentration distributions in 2005 as initial conditions for steady-state flow runs, simulations were executed to

  17. Dimeric Quaternary Structure of Human Laforin*♦

    PubMed Central

    Sankhala, Rajeshwer S.; Koksal, Adem C.; Ho, Lan; Nitschke, Felix; Minassian, Berge A.; Cingolani, Gino

    2015-01-01

    The phosphatase laforin removes phosphate groups from glycogen during biosynthetic activity. Loss-of-function mutations in the gene encoding laforin is the predominant cause of Lafora disease, a fatal form of progressive myoclonic epilepsy. Here, we used hybrid structural methods to determine the molecular architecture of human laforin. We found that laforin adopts a dimeric quaternary structure, topologically similar to the prototypical dual specificity phosphatase VH1. The interface between the laforin carbohydrate-binding module and the dual specificity phosphatase domain generates an intimate substrate-binding crevice that allows for recognition and dephosphorylation of phosphomonoesters of glucose. We identify novel molecular determinants in the laforin active site that help decipher the mechanism of glucan phosphatase activity. PMID:25538239

  18. Fibrillar dimer formation of islet amyloid polypeptides

    SciTech Connect

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  19. Fibrillar dimer formation of islet amyloid polypeptides

    NASA Astrophysics Data System (ADS)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  20. Theoretical studies of transition metal dimers

    NASA Technical Reports Server (NTRS)

    Walch, Stephen P.; Bauschlicher, Charles W., Jr.

    1985-01-01

    The CASSCF approach was used to perform the MCSCF calculations for a number of transition metal dimers, including the Sc2, Ti2, Cr2, Cu2, TiV, Y2, Nb2, and Mo2 molecules; in addition, CASSCF/CI calculations were carried out for Sc2, Ti2, Cu2, and Y2. The CASSCF procedure is shown to provide a consistent set of calculations for these molecules, from which trends and a simple qualitative picture of the electronic structure may be derived. In particular, the calculations confirmed the ground states of the Sc2 and the TiV, and led to predictions for other molecules in this series. In addition to specific predictions, the study provides a simple qualitative picture of the bonding in these dimers.

  1. Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

    PubMed Central

    Trzeciecka, Anna; Klossowski, Szymon; Bajor, Malgorzata; Zagozdzon, Radoslaw; Gaj, Pawel; Muchowicz, Angelika; Malinowska, Agata; Czerwoniec, Anna; Barankiewicz, Joanna; Domagala, Antoni; Chlebowska, Justyna; Prochorec-Sobieszek, Monika; Winiarska, Magdalena; Ostaszewski, Ryszard; Gwizdalska, Iwonna; Golab, Jakub; Nowis, Dominika; Firczuk, Malgorzata

    2016-01-01

    Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease. PMID:26636537

  2. Geochemistry of Volcanic Rocks from International Ocean Discovery Program (IODP) Site 1438, Amami Sankaku Basin: Implications for Izu-Bonin-Mariana (IBM) Arc Initiation

    NASA Astrophysics Data System (ADS)

    Hickey-Vargas, R.; Ishizuka, O.; Yogodzinski, G. M.; Bizimis, M.; Savov, I. P.; McCarthy, A. J.; Arculus, R. J.; Bogus, K.

    2015-12-01

    IODP Expedition 351 drilled 150 m of volcanic basement overlain by 1461 m of sedimentary material at Site 1438 in the Amami Sankaku basin, just west of the Kyushu Palau Ridge, the locus of IBM arc initiation. Age interpretations based on biostratigraphy (Arculus et al., Nat. Geosci., in-press) determined that the age of the basement section is between 64 and 51 Ma, encompassing the age of the earliest volcanic products of the IBM arc. The Site 1438 volcanic basement consists of multiple flows of aphyric microcrystalline to finely crystalline basalts containing plagioclase and clinopyroxene with rare olivine pseudomorphs. New XRF major and ICPMS trace element data confirm findings of shipboard analysis that the basalts are moderately differentiated (6-14 % MgO; Mg# = 51-83; 73-490 ppm Cr and 58-350 ppm Ni) with downcore variations related to flow units. Ti/V and Ti/Sc ratios are 16-27 and 75-152, respectively, with lowest values at the base of the core. One prominent characteristic of the basalts is their depletion of immobile highly incompatible elements compared with MORB. Basalts have MORB-normalized La/Nd of 0.5 to 0.9, and most have Th/La < 0.05. Although all basalts are LREE-depleted, La/Nd ratios increase slightly upcore, and Th enrichment compared with LREE occurs in the uppermost 5 meters. Cs, Rb, K, Ba and U are concomitantly enriched relative to LREE in several intervals as a probable result of seawater alteration, but ratios less than those of MORB are found in other areas. In contrast to basement, andesites from three sills in the lowermost sedimentary unit have arc-like trace element patterns with La/Nb > 3 and primitive mantle normalized La/Yb > 1. Our results suggest that mantle melting at the onset of subduction involved exceptionally depleted sources. Enrichment over time may be related to increasing subduction inputs and/or other processes, such as entrainment of fertile asthenosphere during extension of the overriding plate.

  3. Structural modeling of the catalytic subunit-regulatory subunit dimeric complex of the camp-dependent protein kinase.

    SciTech Connect

    Tung, C-S; Gallagher, S. C.; Walsh, D. A.; Trewhella, J.

    2001-01-01

    The cAMP-dependent protein kinase (PKA) is a multifunctional kinase that serves as a prototype for understanding second messenger signaling and protein phosphorylation. In the absence of a cAMP signal, PKA exists as a dimer of dimers, consisting of two regulatory (R) and two catalystic (C) subunits. Based on experimentally derived data (i.e., crystal structures of the R and C subunits, mutagenesis data identifying points of subunit-subunit contacts), the neutron scattering derived model for the heterodimer (Zhao et al., 1998) and using a set of computational approaches (homology modeling, Monte Carlo simulation), they have developed a high-resolution model of the RII{alpha}-C{alpha} dimer. The nature of the subunit-subunit interface was studied. The model reveals an averaged size dimer interface (2100 Angstrom{sup 2}) that is distant from the pseudo-substrate binding site on the C subunit. The additional contacts made by the pseudosubstrate increases the stability of the dimeric complex. Based on a set of R-C dimer structures derived using a simulated annealing approach, specific interactions (hydrogen bonds) between the two subunits and were identified.

  4. Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor.

    PubMed Central

    Alvarez, E; Gironès, N; Davis, R J

    1989-01-01

    The human transferrin receptor is expressed as a disulfide-linked dimer at the cell surface. The sites of intermolecular disulfide bonds are Cys-89 and Cys-98. We have examined the functional significance of the covalent dimeric structure of the transferrin receptor by substitution of Cys-89 and Cys-98 with serine residues. Wild-type and mutated transferrin receptors were expressed in Chinese hamster ovary cells (clone TF-) that lack detectable endogenous transferrin receptors. The rates of receptor endocytosis and recycling were measured and the accumulation of iron by cells incubated with [59Fe]diferric transferrin was investigated. No significant differences between these rates were observed when cells expressing wild-type and mutated receptors were compared. The structure of the mutant receptor lacking intermolecular disulfide bonds was investigated. The presence of a population of mutant receptors with a non-covalent dimeric structure was indicated by cross-linking studies using diferric [125I]transferrin and the bifunctional reagent disuccinimidyl suberimidate. However, sucrose density gradient sedimentation analysis of Triton X-100 solubilized transferrin receptors demonstrated that the mutant receptor existed as a monomer in the absence of diferric transferrin and as an apparent dimer in the presence of this receptor ligand. We conclude that the covalent dimeric structure of the transferrin receptor is not required for the expression of the dimeric state and functional activity of the receptor. Images PMID:2507316

  5. A Novel Dimer Interface and Conformational Changes Revealed by an X-ray Structure of B. subtilis SecA

    SciTech Connect

    Zimmer,J.; Li, W.; Rapoport, T.

    2006-01-01

    The SecA ATPase moves polypeptides post-translationally across the plasma membrane of eubacteria, but the mechanism of transport is still unclear. We describe the crystal structure of a novel dimeric form of Bacillus subtilis SecA. Dimerization of SecA occurs at the prominent groove formed by the nucleotide binding domain 2 (nbd2) and the preprotein cross-linking (ppx) domain. The dimer interface is very large, burying approximately 5400 {angstrom}{sup 2} of solvent accessible surface per monomer. Single cysteine disulfide cross-linking shows the presence of this novel SecA dimer in solution. In addition, other dimers also exist in solution, arguing that they all are in equilibrium with monomeric SecA and supporting the idea that the monomer may be the functional species. Dimerization of SecA causes an {alpha}-helix of one subunit to convert to a short {beta}-strand that participates in {beta}-sheet formation with strands in the other subunit. This conversion of secondary structure elements occurs close to the connection between the nbd1 and ppx domains, a potential site of interaction with translocation substrate. Comparing the different X-ray structures of B. subtilis SecA suggests that small changes in the nucleotide binding domains could be amplified via helix 1 of the helical scaffold domain (hsd) to generate larger movements of the domains involved in polypeptide binding.

  6. Intermolecular dimerization with pillared layered clay templates.

    SciTech Connect

    Wiederrecht, G. P.; Sandi, G.; Carrado, K. A.; Seifert, S.; Chemistry

    2001-11-19

    Solutions of pyrene in the presence of a pillared, layered montmorillonite clay produce hybrid organic-inorganic materials with substantial molecular loading in the gallery regions between the clay layers. The results are in sharp contrast to other aromatics, such as benzene, naphthalene, or perylene, which show minimal incorporation of the molecules into the gallery regions of the clay. We present evidence that the unusual affinity for pyrene to form intermolecular dimers is the reason for the high loading. Pyrene monomers are easily introduced to the layers. Through steric hindrance, subsequent intermolecular dimer formation is allowed, and they are captured by the pillared, layered structure. CW and time-resolved emission spectra strongly indicate the presence of face-to-face intermolecular dimers (excimers) within the clay galleries. The combination of the ease of high molecular loading into an inorganic, high aspect ratio template and the collective optical properties of the organic layer may be useful as a new means to create hybrid structures.

  7. Multiphoton ionization spectroscopy of the sodium dimer

    NASA Astrophysics Data System (ADS)

    Keller, John; Weiner, John

    1984-07-01

    We report an investigation of the role of molecular multiphoton ionization in the production of Na+2 when sodium vapor is subjected to intense optical radiation. Previous authors attribute the source of much of the ion dimer signal to laser-induced associative ionization of atom sodium. In this experiment, we distinguish the molecular process from atomic collisional mechanisms by producing an intense molecular beam created through free-jet expansion of the metal vapor. The beam of nearly 50% dimers cooled to their low rotational and vibrational states allow us to obtain a simplified three-photon ionization spectrum. We find that the spectrum displays two-photon resonances corresponding to known Rydberg level transitions and that the A state, acting as virtual intermediate, plays a crucial role in the large peak-to-peak intensity variations. We employ a simple model of multiphoton ionization which uses a rate-equation approach to generate a calculated spectrum. Based on the experimental results and the success of the model in reflecting them, we conclude that much of the highly structured component of the dimer ion signal reported previously under different experimental conditions is probably due to molecular multiphoton ionization but that this structure rides on a slowly varying broad signal envelope due to laser-induced associative ionization.

  8. Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site

    PubMed Central

    Pearse, Ian; Zhu, Ying X.; Murray, Eleanor J.; Dudeja, Pradeep K.; Ramaswamy, Krishnamurthy; Malakooti, Jaleh

    2007-01-01

    We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the −40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, −10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5′ flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at −25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5′-UTR (5′-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5′-UTR may co-operate with the 5′-flanking region to achieve the optimal promoter activity. PMID:17561809

  9. Overexpression of eukaryotic initiation factor 5 rescues the translational defect of tpk1w in a manner that necessitates a novel phosphorylation site.

    PubMed

    Bavli-Kertselli, Ira; Melamed, Daniel; Bar-Ziv, Lavi; Volf, Hila; Arava, Yoav

    2015-02-01

    Cells respond to changes in their environment through mechanisms that often necessitate reprogramming of the translation machinery. The fastest and strongest of all tested responses is the translation inhibition observed following abrupt depletion of glucose from the media of yeast cells. The speed of the response suggests a post-translational modification of a key component of the translation machinery. This translation factor is as yet unknown. A cAMP-dependent protein kinase mutant yeast strain (tpk1(w)) that does not respond properly to glucose depletion and maintains translation was described previously. We hypothesized that the inability of tpk1(w) to arrest translation results from abnormal expression of key translation mediators. Genome-wide analysis of steady-state mRNA levels in tpk1(w) revealed underexpression of several candidates. Elevating the cellular levels of eukaryotic initiation factor (eIF) 5 by overexpression rescued the translational defect of tpk1(w). Restoring ribosomal dissociation by eIF5 necessitated an active GAP domain and multiple regions throughout this protein. Phosphoproteomics analysis of wild-type cells overexpressing eIF5 revealed increased phosphorylation in a novel site (Thr191) upon glucose depletion. Mutating this residue and introducing it into tpk1(w) abolished the ability of eIF5 to rescue the translational defect. Intriguingly, introducing this mutation into the wild-type strain did not hamper its translational response. We further show that Thr191 is phosphorylated in vitro by Casein Kinase II (CKII), and yeast cells with a mutated CKII have a reduced response to glucose depletion. These results implicate phosphorylation of eIF5 at Thr191 by CKII as one of the pathways for regulating translation upon glucose depletion.

  10. Prospective study of plasma D-dimer and incident venous thromboembolism: the Atherosclerosis Risk in Communities (ARIC) Study

    PubMed Central

    Folsom, Aaron R.; Alonso, Alvaro; George, Kristen M.; Roetker, Nicholas S.; Tang, Weihong; Cushman, Mary

    2015-01-01

    Introduction Plasma D-dimer is a useful clinical test for acute venous thromboembolism (VTE), and concentrations remain higher in VTE patients after treatment than in controls. Yet, evidence is limited on whether higher basal D-dimer concentrations in the general population are associated with greater risk of first VTE. Objective To assess the prospective association between D-dimer and incident VTE over a long follow-up. Methods We measured plasma D-dimer in 12,097 participants, initially free of VTE, in the Atherosclerosis Risk in Communities Study. Over a median follow-up of 17 years, we identified 521 VTEs. We calculated hazard ratios of VTE using proportional hazards regression. Results The age, race, and sex adjusted hazard ratios of VTE across quintiles of D-dimer were 1, 1.5, 1.8, 2.1, and 3.2 (p for trend <0.0001). For the first 10 years of follow-up, the hazard ratio for the highest versus lowest quintile was 3.5, and was 2.9 after 10 years. In both whites and African Americans, VTE risk remained strongly associated with D-dimer after further adjustment for diabetes, body mass index, kidney function, and several thrombophilia genetic markers. D-dimer was associated with both unprovoked and provoked VTE, but more strongly with unprovoked. Conclusions A higher basal level of plasma D-dimer in the general population, presumably reflecting a predisposition to thrombosis, is a strong, long-term risk factor for a first VTE. PMID:26337932

  11. The time scale for electronic reorganization upon sudden ionization of the water and water-methanol hydrogen bonded dimers and of the weakly bound NO dimer

    NASA Astrophysics Data System (ADS)

    Remacle, F.; Levine, R. D.

    2006-10-01

    When the valence molecular orbital is localized sudden ionization can cause the nascent hole to move rapidly even before any relaxation of the geometry occurs. Hydrogen bonded clusters offer suitable test systems where the hole is initially localized on one moiety. Computational studies are reported for the water dimer and water-methanol bimer. The local ionization potential of water is different in the methanol-water and water-methanol conformers and this difference is very clearly reflected in the dynamics of charge migration. For the NO dimer the results are that its structure is symmetric so that the two NO molecules are equivalent and do not exhibit the required localization. The role of symmetry is also evident in the charge propagation for holes created in different orbitals. Localization of the initial hole distribution even if absent in the bare molecule can still be induced by the intense electric field of a sudden photoionization. This effect is computationally studied for the NO dimer in the presence of a static electric field.

  12. Properties and metathesis activity of monomeric and dimeric Mo centres variously located on γ-alumina A DFT study

    NASA Astrophysics Data System (ADS)

    Handzlik, Jarosław

    2007-05-01

    Ethene metathesis proceeding on monomeric and dimeric Mo species on the (1 0 0) and (1 1 0) γ-alumina is investigated by density functional theory, applying the cluster approach. The calculated vibrational frequencies of the surface OH groups are assigned to the experimental IR bands. It is shown that both monomeric and dimeric Mo forms can be the active sites of olefin metathesis. Metathesis activity and stability of the Mo-methylidene centres depend on their location on alumina. The differences in the sites reactivity are explained on the basis of their geometrical and electronic structure parameters. For the monomeric centres, isomerisation of the trigonal bipyramidal intermediate to the stable square pyramidal molybdacyclobutane is kinetically favoured over the cycloreversal step. The situation is opposite in the case of the dimeric species.

  13. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    Chou, Chi-Yuan; Tong, Liang

    2012-06-19

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  14. Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

    SciTech Connect

    C Chou; L Tong

    2011-12-31

    Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO{sub 2} donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 {angstrom} resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca{sup 2+} ions or two ADP molecules and one Mg{sup 2+} ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca{sup 2+} ion and the Mg{sup 2+} ion are associated with the ADP molecule in the active site, and the other Ca{sup 2+} ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

  15. Spin Configurations of π Electrons and Dimerization in Quasi-One-Dimensional Organic Bipartite Lozenge Chains

    NASA Astrophysics Data System (ADS)

    Duan, Y. F.; Yao, K. L.; Yi, L.

    Based on a theoretical model proposed for an organic bipartite lozenge ferrimagnetic chain, the spin configuration of π electrons and the dimerization are investigated. With the Hartree-Fock approximation, the strong electron-phonon coupling and the electron-electron interaction in the one-dimensional system are taken into account self-consistently. It is shown that around the middle of the chain appears a π electron spin polarization cloud with alternation of sign and amplitude of the spin density extending over a certain distance, which extends all over the chain with no decay when the e-e interaction is larger than a critical value. In the stable ferrimagnetic state, the antiferromagnetic exchange interaction between electrons at site A and site B along the chain will become very strong, and almost zero dimerization happens for the chain.

  16. Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication

    PubMed Central

    de la Torre, Beatriz G.; Valle, Javier; Andreu, David; Sobrino, Francisco

    2015-01-01

    Nonstructural protein 3A is involved in relevant functions in foot-and-mouth disease virus (FMDV) replication. FMDV 3A can form homodimers and preservation of the two hydrophobic α-helices (α1 and α2) that stabilize the dimer interface is essential for virus replication. In this work, small peptides mimicking residues involved in the dimer interface were used to interfere with dimerization and thus gain insight on its biological function. The dimer interface peptides α1, α2 and that spanning the two hydrophobic α-helices, α12, impaired in vitro dimer formation of a peptide containing the two α-helices, this effect being higher with peptide α12. To assess the effect of dimer inhibition in cultured cells, the interfering peptides were N-terminally fused to a heptaarginine (R7) sequence to favor their intracellular translocation. Thus, when fused to R7, interference peptides (100 μM) were able to inhibit dimerization of transiently expressed 3A, the higher inhibitions being found with peptides α1 and α12. The 3A dimerization impairment exerted by the peptides correlated with significant, specific reductions in the viral yield recovered from peptide-treated FMDV infected cells. In this case, α2 was the only peptide producing significant reductions at concentrations lower than 100 μM. Thus, dimer interface peptides constitute a tool to understand the structure-function relationship of this viral protein and point to 3A dimerization as a potential antiviral target. PMID:26505190

  17. Analysis of SecA Dimerization in Solution

    PubMed Central

    2015-01-01

    The Sec pathway mediates translocation of protein across the inner membrane of bacteria. SecA is a motor protein that drives translocation of preprotein through the SecYEG channel. SecA reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in SecA crystal structures. Here, we have used biophysical approaches to address the nature of the SecA dimer that exists in solution. We have taken advantage of the extreme salt sensitivity of SecA dimerization to compare the rates of hydrogen–deuterium exchange of the monomer and dimer and have analyzed the effects of single-alanine substitutions on dimerization affinity. Our results support the antiparallel dimer arrangement observed in one of the crystal structures of Bacillus subtilis SecA. Additional residues lying within the preprotein binding domain and the C-terminus are also protected from exchange upon dimerization, indicating linkage to a conformational transition of the preprotein binding domain from an open to a closed state. In agreement with this interpretation, normal mode analysis demonstrates that the SecA dimer interface influences the global dynamics of SecA such that dimerization stabilizes the closed conformation. PMID:24786965

  18. Dimerization of DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte migration.

    PubMed

    Terasawa, Masao; Uruno, Takehito; Mori, Sayako; Kukimoto-Niino, Mutsuko; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Tanaka, Yoshihiko; Yokoyama, Shigeyuki; Fukui, Yoshinori

    2012-01-01

    The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane. PMID:23050005

  19. On the fitting of binding data when receptor dimerization is suspected

    PubMed Central

    Giraldo, J

    2008-01-01

    Mechanistic and empirical modelling are compared in context of dimeric receptors. In particular, the supposed advantages of the two-state dimer model for fitting of binding data with respect to classical approaches such as the two-independent sites model are investigated. The two models are revisited from both the mechanistic and empirical point of views. The problem of overparameterized models and the benefits of the concurrent use of mechanistic and empirical models for mechanism analysis are discussed. The pros and cons of mathematical models are examined with special emphasis given to the interpretation of the connection between the shapes of the curves and receptor cooperativity. It is shown that a given pharmacological phenotype (curve shape) can be obtained from different receptor genotypes (as, for instance, non-interconvertible monomeric receptor species, receptor-G protein interactions and dimeric receptors), though values of the Hill coefficient greater than one are indicative of receptor oligomerization. The existence of a relationship between the recently defined dimer cooperativity index and the more familiar Hill coefficient is proven. PMID:18536745

  20. The dimerization domain in DapE enzymes is required for catalysis.

    PubMed

    Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena; Gutierrez, Blanca; Sanchez, Stephen; Jedrzejczak, Robert; Mack, Jamey C; Olsen, Kenneth W; Joachimiak, Andrzej; Holz, Richard C

    2014-01-01

    The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

  1. The role of tyrosine sulfation in the dimerization of the CXCR4:SDF-1 complex

    PubMed Central

    Rapp, Chaya; Snow, Sara; Laufer, Talya; McClendon, Christopher L

    2013-01-01

    Oligomerization of G protein-coupled receptors is a recognized mode of regulation of receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine receptor is a G protein-coupled receptor that is post-translationally modified by tyrosine sulfation at three sites on its N-terminus (Y7, Y12, Y21), leading to enhanced affinity for its ligand, stromal cell derived factor (SDF-1, also called CXCL12). The complex has been implicated in cancer metastasis and is a therapeutic target in cancer treatment. Using molecular dynamics simulation of NMR-derived structures of the CXCR4 N-terminus in complex with SDF-1, and calculations of electrostatic binding energies for these complexes, we address the role of tyrosine sulfation in this complex. Our results show that sulfation stabilizes the dimeric state of the CXCR4:SDF-1 complex through hydrogen bonding across the dimer interface, conformational changes in residues at the dimer interface, and an enhancement in electrostatic binding energies associated with dimerization. These findings suggest a mechanism through which post-translational modifications such as tyrosine sulfation might regulate downstream function through modulation of the oligomeric state of the modified system. PMID:23740770

  2. Application of ground-truth for classification and quantification of bird movements on migratory bird habitat initiative sites in southwest Louisiana: final report

    USGS Publications Warehouse

    Barrow, Wylie C.; Baldwin, Michael J.; Randall, Lori A.; Pitre, John; Dudley, Kyle J.

    2013-01-01

    This project was initiated to assess migrating and wintering bird use of lands enrolled in the Natural Resources Conservation Service’s (NRCS) Migratory Bird Habitat Initiative (MBHI). The MBHI program was developed in response to the Deepwater Horizon oil spill in 2010, with the goal of improving/creating habitat for waterbirds affected by the spill. In collaboration with the University of Delaware (UDEL), we used weather surveillance radar data (Sieges 2014), portable marine radar data, thermal infrared images, and visual observations to assess bird use of MBHI easements. Migrating and wintering birds routinely make synchronous flights near dusk (e.g., departure during migration, feeding flights during winter). Weather radars readily detect birds at the onset of these flights and have proven to be useful remote sensing tools for assessing bird-habitat relations during migration and determining the response of wintering waterfowl to wetland restoration (e.g., Wetlands Reserve Program lands). However, ground-truthing is required to identify radar echoes to species or species group. We designed a field study to ground-truth a larger-scale, weather radar assessment of bird use of MBHI sites in southwest Louisiana. We examined seasonal bird use of MBHI fields in fall, winter, and spring of 2011-2012. To assess diurnal use, we conducted total area surveys of MBHI sites in the afternoon, collecting data on bird species composition, abundance, behavior, and habitat use. In the evenings, we quantified bird activity at the MBHI easements and described flight behavior (i.e., birds landing in, departing from, circling, or flying over the MBHI tract). Our field sampling captured the onset of evening flights and spanned the period of collection of the weather radar data analyzed. Pre- and post-dusk surveys were conducted using a portable radar system and a thermal infrared camera. Landbirds, shorebirds, and wading birds were commonly found on MBHI fields during diurnal

  3. Application of ground-truth for classification and quantification of bird movements on migratory bird habitat initiative sites in southwest Louisiana: final report

    USGS Publications Warehouse

    Barrow, Wylie C.; Baldwin, Michael J.; Randall, Lori A.; Pitre, John; Dudley, Kyle J.

    2013-01-01

    This project was initiated to assess migrating and wintering bird use of lands enrolled in the Natural Resources Conservation Service’s (NRCS) Migratory Bird Habitat Initiative (MBHI). The MBHI program was developed in response to the Deepwater Horizon oil spill in 2010, with the goal of improving/creating habitat for waterbirds affected by the spill. In collaboration with the University of Delaware (UDEL), we used weather surveillance radar data (Sieges 2014), portable marine radar data, thermal infrared images, and visual observations to assess bird use of MBHI easements. Migrating and wintering birds routinely make synchronous flights near dusk (e.g., departure during migration, feeding flights during winter). Weather radars readily detect birds at the onset of these flights and have proven to be useful remote sensing tools for assessing bird-habitat relations during migration and determining the response of wintering waterfowl to wetland restoration (e.g., Wetlands Reserve Program lands). However, ground-truthing is required to identify radar echoes to species or species group. We designed a field study to ground-truth a larger-scale, weather radar assessment of bird use of MBHI sites in southwest Louisiana. We examined seasonal bird use of MBHI fields in fall, winter, and spring of 2011-2012. To assess diurnal use, we conducted total area surveys of MBHI sites in the afternoon, collecting data on bird species composition, abundance, behavior, and habitat use. In the evenings, we quantified bird activity at the MBHI easements and described flight behavior (i.e., birds landing in, departing from, circling, or flying over the MBHI tract). Our field sampling captured the onset of evening flights and spanned the period of collection of the weather radar data analyzed. Pre- and post-dusk surveys were conducted using a portable radar system and a thermal infrared camera. Landbirds, shorebirds, and wading birds were commonly found on MBHI fields during diurnal

  4. Reactive Pathways in the Chlorobenzene-Ammonia Dimer Cation Radical: New Insights from Experiment and Theory

    NASA Astrophysics Data System (ADS)

    Nyambo, Silver; Uhler, Brandon; Kalume, Aimable; Muzangwa, Lloyd; Reid, Scott

    2014-06-01

    Previously, we have studied non-covalent interactions in mono-halogenated benzene clusters using mass selected resonant 2-photon ionization methods. We have extended our studies by investigating the interaction between these mono-halobenzenes with a prototypical N atom donor (NH_3). Thus, we have obtained electronic spectra of PhX…(NH_3)n ( X=F, Cl, Br and n=1,2….) complexes in the region of the PhX monomer S_0-S_1 (ππ*) transition. Here we are mainly focusing on PhCl…NH_3 dimer. We found that upon ionization of the dimer, three reactive pathways of the [PhCl…NH_3]+. have been evidenced. The primary pathway is the Cl atom elimination, previously evidenced. The second and third pathways, HCl elimination and H atom elimination are identified for the first time in the R2PI studies of the dimer. Electronic spectra obtained for the three pathways shows that they originate from a common precursor. The reactive pathways in this system were extensively characterized computationally. We used DFT and post-Hartree Fock electronic structure calculations, Frank-Condon analysis to support our experimental findings. The results were consistent with previous direct ab initio molecular dynamics calculations, we found two nearly iso-energetic Wheland intermediates which lie significantly lower in energy than the initially formed dimer cation radical [PhCl…NH_3]+..

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

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

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

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

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

    PubMed

    Whitney, John C; Whitfield, Gregory B; Marmont, Lindsey S; Yip, Patrick; Neculai, A Mirela; Lobsanov, Yuri D; Robinson, Howard; Ohman, Dennis E; Howell, P Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  8. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

  9. Induction of pyrimidine dimers and unscheduled DNA synthesis in cultured mouse epithelial cells exposed to 254-nm- and u. v. -B radiation

    SciTech Connect

    Yotti, L.P.; Ley, R.D.

    1983-01-01

    The induction and fate of pyrimidine dimers and unscheduled DNA synthesis were measured in u.v.-irradiated primary, newborn SENCAR mouse epithelial cells. Unscheduled DNA synthesis was induced in a dose responsive manner by two u.v. sources, a germicidal lamp (254 nm) and an FS40 sunlamp (280--400 nm). Using the endonuclease-sensitive site assay to detect pyrimidine dimer production and excision, we examined the response of the newborn mouse cells to both u.v. sources. We were unable to detect the removal of pyrimidine dimers with either of the two sources of u.v. The speculation is made that primary, newborn mouse epidermal cells excise u.v.-induced pyrimidine dimers to an extent below the level of detection of the endonuclease-sensitive site assay but to an extent sufficient to induce unscheduled DNA synthesis.

  10. Dimerization of FIR Upon FUSE DNA Binding Suggests Mechanism of c-myc Inhibition

    SciTech Connect

    Crichlow,G.; Zhou, H.; Hsiao, H.; Frederick, K.; Debrosse, M.; Yang, Y.; Folta-Stogniew, E.; Chung, H.; Fan, C.; et al

    2008-01-01

    c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control.

  11. Suppression of cell-cycle progression by Jun dimerization protein-2 (JDP2) involves downregulation of cyclin-A2

    PubMed Central

    Pan, J; Nakade, K; Huang, Y-C; Zhu, Z-W; Masuzaki, S; Hasegawa, H; Murata, T; Yoshiki, A; Yamaguchi, N; Lee, C-H; Yang, W-C; Tsai, E-M; Obata, Y; Yokoyama, K K

    2010-01-01

    We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2-knockout (Jdp2KO) mice by targeting exon-1 to disrupt the site of initiation of transcription. The epidermal thickening of skin from the Jdp2KO mice after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) proceeded more rapidly than that of control mice, and more proliferating cells were found at the epidermis. Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation. PMID:20802531

  12. Caffeine dimerization: effects of sugar, salts, and water structure.

    PubMed

    Shimizu, Seishi

    2015-10-01

    Sugars and salts strongly affect the dimerization of caffeine in water. Such a change of dimerization, considered to be crucial for bitter taste suppression, has long been rationalized by the change of "water structure" induced by the additives; "kosmotropic" (water structure enhancing) salts and sugars promote dimerization, whereas "chaotropic" (water structure breaking) salts suppress dimerization. Based on statistical thermodynamics, here we challenge this consensus; we combine the rigorous Kirkwood-Buff theory of solution with the classical isodesmic model of caffeine association. Instead of the change of water structure, we show that the enhancement of caffeine dimerization is due to the exclusion of additives from caffeine, and that the weakening of dimerization is due to the binding of additives on caffeine.

  13. Graded-index optical dimer formed by optical force.

    PubMed

    Akbarzadeh, Alireza; Koschny, Thomas; Kafesaki, Maria; Economou, Eleftherios N; Soukoulis, Costas M

    2016-05-30

    We propose an optical dimer formed from two spherical lenses bound by the pressure that light exerts on matter. With the help of the method of force tracing, we find the required graded-index profiles of the lenses for the existence of the dimer. We study the dynamics of the opto-mechanical interaction of lenses under the illumination of collimated light beams and quantitatively validate the performance of proposed dimer. We also examine the stability of dimer due to the lateral misalignments and we show how restoring forces bring the dimer into lateral equilibrium. The dimer can be employed in various practical applications such as optical manipulation, sensing and imaging. PMID:27410066

  14. Cytochrome P450 as dimerization catalyst in diketopiperazine alkaloid biosynthesis.

    PubMed

    Saruwatari, Takayoshi; Yagishita, Fumitoshi; Mino, Takashi; Noguchi, Hiroshi; Hotta, Kinya; Watanabe, Kenji

    2014-03-21

    As dimeric natural products frequently exhibit useful biological activities, identifying and understanding their mechanisms of dimerization is of great interest. One such compound is (−)-ditryptophenaline, isolated from Aspergillus flavus, which inhibits substance P receptor for potential analgesic and anti-inflammatory activity. Through targeted gene knockout in A. flavus and heterologous yeast gene expression, we determined for the first time the gene cluster and pathway for the biosynthesis of a dimeric diketopiperazine alkaloid. We also determined that a single cytochrome P450, DtpC, is responsible not only for pyrroloindole ring formation but also for concurrent dimerization of N-methylphenylalanyltryptophanyl diketopiperazine monomers into a homodimeric product. Furthermore, DtpC exhibits relaxed substrate specificity, allowing the formation of two new dimeric compounds from a non-native monomeric precursor, brevianamide F. A radical-mediated mechanism of dimerization is proposed.

  15. Dimerize RACK1 upon transformation with oncogenic ras

    SciTech Connect

    Chu, L.-Y.; Chen, Y.-H.; Chuang, N.-N. . E-mail: zonnc@sinica.edu.tw

    2005-05-06

    From our previous studies, we learned that syndecan-2/p120-GAP complex provided docking site for Src to prosecute tyrosine kinase activity upon transformation with oncogenic ras. And, RACK1 protein was reactive with syndecan-2 to keep Src inactivated, but not when Ras was overexpressed. In the present study, we characterized the reaction between RACK1 protein and Ras. RACK1 was isolated from BALB/3T3 cells transfected with plasmids pcDNA3.1-[S-ras(Q{sub 61}K)] of shrimp Penaeus japonicus and RACK1 was revealed to react with GTP-K{sub B}-Ras(Q{sub 61}K), not GDP-K{sub B}-Ras(Q{sub 61}K). This selective interaction between RACK1 and GTP-K{sub B}-Ras(Q{sub 61}K) was further confirmed with RACK1 of human placenta and mouse RACK1-encoded fusion protein. We found that RACK1 was dimerized upon reaction with GTP-K{sub B}-Ras(Q{sub 61}K), as well as with 14-3-3{beta} and geranylgeranyl pyrophosphate, as revealed by phosphorylation with Src tyrosine kinase. We reported the complex of RACK1/GTP-K{sub B}-Ras(Q{sub 61}K) reacted selectively with p120-GAP. This interaction was sufficient to dissemble RACK1 into monomers, a preferred form to compete for the binding of syndecan-2. These data indicate that the reaction of GTP-K{sub B}-Ras(Q{sub 61}K) with RACK1 in dimers may operate a mechanism to deplete RACK1 from reaction with syndecan-2 upon transformation by oncogenic ras and the RACK1/GTP-Ras complex may provide a route to react with p120-GAP and recycle monomeric RACK1 to syndecan-2.

  16. Porphyrin dimers as photosensitizers in photodynamic therapy

    SciTech Connect

    Pandey, R.K.; Smith, K.M.; Dougherty, T.J. )

    1990-07-01

    Porphyrin dimers 9 with either linkages and possible isomers bis(1-(6,7-bis(2-(methoxycarbonyl)ethyl)-1,3,5,8-tetramethyl-2- vinylporphin-4-yl)ethyl) ether (10) bis(1-(6,7-bis(2-(methoxycarbonyl)ethyl)-1,3,5,8-tetramethyl-4- vinylporphin-2-yl)ethyl) ether (11), and 1-(6,7-bis(2-(methoxycarbonyl)ethyl)-1,3,5,8-tetramethyl-2-vinylporph in- 4-yl)ethyl 1-(6,7-bis(2-(methoxycarbonyl)ethyl)-1,3,5,8-tetramethyl-4-vinylporph in- 2-yl)ethyl ether (12) were synthesized from the corresponding (1-hydroxyethyl)vinyldeuteroporphyrin IX dimethyl esters (Hvd). The pure Hvd isomers 2-(1-hydroxyethyl)-4-vinyldeuteroporphyrin IX dimethyl ester (7) and 4-(1-hydroxyethyl)-2-vinyldeuteroporphyrin IX dimethyl ester (8) were obtained from 2-acetyl-4-(1-hydroxyethyl) deuteroporphyrin IX dimethyl ester (3) and 4-acetyl-2-(1-hydroxyethyl)deuteroporphyrin IX dimethyl ester (4). Porphyrins 3 and 4 were prepared either by partial reduction of 2,4-diacetyldeuteroporphyrin IX dimethyl ester (2) or by oxidation of hematoporphyrin IX dimethyl ester (1) by using tetra-n-propylammonium perruthenate (Prn4N)(RuO4) with N-methylmorpholine N-oxide as an oxidizing agent. The in vivo photosensitizing ability and therapeutic ratios of dimers 9-12 were compared with that of Photofrin II in the SMT-F tumor growing subcutaneously in DBA/2 Ha mice. These dimers were found to have better tumoricidal activity than Photofrin II with reduced skin phototoxicity.

  17. RING DOMAIN DIMERIZATION IS ESSENTIAL FOR RNF4 FUNCTION

    PubMed Central

    Liew, Chu Wai; Sun, Huaiyu; Hunter, Tony; Day, Catherine L.

    2011-01-01

    SYNOPSIS RNF4 family ubiquitin ligases are RING E3 ligases that regulate the homeostasis of SUMOylated proteins by promoting their ubiquitylation. Here we report that the RING domain of RNF4 forms a stable dimer, and that dimerization is required for ubiquitin transfer. Our data suggests that the stability of the E2~ubiquitin thioester bond is regulated by RING domain dimerization. PMID:20681948

  18. The properties of dimers confined between two charged plates.

    PubMed

    Hatlo, Marius M; Bohinc, Klemen; Lue, Leo

    2010-03-21

    We consider two like-charged planar surfaces immersed in solution of oppositely charged dimer counterions with a bond length l. To analyze this system, we extend and employ a self-consistent field theory that has been shown to be accurate from the weak to the intermediate through to the strong coupling regimes. In the limit of very short dimers, the results converge to the results for pointlike divalent ions. Near the surfaces, the dimers lie parallel to the charged plates. In the intermediate coupling regime, the dimers are aligned perpendicularly to the surface when they are a distance l from a surface. In the weak coupling regime, the interactions are only repulsive. At slightly higher couplings, there is a minimum in the variation of the free energy with distance at approximately the bond length of the dimers, which arises from bridging conformations of the dimers. In the intermediate coupling regime, an additional minimum in the free energy is observed at much smaller distances, which is due to the correlations between the dimers. For large dimer bond lengths, this minimum is metastable with respect to the previous minimum. However, as the bond length decreases, this minimum becomes the stable, while the minimum associated with the dimer bond length becomes metastable and eventually disappears. For shorter dimer bond length the attractive interaction is the result of correlations between counterions and charges on the surfaces. We find that dimers can mediate attractive interaction between like-charged surfaces in the intermediate coupling regime. The analysis of orientations confirms the bridging mechanism for sufficiently long dimers, whereas at high electrostatic couplings charge correlations contribute to the attraction. PMID:20331276

  19. The properties of dimers confined between two charged plates.

    PubMed

    Hatlo, Marius M; Bohinc, Klemen; Lue, Leo

    2010-03-21

    We consider two like-charged planar surfaces immersed in solution of oppositely charged dimer counterions with a bond length l. To analyze this system, we extend and employ a self-consistent field theory that has been shown to be accurate from the weak to the intermediate through to the strong coupling regimes. In the limit of very short dimers, the results converge to the results for pointlike divalent ions. Near the surfaces, the dimers lie parallel to the charged plates. In the intermediate coupling regime, the dimers are aligned perpendicularly to the surface when they are a distance l from a surface. In the weak coupling regime, the interactions are only repulsive. At slightly higher couplings, there is a minimum in the variation of the free energy with distance at approximately the bond length of the dimers, which arises from bridging conformations of the dimers. In the intermediate coupling regime, an additional minimum in the free energy is observed at much smaller distances, which is due to the correlations between the dimers. For large dimer bond lengths, this minimum is metastable with respect to the previous minimum. However, as the bond length decreases, this minimum becomes the stable, while the minimum associated with the dimer bond length becomes metastable and eventually disappears. For shorter dimer bond length the attractive interaction is the result of correlations between counterions and charges on the surfaces. We find that dimers can mediate attractive interaction between like-charged surfaces in the intermediate coupling regime. The analysis of orientations confirms the bridging mechanism for sufficiently long dimers, whereas at high electrostatic couplings charge correlations contribute to the attraction.

  20. Dynamic combinatorial enrichment of polyconformational D-/L-peptide dimers.

    PubMed

    Jadhav, Kirtikumar B; Lichtenecker, Roman J; Bullach, Anke; Mandal, Bhubaneswar; Arndt, Hans-Dieter

    2015-04-01

    D-/L-peptides such as gramicidin A (gA) adopt unique dimeric β-helical structures of different topologies. To overcome their conformational promiscuity and enrich individual components, a dynamic combinatorial approach assisted by thiol tags was developed. This method led to identification of the preferential formation of antiparallel dimers under a broad range of conditions, which was independent of peptide side-chain polarity. Exclusive formation of an antiparallel cyclic dimer was achieved in the presence of cesium ions.

  1. Oligomerization of deoxynucleoside-bisphosphate dimers: template and linkage specificity

    NASA Technical Reports Server (NTRS)

    Visscher, J.; van der Woerd, R.; Bakker, C. G.; Schwartz, A. W.; Orgel, L. E. (Principal Investigator)

    1989-01-01

    Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3'-5' pyrophosphate-linked dimer pdAppdAp, in comparison with the 3'-3' and 5'-5' linked dimers. In the absence of poly(U), the 5'-5' linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.

  2. A high-power xenon dimer excilamp

    NASA Astrophysics Data System (ADS)

    Lomaev, M. I.; Skakun, V. S.; Tarasenko, V. F.; Shitts, D. V.

    2006-06-01

    A high-power sealed-off excilamp operating on xenon dimers excited by a barrier discharge and emitting in the vacuum ultraviolet spectral range (λ ˜ 172 nm) has been designed, constructed, and tested. The lamp comprises six quartz tubes (emitters) and has a total radiating surface area of 20 × 20 = 400 cm2. The average output power density radiated from the surface of each emitter exceeds 120 mW/cm2. The total output power of the excilamp immediately upon discharge ignition exceeds 50 W.

  3. Absolute Ligand Discrimination by Dimeric Signaling Receptors.

    PubMed

    Fathi, Sepehr; Nayak, Chitra R; Feld, Jordan J; Zilman, Anton G

    2016-09-01

    Many signaling pathways act through shared components, where different ligand molecules bind the same receptors or activate overlapping sets of response regulators downstream. Nevertheless, different ligands acting through cross-wired pathways often lead to different outcomes in terms of the target cell behavior and function. Although a number of mechanisms have been proposed, it still largely remains unclear how cells can reliably discriminate different molecular ligands under such circumstances. Here we show that signaling via ligand-induced receptor dimerization-a very common motif in cellular signaling-naturally incorporates a mechanism for the discrimination of ligands acting through the same receptor. PMID:27602720

  4. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Kolthoff Landfill in Cleveland, Ohio. 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.

    2013-06-01

    The U.S. Environmental Protection Agency (EPA), Region 5, in accordance with the RE-Powering America's Land initiative, selected the Kolthoff Landfill site in Cleveland, Ohio, 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.

  5. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Tronox Facility in Savannah, Georgia. 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

    Kiatreungwattana, K.; 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 Tronox Facility site in Savannah, Georgia, 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.

  6. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Peru Mill Industrial Park in the City of Deming, New Mexico. 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

    Kiatreungwattana, K.; Geiger, J.; Healey, V.; Mosey, G.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Peru Mill Industrial Park site in the City of Deming, New Mexico, 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.

  7. Feasibility Study of Economics and Performance of Solar Photovoltaics at the Chino Mine in Silver City, New Mexico. 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

    Kiatreungwattana, K.; Geiger, J.; Healey, V.; Mosey, G.

    2013-04-01

    The U.S. Environmental Protection Agency (EPA), in accordance with the RE-Powering America's Land initiative, selected the Chino Mine site in Silver City, New Mexico, 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.

  8. Oligomerization of deoxynucleoside-biphosphate dimers - Template and linkage specificity

    NASA Technical Reports Server (NTRS)

    Visscher, J.; Van Der Woerd, R.; Bakker, C. G.; Schwartz, Alan W.

    1989-01-01

    The oligomerization of the activated 3-prime-5-prime pyrophosphate-linked dimer, pdAppdAp, is presently noted to be selectively favored by a poly(U) template over the 3-prime-3-prime and 5-prime-5-prime linked dimers. Both overall yields and the production of the longest oligomers were markedly stimulated by poly(U)'s presence; in its absence, the 5-prime-5-prime linked dimer became the most reactive, yielding chains of the order of 60 monomer-unit lengths. Remarkable self-organization properties are noted for the 5-prime-5-prime dimer of pdAp.

  9. Rotational spectra of propargyl alcohol dimer: A dimer bound with three different types of hydrogen bonds

    SciTech Connect

    Mani, Devendra; Arunan, E.

    2014-10-28

    Pure rotational spectra of the propargyl alcohol dimer and its three deuterium isotopologues have been observed in the 4 to 13 GHz range using a pulsed-nozzle Fourier transform microwave spectrometer. For the parent dimer, a total of 51 transitions could be observed and fitted within experimental uncertainty. For two mono-substituted and one bi-substituted deuterium isotopologues, a total of 14, 17, and 19 transitions were observed, respectively. The observed rotational constants for the parent dimer [A = 2321.8335(4) MHz, B = 1150.4774(2) MHz, and C = 1124.8898(2) MHz] are close to those of the most stable structure predicted by ab initio calculations. Spectra of the three deuterated isotopologues and Kraitchman analysis positively confirm this structure. Geometrical parameters and “Atoms in Molecules” analysis on the observed structure reveal that the two propargyl alcohol units in the dimer are bound by three different types of hydrogen bonds: O–H⋯O, O–H⋯π, and C–H⋯π. To the best of our knowledge, propargyl alcohol seems to be the smallest molecule forming a homodimer with three different points of contact.

  10. Pre-Feasibility Analysis of Pellet Manufacturing on the Former Loring Air Force Base Site. 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

    Hunsberger, R.; Mosey, G.

    2014-04-01

    The U.S. Environmental Protection Agency (EPA) Office of Solid Waste and Emergency Response, in accordance with the RE-Powering America's Lands initiative, engaged the U.S. Department of Energy's (DOE) National Renewable Energy Laboratory (NREL) to conduct feasibility studies to assess the viability of developing renewable energy generating facilities on contaminated sites. This site, in Limestone, Maine -- formerly the location of the Loring Air Force Base but now owned by the Aroostook Band of Micmac -- was selected for the potential to produce heating pellets from woody feedstock. Biomass was chosen as the renewable energy resource to evaluate based on abundant woody-biomass resources available in the area. NREL also evaluates potential savings from converting existing Micmac property from oil-fired heating to pellet heating.

  11. Equilibrium of sortase A dimerization on Staphylococcus aureus cell surface mediates its cell wall sorting activity

    PubMed Central

    Zhu, Jie; Xiang, Liang; Jiang, Faqin

    2015-01-01

    Staphylococcus aureus sortase A (SrtA) transpeptidase is a therapeutically important membrane-bound enzyme in Gram-positive bacteria, which organizes the covalently attached cell surface proteins on the peptidoglycan cell wall of the organism. Here, we report the direct observation of the highly selective homo-dimerization of SrtA on the cell membrane. To address the biological significance of the dimerization towards enzyme function, site-directed mutagenesis was performed to generate a SrtA mutant, which exists as monomer on the cell membrane. We observed that the cell surface display of adhesive proteins in S. aureus cells expressing monomeric SrtA mutant is more prominent than the cells expressing the wild-type enzyme. A cell-based invasion assay was also performed to evaluate the activities of wild-type SrtA and its monomeric mutant as well. Our data demonstrated that S. aureus cells expressing SrtA in monomeric form invade host mammalian cells more efficiently than those expressing wild-type SrtA in dimer-monomer equilibrium. The results suggested that the monomeric form of SrtA is more active than the dimeric form of the enzyme in terms of cell surface display of virulence factors for infection. This is the first study to present the oligomerization of SrtA and its related biological function on the cell membrane. Study of SrtA dimerization has implications for understanding its catalytic mechanism at the cellular level as well as the development of novel anti-infective agents. PMID:26129884

  12. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    PubMed Central

    McMillan, Kirk; Adler, Marc; Auld, Douglas S.; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.; Ohlmeyer, Michael H. J.; Pan, Gonghua; Parkinson, John F.; Phillips, Gary B.; Polokoff, Mark A.; Sigal, Nolan H.; Vergona, Ronald; Whitlow, Marc; Young, Tish A.; Devlin, James J.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies. PMID:10677491

  13. The Ras G Domain Lacks the Intrinsic Propensity to Form Dimers

    PubMed Central

    Kovrigina, Elizaveta A.; Galiakhmetov, Azamat R.; Kovrigin, Evgenii L.

    2015-01-01

    Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm. One proposed mechanism of dimerization involves formation of salt bridges between the two GTPase domains (G domains) leading to formation of a compact dimer as observed in Ras crystal structures. In this work, we interrogated the intrinsic ability of Ras to self-associate in solution by creating conditions of high local concentration through irreversibly tethering the two G domains together at their unstructured C-terminal tails. We evaluated possible self-association in this inverted tandem conjugate via analysis of the time-domain fluorescence anisotropy and NMR chemical shift perturbations. We did not observe the increased rotational correlation time expected for the G domain dimer. Variation of the ionic strength (to modulate stability of the salt bridges) did not affect the rotational correlation time in the tandem further supporting independent rotational diffusion of two G domains. In a parallel line of experiments to detect and map weak self-association of the G domains, we analyzed NMR chemical shifts perturbations at a number of sites near the crystallographic dimer interface. The nearly complete lack of chemical shift perturbations in the tandem construct supported a simple model with the independent G domains repelled from each other by their overall negative charge. These results lead us to the conclusion that self-association of the G domains cannot be responsible for homodimerization of Ras reported in the literature. PMID:26331257

  14. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    SciTech Connect

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with

  15. CO dimer: the infrared spectrum revisited.

    PubMed

    Rezaei, Mojtaba; Sheybani-Deloui, S; Moazzen-Ahmadi, N; Michaelian, K H; McKellar, A R W

    2013-10-01

    A broad-band (2135-2200 cm(-1)) infrared spectrum of the CO dimer is recorded using a tunable quantum cascade laser to probe a supersonic jet expansion with an effective rotational temperature of about 2.5 K. Analysis of the spectrum reveals the first known levels of the excited state (vCO = 1) with A(+) symmetry and establishes that resonant vibrational splittings are small (<0.2 cm(-1)) for both the C-bonded and O-bonded dimer isomers. The spectrum extends over a surprisingly large range, with somewhat reduced intensity above 2150 cm(-1). A total of 28 new "stacks" of rotational levels having A(-) symmetry are assigned for vCO = 1 on the basis of combination differences, adding to the 8 stacks previously known, and extending up to 51 cm(-1) above the vCO = 1 origin. Assignments are given for the first 13 stacks of vCO = 1 in terms of the very low frequency geared bending intermolecular vibration. PMID:23413960

  16. Rotational Spectrum of Propargyl Alcohol Dimer

    NASA Astrophysics Data System (ADS)

    Mani, Devendra; Arunan, E.

    2013-06-01

    Propargyl alcohol is a molecule of interest to astrophysics as well as combustion studies. Rotational-tunneling spectra of propargyl alcohol monomer is well known and shows that the molecule exists in gauche form. Recently we reported microwave spectra of Ar...propargyl alcohol complex. Propargyl alcochol exists in gauche form in the complex as well. In this study we have recorded pure rotational spectra of propargyl alcohol dimer between 4-13 GHz range.A total of 47 transitions, 24 a-type, 16 b-type and 7 c-type, have been observed and fitted with semi rigid rotor asymmetric top hamiltonian. The fitted rotational constants are: A = 2321.83323(47) MHz, B = 1150.47726(24) MHz and C = 1124.89000(20) MHz. The standard deviation for the fit is 2.5 kHz. The experimental rotational constants are very close to the structure predicted by ab-initio calculations in which two gauche-propargyl alcohol moieties are in three point contact stabilized by O-H...O, O-H...pi and C-H...pi interactions. Few transitions for duterated isotopologues of the dimer have also been observed and search for the remaining transitions is in progress. Details will be presented in the talk. E. Hirota,J. Mol. Spectrosc. 26 (1968) 335-350. J.C. Pearson, B.J. Drouin, J. Mol. Spectrosc. 234 (2005) 149-156. D. Mani, E. Arunan, ChemPhysChem 14 (2013) 754-763.

  17. Fibrillar dimer formation of islet amyloid polypeptides

    DOE PAGES

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less