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Sample records for acid substitution rates

  1. 40 CFR 721.10126 - Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... substituted benezenesulfonic acid reaction product with naphthalenesulfonato azo substituted phenyl azo... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10126 Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with naphthalenesulfonato...

  2. 40 CFR 721.10126 - Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... substituted benezenesulfonic acid reaction product with naphthalenesulfonato azo substituted phenyl azo... CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10126 Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with naphthalenesulfonato...

  3. 40 CFR 721.10126 - Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... substituted benzenesulfonic acid copper compound (generic). 721.10126 Section 721.10126 Protection of... substituted phenyl azo substituted benzenesulfonic acid copper compound (generic). (a) Chemical substance and... substituted phenyl azo substituted benzenesulfonic acid copper compound (PMN P-06-689) is subject to...

  4. 40 CFR 721.10126 - Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... substituted benzenesulfonic acid copper compound (generic). 721.10126 Section 721.10126 Protection of... substituted phenyl azo substituted benzenesulfonic acid copper compound (generic). (a) Chemical substance and... substituted phenyl azo substituted benzenesulfonic acid copper compound (PMN P-06-689) is subject to...

  5. 40 CFR 721.10126 - Alkyl amino substituted triazine amino substituted benezenesulfonic acid reaction product with...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... substituted benzenesulfonic acid copper compound (generic). 721.10126 Section 721.10126 Protection of... substituted phenyl azo substituted benzenesulfonic acid copper compound (generic). (a) Chemical substance and... substituted phenyl azo substituted benzenesulfonic acid copper compound (PMN P-06-689) is subject to...

  6. Are substitution rates and RNA editing correlated?

    PubMed Central

    2010-01-01

    Background RNA editing is a post-transcriptional process that, in seed plants, involves a cytosine to uracil change in messenger RNA, causing the translated protein to differ from that predicted by the DNA sequence. RNA editing occurs extensively in plant mitochondria, but large differences in editing frequencies are found in some groups. The underlying processes responsible for the distribution of edited sites are largely unknown, but gene function, substitution rate, and gene conversion have been proposed to influence editing frequencies. Results We studied five mitochondrial genes in the monocot order Alismatales, all showing marked differences in editing frequencies among taxa. A general tendency to lose edited sites was observed in all taxa, but this tendency was particularly strong in two clades, with most of the edited sites lost in parallel in two different areas of the phylogeny. This pattern is observed in at least four of the five genes analyzed. Except in the groups that show an unusually low editing frequency, the rate of C-to-T changes in edited sites was not significantly higher that in non-edited 3rd codon positions. This may indicate that selection is not actively removing edited sites in nine of the 12 families of the core Alismatales. In all genes but ccmB, a significant correlation was found between frequency of change in edited sites and synonymous substitution rate. In general, taxa with higher substitution rates tend to have fewer edited sites, as indicated by the phylogenetically independent correlation analyses. The elimination of edited sites in groups that lack or have reduced levels of editing could be a result of gene conversion involving a cDNA copy (retroprocessing). If so, this phenomenon could be relatively common in the Alismatales, and may have affected some groups recurrently. Indirect evidence of retroprocessing without a necessary correlation with substitution rate was found mostly in families Alismataceae and Hydrocharitaceae

  7. Capillary Electrophoresis of Substituted Benzoic Acids

    ERIC Educational Resources Information Center

    Mills, Nancy S.; Spence, John D.; Bushey, Michelle M.

    2005-01-01

    A series of substituted benzoic acids (SBAs) are prepared by students. The pKa shift, a result of the electron-withdrawing or electron-donating characteristics of the subsistent is examined in reference to the electrophoretic migration behavior of benzoic acid.

  8. 40 CFR 721.1680 - Substituted benzoic acid (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted benzoic acid (generic... Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as substituted benzoic acid (PMN...

  9. 40 CFR 721.1680 - Substituted benzoic acid (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted benzoic acid (generic... Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as substituted benzoic acid (PMN...

  10. 40 CFR 721.1680 - Substituted benzoic acid (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted benzoic acid (generic... Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as substituted benzoic acid (PMN...

  11. 40 CFR 721.1680 - Substituted benzoic acid (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Substituted benzoic acid (generic... Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as substituted benzoic acid (PMN...

  12. 40 CFR 721.1680 - Substituted benzoic acid (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted benzoic acid (generic... Substances § 721.1680 Substituted benzoic acid (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as substituted benzoic acid (PMN...

  13. 40 CFR 721.1643 - Benzenesulfonic acid, amino substituted phenylazo-.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzenesulfonic acid, amino... Specific Chemical Substances § 721.1643 Benzenesulfonic acid, amino substituted phenylazo-. (a) Chemical... as a benzenesulfonic acid, amino substituted phenylazo- (PMN P-95-86) is subject to reporting...

  14. 40 CFR 721.1643 - Benzenesulfonic acid, amino substituted phenylazo-.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzenesulfonic acid, amino... Specific Chemical Substances § 721.1643 Benzenesulfonic acid, amino substituted phenylazo-. (a) Chemical... as a benzenesulfonic acid, amino substituted phenylazo- (PMN P-95-86) is subject to reporting...

  15. 40 CFR 721.1643 - Benzenesulfonic acid, amino substituted phenylazo-.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Benzenesulfonic acid, amino... Specific Chemical Substances § 721.1643 Benzenesulfonic acid, amino substituted phenylazo-. (a) Chemical... as a benzenesulfonic acid, amino substituted phenylazo- (PMN P-95-86) is subject to reporting...

  16. 40 CFR 721.1643 - Benzenesulfonic acid, amino substituted phenylazo-.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Benzenesulfonic acid, amino... Specific Chemical Substances § 721.1643 Benzenesulfonic acid, amino substituted phenylazo-. (a) Chemical... as a benzenesulfonic acid, amino substituted phenylazo- (PMN P-95-86) is subject to reporting...

  17. 40 CFR 721.1643 - Benzenesulfonic acid, amino substituted phenylazo-.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzenesulfonic acid, amino... Specific Chemical Substances § 721.1643 Benzenesulfonic acid, amino substituted phenylazo-. (a) Chemical... as a benzenesulfonic acid, amino substituted phenylazo- (PMN P-95-86) is subject to reporting...

  18. 40 CFR 721.1700 - Halonitrobenzoic acid, substituted (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Halonitrobenzoic acid, substituted (generic name). 721.1700 Section 721.1700 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1700 Halonitrobenzoic acid, substituted (generic name). (a)...

  19. 40 CFR 721.1700 - Halonitrobenzoic acid, substituted (generic name).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Halonitrobenzoic acid, substituted (generic name). 721.1700 Section 721.1700 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1700 Halonitrobenzoic acid, substituted (generic name). (a)...

  20. 40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.5278 Substituted naphthalenesulfonic acid, alkali salt. (a) Chemical... as a substituted naphthalenesulfonic acid, alkali salt (PMN P-95-85) is subject to reporting...

  1. 40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Substituted benzenesulfonic acid salt (generic). 721.1648 Section 721.1648 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a)...

  2. 40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ..., alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.5278 Substituted naphthalenesulfonic acid, alkali salt. (a) Chemical... as a substituted naphthalenesulfonic acid, alkali salt (PMN P-95-85) is subject to reporting...

  3. 40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ..., alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.5278 Substituted naphthalenesulfonic acid, alkali salt. (a) Chemical... as a substituted naphthalenesulfonic acid, alkali salt (PMN P-95-85) is subject to reporting...

  4. 40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted benzenesulfonic acid salt (generic). 721.1648 Section 721.1648 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a)...

  5. 40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.5278 Substituted naphthalenesulfonic acid, alkali salt. (a) Chemical... as a substituted naphthalenesulfonic acid, alkali salt (PMN P-95-85) is subject to reporting...

  6. 40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted benzenesulfonic acid salt (generic). 721.1648 Section 721.1648 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a)...

  7. 40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted benzenesulfonic acid salt (generic). 721.1648 Section 721.1648 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a)...

  8. 40 CFR 721.5278 - Substituted naphthalenesulfonic acid, alkali salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., alkali salt. 721.5278 Section 721.5278 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.5278 Substituted naphthalenesulfonic acid, alkali salt. (a) Chemical... as a substituted naphthalenesulfonic acid, alkali salt (PMN P-95-85) is subject to reporting...

  9. 40 CFR 721.1648 - Substituted benzenesulfonic acid salt (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted benzenesulfonic acid salt (generic). 721.1648 Section 721.1648 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED... Specific Chemical Substances § 721.1648 Substituted benzenesulfonic acid salt (generic). (a)...

  10. FRAGS: estimation of coding sequence substitution rates from fragmentary data

    PubMed Central

    Swart, Estienne C; Hide, Winston A; Seoighe, Cathal

    2004-01-01

    Background Rates of substitution in protein-coding sequences can provide important insights into evolutionary processes that are of biomedical and theoretical interest. Increased availability of coding sequence data has enabled researchers to estimate more accurately the coding sequence divergence of pairs of organisms. However the use of different data sources, alignment protocols and methods to estimate substitution rates leads to widely varying estimates of key parameters that define the coding sequence divergence of orthologous genes. Although complete genome sequence data are not available for all organisms, fragmentary sequence data can provide accurate estimates of substitution rates provided that an appropriate and consistent methodology is used and that differences in the estimates obtainable from different data sources are taken into account. Results We have developed FRAGS, an application framework that uses existing, freely available software components to construct in-frame alignments and estimate coding substitution rates from fragmentary sequence data. Coding sequence substitution estimates for human and chimpanzee sequences, generated by FRAGS, reveal that methodological differences can give rise to significantly different estimates of important substitution parameters. The estimated substitution rates were also used to infer upper-bounds on the amount of sequencing error in the datasets that we have analysed. Conclusion We have developed a system that performs robust estimation of substitution rates for orthologous sequences from a pair of organisms. Our system can be used when fragmentary genomic or transcript data is available from one of the organisms and the other is a completely sequenced genome within the Ensembl database. As well as estimating substitution statistics our system enables the user to manage and query alignment and substitution data. PMID:15005802

  11. Rate of amino acid substitution is influenced by the degree and conservation of male-biased transcription over 50 myr of Drosophila evolution.

    PubMed

    Grath, Sonja; Parsch, John

    2012-01-01

    Sex-biased gene expression (i.e., the differential expression of genes between males and females) is common among sexually reproducing species. However, genes often differ in their sex-bias classification or degree of sex bias between species. There is also an unequal distribution of sex-biased genes (especially male-biased genes) between the X chromosome and the autosomes. We used whole-genome expression data and evolutionary rate estimates for two different Drosophilid lineages, melanogaster and obscura, spanning an evolutionary time scale of around 50 Myr to investigate the influence of sex-biased gene expression and chromosomal location on the rate of molecular evolution. In both lineages, the rate of protein evolution correlated positively with the male/female expression ratio. Genes with highly male-biased expression, genes expressed specifically in male reproductive tissues, and genes with conserved male-biased expression over long evolutionary time scales showed the fastest rates of evolution. An analysis of sex-biased gene evolution in both lineages revealed evidence for a "fast-X" effect in which the rate of evolution was greater for X-linked than for autosomal genes. This pattern was particularly pronounced for male-biased genes. Genes located on the obscura "neo-X" chromosome, which originated from a recent X-autosome fusion, showed rates of evolution that were intermediate between genes located on the ancestral X-chromosome and the autosomes. This suggests that the shift to X-linkage led to an increase in the rate of molecular evolution. PMID:22321769

  12. Rate of Amino Acid Substitution Is Influenced by the Degree and Conservation of Male-Biased Transcription Over 50 Myr of Drosophila Evolution

    PubMed Central

    Grath, Sonja; Parsch, John

    2012-01-01

    Sex-biased gene expression (i.e., the differential expression of genes between males and females) is common among sexually reproducing species. However, genes often differ in their sex-bias classification or degree of sex bias between species. There is also an unequal distribution of sex-biased genes (especially male-biased genes) between the X chromosome and the autosomes. We used whole-genome expression data and evolutionary rate estimates for two different Drosophilid lineages, melanogaster and obscura, spanning an evolutionary time scale of around 50 Myr to investigate the influence of sex-biased gene expression and chromosomal location on the rate of molecular evolution. In both lineages, the rate of protein evolution correlated positively with the male/female expression ratio. Genes with highly male-biased expression, genes expressed specifically in male reproductive tissues, and genes with conserved male-biased expression over long evolutionary time scales showed the fastest rates of evolution. An analysis of sex-biased gene evolution in both lineages revealed evidence for a “fast-X” effect in which the rate of evolution was greater for X-linked than for autosomal genes. This pattern was particularly pronounced for male-biased genes. Genes located on the obscura “neo-X” chromosome, which originated from a recent X-autosome fusion, showed rates of evolution that were intermediate between genes located on the ancestral X-chromosome and the autosomes. This suggests that the shift to X-linkage led to an increase in the rate of molecular evolution. PMID:22321769

  13. Microbial desulfonation of substituted naphthalenesulfonic acids and benzenesulfonic acids.

    PubMed Central

    Zürrer, D; Cook, A M; Leisinger, T

    1987-01-01

    Sulfur-limited batch enrichment cultures containing one of nine multisubstituted naphthalenesulfonates and an inoculum from sewage yielded several taxa of bacteria which could quantitatively utilize 19 sulfonated aromatic compounds as the sole sulfur source for growth. Growth yields were about 4 kg of protein per mol of sulfur. Specific degradation rates were about 4 to 14 mu kat/kg of protein. A Pseudomonas sp., an Arthrobacter sp., and an unidentified bacterium were examined. Each desulfonated at least 16 aromatic compounds, none of which served as a carbon source. Pseudomonas sp. strain S-313 converted 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, 5-amino-1-naphthalenesulfonic acid, benzenesulfonic acid, and 3-aminobenzenesulfonic acid to 1-naphthol, 2-naphthol, 5-amino-1-naphthol, phenol, and 3-aminophenol, respectively. Experiments with 18O2 showed that the hydroxyl group was derived from molecular oxygen. PMID:3662502

  14. Gene Tree Discordance Causes Apparent Substitution Rate Variation.

    PubMed

    Mendes, Fábio K; Hahn, Matthew W

    2016-07-01

    Substitution rates are known to be variable among genes, chromosomes, species, and lineages due to multifarious biological processes. Here, we consider another source of substitution rate variation due to a technical bias associated with gene tree discordance. Discordance has been found to be rampant in genome-wide data sets, often due to incomplete lineage sorting (ILS). This apparent substitution rate variation is caused when substitutions that occur on discordant gene trees are analyzed in the context of a single, fixed species tree. Such substitutions have to be resolved by proposing multiple substitutions on the species tree, and we therefore refer to this phenomenon as Substitutions Produced by ILS (SPILS). We use simulations to demonstrate that SPILS has a larger effect with increasing levels of ILS, and on trees with larger numbers of taxa. Specific branches of the species trees are consistently, but erroneously, inferred to be longer or shorter, and we show that these branches can be predicted based on discordant tree topologies. Moreover, we observe that fixing a species tree topology when performing tests of positive selection increases the false positive rate, particularly for genes whose discordant topologies are most affected by SPILS. Finally, we use data from multiple Drosophila species to show that SPILS can be detected in nature. Although the effects of SPILS are modest per gene, it has the potential to affect substitution rate variation whenever high levels of ILS are present, particularly in rapid radiations. The problems outlined here have implications for character mapping of any type of trait, and for any biological process that causes discordance. We discuss possible solutions to these problems, and areas in which they are likely to have caused faulty inferences of convergence and accelerated evolution. PMID:26927960

  15. FLU, an amino acid substitution model for influenza proteins

    PubMed Central

    2010-01-01

    Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We propose an influenza-specific amino acid substitution model to enhance the understanding of the evolution of influenza viruses. Results A maximum likelihood approach was applied to estimate an amino acid substitution model (FLU) from ~113, 000 influenza protein sequences, consisting of ~20 million residues. FLU outperforms 14 widely used models in constructing maximum likelihood phylogenetic trees for the majority of influenza protein alignments. On average, FLU gains ~42 log likelihood points with an alignment of 300 sites. Moreover, topologies of trees constructed using FLU and other models are frequently different. FLU does indeed have an impact on likelihood improvement as well as tree topologies. It was implemented in PhyML and can be downloaded from ftp://ftp.sanger.ac.uk/pub/1000genomes/lsq/FLU or included in PhyML 3.0 server at http://www.atgc-montpellier.fr/phyml/. Conclusions FLU should be useful for any influenza protein analysis system which requires an accurate description of amino acid substitutions. PMID:20384985

  16. Silver-catalysed protodecarboxylation of ortho-substituted benzoic acids.

    PubMed

    Cornella, Josep; Sanchez, Carolina; Banawa, David; Larrosa, Igor

    2009-12-14

    Catalytic amounts of Ag(I) salts in DMSO have been found to promote the protodecarboxylation of a wide variety of ortho-substituted benzoic acids under mild conditions and in excellent yields, highlighting a possible role for silver in decarboxylative cross-couplings. PMID:19921021

  17. In Vitro Investigation of Self-Assembled Nanoparticles Based on Hyaluronic Acid-Deoxycholic Acid Conjugates for Controlled Release Doxorubicin: Effect of Degree of Substitution of Deoxycholic Acid

    PubMed Central

    Wei, Wen-Hao; Dong, Xue-Meng; Liu, Chen-Guang

    2015-01-01

    Self-assembled nanoparticles based on a hyaluronic acid-deoxycholic acid (HD) chemical conjugate with different degree of substitution (DS) of deoxycholic acid (DOCA) were prepared. The degree of substitution (DS) was determined by titration method. The nanoparticles were loaded with doxorubicin (DOX) as the model drug. The human cervical cancer (HeLa) cell line was utilized for in vitro studies and cell cytotoxicity of DOX incorporated in the HD nanoparticles was accessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, cellular uptake of fluorescently labeled nanoparticles was also investigated. An increase in the degree of deoxycholic acid substitution reduced the size of the nanoparticles and also enhanced their drug encapsulation efficiency (EE), which increased with the increase of DS. A higher degree of deoxycholic acid substitution also lead to a lower release rate and an initial burst release of doxorubicin from the nanoparticles. In summary, the degree of substitution allows the modulation of the particle size, drug encapsulation efficiency, drug release rate, and cell uptake efficiency of the nanoparticles. The herein developed hyaluronic acid-deoxycholic acid conjugates are a good candidate for drug delivery and could potentiate therapeutic formulations for doxorubicin–mediated cancer therapy. PMID:25837468

  18. Detection of nonneutral substitution rates on mammalian phylogenies

    PubMed Central

    Pollard, Katherine S.; Hubisz, Melissa J.; Rosenbloom, Kate R.; Siepel, Adam

    2010-01-01

    Methods for detecting nucleotide substitution rates that are faster or slower than expected under neutral drift are widely used to identify candidate functional elements in genomic sequences. However, most existing methods consider either reductions (conservation) or increases (acceleration) in rate but not both, or assume that selection acts uniformly across the branches of a phylogeny. Here we examine the more general problem of detecting departures from the neutral rate of substitution in either direction, possibly in a clade-specific manner. We consider four statistical, phylogenetic tests for addressing this problem: a likelihood ratio test, a score test, a test based on exact distributions of numbers of substitutions, and the genomic evolutionary rate profiling (GERP) test. All four tests have been implemented in a freely available program called phyloP. Based on extensive simulation experiments, these tests are remarkably similar in statistical power. With 36 mammalian species, they all appear to be capable of fairly good sensitivity with low false-positive rates in detecting strong selection at individual nucleotides, moderate selection in 3-bp elements, and weaker or clade-specific selection in longer elements. By applying phyloP to mammalian multiple alignments from the ENCODE project, we shed light on patterns of conservation/acceleration in known and predicted functional elements, approximate fractions of sites subject to constraint, and differences in clade-specific selection in the primate and glires clades. We also describe new “Conservation” tracks in the UCSC Genome Browser that display both phyloP and phastCons scores for genome-wide alignments of 44 vertebrate species. PMID:19858363

  19. Selective Constraints on Amino Acids Estimated by a Mechanistic Codon Substitution Model with Multiple Nucleotide Changes

    PubMed Central

    Miyazawa, Sanzo

    2011-01-01

    Background Empirical substitution matrices represent the average tendencies of substitutions over various protein families by sacrificing gene-level resolution. We develop a codon-based model, in which mutational tendencies of codon, a genetic code, and the strength of selective constraints against amino acid replacements can be tailored to a given gene. First, selective constraints averaged over proteins are estimated by maximizing the likelihood of each 1-PAM matrix of empirical amino acid (JTT, WAG, and LG) and codon (KHG) substitution matrices. Then, selective constraints specific to given proteins are approximated as a linear function of those estimated from the empirical substitution matrices. Results Akaike information criterion (AIC) values indicate that a model allowing multiple nucleotide changes fits the empirical substitution matrices significantly better. Also, the ML estimates of transition-transversion bias obtained from these empirical matrices are not so large as previously estimated. The selective constraints are characteristic of proteins rather than species. However, their relative strengths among amino acid pairs can be approximated not to depend very much on protein families but amino acid pairs, because the present model, in which selective constraints are approximated to be a linear function of those estimated from the JTT/WAG/LG/KHG matrices, can provide a good fit to other empirical substitution matrices including cpREV for chloroplast proteins and mtREV for vertebrate mitochondrial proteins. Conclusions/Significance The present codon-based model with the ML estimates of selective constraints and with adjustable mutation rates of nucleotide would be useful as a simple substitution model in ML and Bayesian inferences of molecular phylogenetic trees, and enables us to obtain biologically meaningful information at both nucleotide and amino acid levels from codon and protein sequences. PMID:21445250

  20. Hydroxyl radical substitution in halogenated carbonyls: oxalic acid formation.

    PubMed

    Christiansen, Carrie J; Dalal, Shakeel S; Francisco, Joseph S; Mebel, Alexander M; Gaffney, Jeffrey S

    2010-03-01

    An ab initio study of OH radical substitution reactions in halogenated carbonyls is conducted. Hydroxyl radical substitution into oxalyl dichloride [ClC(O)C(O)Cl] and oxalyl dibromide [BrC(O)C(O)Br], resulting in the formation of oxalic acid, is presented. Analogous substitution reactions in formyl chloride [ClCH(O)], acetyl chloride [ClC(O)CH(3)], formyl bromide [BrCH(O)], and acetyl bromide [BrC(O)CH(3)] are considered. Energetics of competing hydrogen abstraction reactions for all applicable species are computed for comparison. Geometry optimizations and frequency computations are performed using the second-order Møller-Plesset perturbation theory (MP2) and the 6-31G(d) basis set for all minimum species and transition states. Single point energy computations are performed using fourth-order Møller-Plesset perturbation theory (MP4) and coupled cluster theory [CCSD(T)]. Potential energy surfaces, including activation energies and enthalpies, are determined from the computations. These potential energy surfaces show that OH substitution into ClC(O)C(O)Cl and BrC(O)C(O)Br, resulting in the formation of oxalic acid and other minor products, is energetically favorable. Energetics of analogous reactions with ClCH(O), BrCH(O), ClC(O)CH(3), and BrC(O)CH(3) are also computed. PMID:20131850

  1. 40 CFR 721.10679 - Carboxylic acid, substituted alkylstannylene ester, reaction products with inorganic acid tetra...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... alkylstannylene ester, reaction products with inorganic acid tetra alkyl ester (generic). 721.10679 Section 721... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10679 Carboxylic acid, substituted alkylstannylene ester, reaction products with inorganic acid tetra alkyl...

  2. Estimating divergence dates and substitution rates in the Drosophila phylogeny.

    PubMed

    Obbard, Darren J; Maclennan, John; Kim, Kang-Wook; Rambaut, Andrew; O'Grady, Patrick M; Jiggins, Francis M

    2012-11-01

    An absolute timescale for evolution is essential if we are to associate evolutionary phenomena, such as adaptation or speciation, with potential causes, such as geological activity or climatic change. Timescales in most phylogenetic studies use geologically dated fossils or phylogeographic events as calibration points, but more recently, it has also become possible to use experimentally derived estimates of the mutation rate as a proxy for substitution rates. The large radiation of drosophilid taxa endemic to the Hawaiian islands has provided multiple calibration points for the Drosophila phylogeny, thanks to the "conveyor belt" process by which this archipelago forms and is colonized by species. However, published date estimates for key nodes in the Drosophila phylogeny vary widely, and many are based on simplistic models of colonization and coalescence or on estimates of island age that are not current. In this study, we use new sequence data from seven species of Hawaiian Drosophila to examine a range of explicit coalescent models and estimate substitution rates. We use these rates, along with a published experimentally determined mutation rate, to date key events in drosophilid evolution. Surprisingly, our estimate for the date for the most recent common ancestor of the genus Drosophila based on mutation rate (25-40 Ma) is closer to being compatible with independent fossil-derived dates (20-50 Ma) than are most of the Hawaiian-calibration models and also has smaller uncertainty. We find that Hawaiian-calibrated dates are extremely sensitive to model choice and give rise to point estimates that range between 26 and 192 Ma, depending on the details of the model. Potential problems with the Hawaiian calibration may arise from systematic variation in the molecular clock due to the long generation time of Hawaiian Drosophila compared with other Drosophila and/or uncertainty in linking island formation dates with colonization dates. As either source of error will

  3. Estimating Divergence Dates and Substitution Rates in the Drosophila Phylogeny

    PubMed Central

    Obbard, Darren J.; Maclennan, John; Kim, Kang-Wook; Rambaut, Andrew; O’Grady, Patrick M.; Jiggins, Francis M.

    2012-01-01

    An absolute timescale for evolution is essential if we are to associate evolutionary phenomena, such as adaptation or speciation, with potential causes, such as geological activity or climatic change. Timescales in most phylogenetic studies use geologically dated fossils or phylogeographic events as calibration points, but more recently, it has also become possible to use experimentally derived estimates of the mutation rate as a proxy for substitution rates. The large radiation of drosophilid taxa endemic to the Hawaiian islands has provided multiple calibration points for the Drosophila phylogeny, thanks to the "conveyor belt" process by which this archipelago forms and is colonized by species. However, published date estimates for key nodes in the Drosophila phylogeny vary widely, and many are based on simplistic models of colonization and coalescence or on estimates of island age that are not current. In this study, we use new sequence data from seven species of Hawaiian Drosophila to examine a range of explicit coalescent models and estimate substitution rates. We use these rates, along with a published experimentally determined mutation rate, to date key events in drosophilid evolution. Surprisingly, our estimate for the date for the most recent common ancestor of the genus Drosophila based on mutation rate (25–40 Ma) is closer to being compatible with independent fossil-derived dates (20–50 Ma) than are most of the Hawaiian-calibration models and also has smaller uncertainty. We find that Hawaiian-calibrated dates are extremely sensitive to model choice and give rise to point estimates that range between 26 and 192 Ma, depending on the details of the model. Potential problems with the Hawaiian calibration may arise from systematic variation in the molecular clock due to the long generation time of Hawaiian Drosophila compared with other Drosophila and/or uncertainty in linking island formation dates with colonization dates. As either source of error will

  4. 40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to...

  5. 40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to...

  6. 40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to...

  7. 40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to...

  8. 40 CFR 721.1875 - Boric acid, alkyl and substituted alkyl esters (generic name).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Boric acid, alkyl and substituted... Significant New Uses for Specific Chemical Substances § 721.1875 Boric acid, alkyl and substituted alkyl... chemical substance boric acid, alkyl and substituted alkyl esters (PMN P-86-1252) is subject to...

  9. 40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting...

  10. 40 CFR 721.10474 - Substituted amino ethane sulfonic acid salt (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted amino ethane sulfonic acid... Specific Chemical Substances § 721.10474 Substituted amino ethane sulfonic acid salt (generic). (a... generically as substituted amino ethane sulfonic acid salt (PMN P-04-107) is subject to reporting under...

  11. 40 CFR 721.10474 - Substituted amino ethane sulfonic acid salt (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted amino ethane sulfonic acid... Specific Chemical Substances § 721.10474 Substituted amino ethane sulfonic acid salt (generic). (a... generically as substituted amino ethane sulfonic acid salt (PMN P-04-107) is subject to reporting under...

  12. 40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting...

  13. 40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical... as oxo-substituted amino al-kan-oic acid derivative (PMN No. P-92-692) is subject to reporting...

  14. 40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting...

  15. 40 CFR 721.6560 - Acrylic acid, polymer with substituted ethene.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.6560 Acrylic acid, polymer with substituted ethene. (a) Chemical... as acrylic acid, polymer with substituted ethene (PMN P-91-521) is subject to reporting under...

  16. 40 CFR 721.6560 - Acrylic acid, polymer with substituted ethene.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.6560 Acrylic acid, polymer with substituted ethene. (a) Chemical... as acrylic acid, polymer with substituted ethene (PMN P-91-521) is subject to reporting under...

  17. 40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting...

  18. 40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting...

  19. 40 CFR 721.6560 - Acrylic acid, polymer with substituted ethene.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.6560 Acrylic acid, polymer with substituted ethene. (a) Chemical... as acrylic acid, polymer with substituted ethene (PMN P-91-521) is subject to reporting under...

  20. 40 CFR 721.6560 - Acrylic acid, polymer with substituted ethene.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.6560 Acrylic acid, polymer with substituted ethene. (a) Chemical... as acrylic acid, polymer with substituted ethene (PMN P-91-521) is subject to reporting under...

  1. 40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting...

  2. 40 CFR 721.10399 - Benzoic acid azo-substituted pyridine (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzoic acid azo-substituted pyridine... Specific Chemical Substances § 721.10399 Benzoic acid azo-substituted pyridine (generic). (a) Chemical... as benzoic acid azo-substituted pyridine (PMN P-10-501) is subject to reporting under this...

  3. 40 CFR 721.10399 - Benzoic acid azo-substituted pyridine (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Benzoic acid azo-substituted pyridine... Specific Chemical Substances § 721.10399 Benzoic acid azo-substituted pyridine (generic). (a) Chemical... as benzoic acid azo-substituted pyridine (PMN P-10-501) is subject to reporting under this...

  4. 40 CFR 721.10399 - Benzoic acid azo-substituted pyridine (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Benzoic acid azo-substituted pyridine... Specific Chemical Substances § 721.10399 Benzoic acid azo-substituted pyridine (generic). (a) Chemical... as benzoic acid azo-substituted pyridine (PMN P-10-501) is subject to reporting under this...

  5. 40 CFR 721.6560 - Acrylic acid, polymer with substituted ethene.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.6560 Acrylic acid, polymer with substituted ethene. (a) Chemical... as acrylic acid, polymer with substituted ethene (PMN P-91-521) is subject to reporting under...

  6. 40 CFR 721.10032 - Acrylic acid, polymer with substituted acrylamides (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Acrylic acid, polymer with substituted... Specific Chemical Substances § 721.10032 Acrylic acid, polymer with substituted acrylamides (generic). (a... generically as acrylic acid, polymer with substituted acrylamides (PMN P-02-269) is subject to reporting...

  7. Stochastic Demography and the Neutral Substitution Rate in Class-Structured Populations

    PubMed Central

    Lehmann, Laurent

    2014-01-01

    The neutral rate of allelic substitution is analyzed for a class-structured population subject to a stationary stochastic demographic process. The substitution rate is shown to be generally equal to the effective mutation rate, and under overlapping generations it can be expressed as the effective mutation rate in newborns when measured in units of average generation time. With uniform mutation rate across classes the substitution rate reduces to the mutation rate. PMID:24594520

  8. New substituted amides and hydrazides of pectic acid

    SciTech Connect

    Lapenko, V.L.; Potapova, L.B.; Slivkin, A.I.; Razumnaya, Z.A.

    1988-05-10

    Structural variants of pectin amides and hydrazides are of practical value as flocculants in water treatment. The purpose of this work was to further investigate the synthesis of substituted amides and hydrazides of pectic acid and to study their activity as flocculants. They used pectin, methylation products of pectin, pectic acid, and methyl pectates. The synthesized analogs of pectinic materials containing nitrogen are essentially copolymers of hydrazido (amido) and carboxyl (methoxyl) derivatives of D-galacturonic acid. The flocculant activity of the new polymers was monitored with simulated drainage water containing kaolin or abrasive powder (for glass manufacture) in the presence of polyvalent metal ions. The use of the new ampholytic flocculants in the purification of water from suspended impurities permits a high degree of clarification with a sharp decrease in reagent consumption.

  9. 40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... substituted oxirane, formaldehyde-phenol polymer glycidyl ether, substituted proplyamine and...-phenol polymer glycidyl ether, substituted proplyamine and polyethylenepolyamines (generic). (a) Chemical... as fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl...

  10. 40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... substituted oxirane, formaldehyde-phenol polymer glycidyl ether, substituted proplyamine and...-phenol polymer glycidyl ether, substituted proplyamine and polyethylenepolyamines (generic). (a) Chemical... as fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl...

  11. 40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted acrylamides and acrylic... New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer... identified generically as substituted acrylamides and acrylic acid copolymer (PMN P-00-0490) is subject...

  12. 40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Substituted acrylamides and acrylic... New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer... identified generically as substituted acrylamides and acrylic acid copolymer (PMN P-00-0490) is subject...

  13. 40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted acrylamides and acrylic... New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer... identified generically as substituted acrylamides and acrylic acid copolymer (PMN P-00-0490) is subject...

  14. A Marcus Treatment of Rate Constants for Protonation of Ring-Substituted α-Methoxystyrenes

    PubMed Central

    Richard, John P.; Williams, Kathleen B.

    2008-01-01

    Rate and equilibrium constants were determined for protonation of ring-substituted α-methoxystyrenes by hydronium ion and by carboxylic acids to form the corresponding ring-substituted α-methyl α-methoxybenzyl carbocations at 25 ° C and I = 1.0 (KCl). The thermodynamic barrier to carbocation formation increases by14.5 kcal/mol as the phenyl ring substituent(s) is changed from 4-MeO- to 3,5-di-NO2-, and as the carboxylic acid is changed from dichloroacetic to acetic acid. The Brønsted coefficient α for protonation by carboxylic acids increases from 0.67 to 0.77 over this range of phenyl ring substituents and the Brønsted coefficient β for proton transfer increases from 0.63 to 0.69 as the carboxylic acid is changed from dichloroacetic to acetic acid. The change in these Brønsted coefficients with changing reaction driving force, ∂α∕∂ΔGavo=∂β∕∂ΔGavo=1∕8Λ=0.011 is used to calculate a Marcus intrinsic reaction barrier of Λ = 11 kcal/mol which is close to the barrier of 13 kcal/mol for thermoneutral proton transfer between this series of acids and bases. The value of α = 0.66 for thermoneutral proton transfer is greater than α = 0.50 required by a reaction that follows the Marcus equation. This elevated value of β may be due to an asymmetry in the reaction coordinate that arises from the difference in the intrinsic barriers for proton transfer at the oxygen acid reactant and resonance stabilized carbon acid product. PMID:17488079

  15. Evaluating Rare Amino Acid Substitutions (RGC_CAMs) in a Yeast Model Clade

    PubMed Central

    Polzin, Kenneth; Rokas, Antonis

    2014-01-01

    When inferring phylogenetic relationships, not all sites in a sequence alignment are equally informative. One recently proposed approach that takes advantage of this inequality relies on sites that contain amino acids whose replacement requires multiple substitutions. Identifying these so-called RGC_CAM substitutions (after Rare Genomic Changes as Conserved Amino acids-Multiple substitutions) requires that, first, at any given site in the amino acid sequence alignment, there must be a minimum of two different amino acids; second, each amino acid must be present in at least two taxa; and third, the amino acids must require a minimum of two nucleotide substitutions to replace each other. Although theory suggests that RGC_CAM substitutions are expected to be rare and less likely to be homoplastic, the informativeness of RGC_CAM substitutions has not been extensively evaluated in biological data sets. We investigated the quality of RGC_CAM substitutions by examining their degree of homoplasy and internode certainty in nearly 2.7 million aligned amino acid sites from 5,261 proteins from five species belonging to the yeast Saccharomyces sensu stricto clade whose phylogeny is well-established. We identified 2,647 sites containing RGC_CAM substitutions, a number that contrasts sharply with the 100,887 sites containing RGC_non-CAM substitutions (i.e., changes between amino acids that require only a single nucleotide substitution). We found that RGC_CAM substitutions had significantly lower homoplasy than RGC_non-CAM ones; specifically RGC_CAM substitutions showed a per-site average homoplasy index of 0.100, whereas RGC_non-CAM substitutions had a homoplasy index of 0.215. Internode certainty values were also higher for sites containing RGC_CAM substitutions than for RGC_non-CAM ones. These results suggest that RGC_CAM substitutions possess a strong phylogenetic signal and are useful markers for phylogenetic inference despite their rarity. PMID:24637883

  16. Direct ortho-arylation of ortho-substituted benzoic acids: overriding Pd-catalyzed protodecarboxylation.

    PubMed

    Arroniz, Carlos; Ironmonger, Alan; Rassias, Gerry; Larrosa, Igor

    2013-02-15

    ortho-Arylation of ortho-substituted benzoic acids is a challenging process due to the tendency of the reaction products toward Pd-catalyzed protodecarboxylation. A simple method for preventing decarboxylation in sterically hindered benzoic acids is reported. The method described represents a reliable and broadly applicable entry to 2-aryl-6-substituted benzoic acids. PMID:23373630

  17. 40 CFR 721.2900 - Substituted aminobenzoic acid ester (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted aminobenzoic acid ester... Specific Chemical Substances § 721.2900 Substituted aminobenzoic acid ester (generic name). (a) Chemical... acid ester (PMN P-84-951) is subject to reporting under this section for the significant new...

  18. 40 CFR 721.2900 - Substituted aminobenzoic acid ester (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Substituted aminobenzoic acid ester... Specific Chemical Substances § 721.2900 Substituted aminobenzoic acid ester (generic name). (a) Chemical... acid ester (PMN P-84-951) is subject to reporting under this section for the significant new...

  19. SYNTHESIS AND CHARACTERIZATION OF SUBSTITUTED POLY(STYRENE)-b-POLY(ACRYLIC ACID) BLOCK COPOLYMER MICELLES

    SciTech Connect

    Pickel, Deanna L; Pickel, Joseph M; Devenyi, Jozsef; Britt, Phillip F

    2009-01-01

    Block copolymer micelle synthesis and characterization has been extensively studied. In particular, most studies have focused on the properties of the hydrophilic corona due to the micelle corona structure s impact on the biodistribution and biocompatibility. Unfortunately, less attention has been given to the effect of the core block on the micelle stability, morphology, and the rate of diffusion of small molecules from the core. This investigation is focused on the synthesis of block copolymers composed of meta-substituted styrenes and acrylic acid by Atom Transfer Radical Polymerization. Micelles with cores composed of substituted styrenes having Tgs ranging from -30 to 100 oC have been prepared and the size and shape of these micelles were characterized by Static and Dynamic Light Scattering and TEM. In addition, the critical micelle concentration and rate of diffusion of small molecules from the core were determined by fluorimetry using pyrene as the probe.

  20. 4-Hydroxybenzyl-substituted amino acid derivatives from Gastrodia elata

    PubMed Central

    Guo, Qinglan; Wang, Yanan; Lin, Sheng; Zhu, Chenggen; Chen, Minghua; Jiang, Zhibo; Xu, Chengbo; Zhang, Dan; Wei, Huailing; Shi, Jiangong

    2015-01-01

    Seven new 4-hydroxybenzyl-substituted amino acid derivatives (1−7), together with 11 known compounds, were isolated from an aqueous extract of the rhizomes of Gastrodia elata Blume. Their structures were determined by spectroscopic and chemical methods. Compounds 1−3 are pyroglutamate derivatives containing 4-hydroxybenzyl units at the N atom and 4−7 are the first examples of natural products with the 4-hydroxybenzyl unit linked via a thioether bond to 2-hydroxy-3-mercaptopropanoic acid (4−6) and 2-hydroxy-4-mercaptobutanoic acid (7), which would be biogenetically derived from cysteine and homocysteine, respectively. The structures of 1 and 2 were verified by synthesis, while the absolute configurations of 4, 5 and 7 were assigned using Mosher’s method based on the MPA determination rule of ΔδRS values. The known compound 4-(hydroxymethyl)-5-nitrobenzene-1,2-diol (8) exhibited activity against Fe2+-cysteine induced rat liver microsomal lipid peroxidation with IC50 values of 9.99×10−6 mol/L. PMID:26579466

  1. Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids.

    PubMed

    Cortinas, Irail; Field, Jim A; Kopplin, Mike; Garbarino, John R; Gandolfi, A Jay; Sierra-Alvarez, Reyes

    2006-05-01

    Large quantities of arsenic are introduced into the environment through land application of poultry litter containing the organoarsenical feed additive roxarsone (3-nitro-4-hydroxyphenylarsonic acid). The objective of this study was to evaluate the bioconversion of roxarsone and related N-substituted phenylarsonic acid derivatives under anaerobic conditions. The results demonstrate that roxarsone is rapidly transformed in the absence of oxygen to the corresponding aromatic amine, 4-hydroxy-3-aminophenylarsonic acid (HAPA). The formation of HAPA is attributable to the facile reduction of the nitro group. Electron-donating substrates, such as hydrogen gas, glucose, and lactate, stimulated the rate of nitro group reduction, indicating a microbial role. During long-term incubations, HAPA and the closely related 4-aminophenylarsonic acid (4-APA) were slowly biologically eliminated by up to 99% under methanogenic and sulfate-reducing conditions, whereas little or no removal occurred in heat-killed inoculum controls. Arsenite and, to a lesser extent, arsenate were observed as products of the degradation. Freely soluble forms of the inorganic arsenical species accounted for 19-28% of the amino-substituted phenylarsonic acids removed. This constitutes the first report of a biologically catalyzed rupture of the phenylarsonic group under anaerobic conditions. PMID:16719096

  2. Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids

    USGS Publications Warehouse

    Cortinas, I.; Field, J.A.; Kopplin, M.; Garbarino, J.R.; Gandolfi, A.J.; Sierra-Alvarez, R.

    2006-01-01

    Large quantities of arsenic are introduced into the environment through land application of poultry litter containing the organoarsenical feed additive roxarsone (3-nitro-4-hydroxyphenylarsonic acid). The objective of this study was to evaluate the bioconversion of roxarsone and related N-substituted phenylarsonic acid derivatives under anaerobic conditions. The results demonstrate that roxarsone is rapidly transformed in the absence of oxygen to the corresponding aromatic amine, 4-hydroxy-3-aminophenylarsonic acid (HAPA). The formation of HAPA is attributable to the facile reduction of the nitro group. Electron-donating substrates, such as hydrogen gas, glucose, and lactate, stimulated the rate of nitro group reduction, indicating a microbial role. During long-term incubations, HAPA and the closely related 4-aminophenylarsonic acid (4-APA) were slowly biologically eliminated by up to 99% under methanogenic and sulfate-reducing conditions, whereas little or no removal occurred in heat-killed inoculum controls. Arsenite and, to a lesser extent, arsenate were observed as products of the degradation. Freely soluble forms of the inorganic arsenical species accounted for 19-28% of the amino-substituted phenylarsonic acids removed. This constitutes the first report of a biologically catalyzed rupture of the phenylarsonic group under anaerobic conditions. ?? 2006 American Chemical Society.

  3. 40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified...

  4. 40 CFR 721.430 - Oxo-substituted amino-al-kanoic acid derivative.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Oxo-substituted amino-al-kanoic acid... Specific Chemical Substances § 721.430 Oxo-substituted amino-al-kanoic acid derivative. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified...

  5. Rates of molecular evolution and diversification in plants: chloroplast substitution rates correlate with species-richness in the Proteaceae

    PubMed Central

    2013-01-01

    Background Many factors have been identified as correlates of the rate of molecular evolution, such as body size and generation length. Analysis of many molecular phylogenies has also revealed correlations between substitution rates and clade size, suggesting a link between rates of molecular evolution and the process of diversification. However, it is not known whether this relationship applies to all lineages and all sequences. Here, in order to investigate how widespread this phenomenon is, we investigate patterns of substitution in chloroplast genomes of the diverse angiosperm family Proteaceae. We used DNA sequences from six chloroplast genes (6278bp alignment with 62 taxa) to test for a correlation between diversification and the rate of substitutions. Results Using phylogenetically-independent sister pairs, we show that species-rich lineages of Proteaceae tend to have significantly higher chloroplast substitution rates, for both synonymous and non-synonymous substitutions. Conclusions We show that the rate of molecular evolution in chloroplast genomes is correlated with net diversification rates in this large plant family. We discuss the possible causes of this relationship, including molecular evolution driving diversification, speciation increasing the rate of substitutions, or a third factor causing an indirect link between molecular and diversification rates. The link between the synonymous substitution rate and clade size is consistent with a role for the mutation rate of chloroplasts driving the speed of reproductive isolation. We find no significant differences in the ratio of non-synonymous to synonymous substitutions between lineages differing in net diversification rate, therefore we detect no signal of population size changes or alteration in selection pressures that might be causing this relationship. PMID:23497266

  6. An injectable oxidated hyaluronic acid/adipic acid dihydrazide hydrogel as a vitreous substitute.

    PubMed

    Su, Wen-Yu; Chen, Ko-Hua; Chen, Yu-Chun; Lee, Yen-Hsien; Tseng, Ching-Li; Lin, Feng-Huei

    2011-01-01

    Vitrectomy is a common procedure for treating ocular-related diseases. The surgery involves removing the vitreous humor from the center of the eye, and vitreous substitutes are needed to replace the vitreous humor after vitrectomy. In the present study, we developed a colorless, transparent and injectable hydrogel with appropriate refractive index as a vitreous substitute. The hydrogel is formed by oxidated hyaluronic acid (oxi-HA) cross-linked with adipic acid dihydrazide (ADH). Hyaluronic acid (HA) was oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by ADH. The refractive index of this hydrogel ranged between 1.3420 and 1.3442, which is quite similar to human vitreous humor (1.3345). The degradation tests demonstrated that the hydrogel could maintain the gel matrix over 35 days, depending on the ADH concentration. In addition, the cytotoxicity was evaluated on retina pigmented epithelium (RPE) cells cultivated following the ISO standard (tests for in vitro cytotoxicity), and the hydrogel was found to be non-toxic. In a preliminary animal study, the oxi-HA/ADH hydrogel was injected into the vitreous cavity of rabbit eyes. The evaluations of slit-lamp observation, intraocular pressure, cornea thickness and histological examination showed no significant abnormal biological reactions for 3 weeks. This study suggests that the injectable oxi-HA/ADH hydrogel should be a potential vitreous substitute. PMID:20843434

  7. Correlated mutations: a hallmark of phenotypic amino acid substitutions.

    PubMed

    Kowarsch, Andreas; Fuchs, Angelika; Frishman, Dmitrij; Pagel, Philipp

    2010-01-01

    Point mutations resulting in the substitution of a single amino acid can cause severe functional consequences, but can also be completely harmless. Understanding what determines the phenotypical impact is important both for planning targeted mutation experiments in the laboratory and for analyzing naturally occurring mutations found in patients. Common wisdom suggests using the extent of evolutionary conservation of a residue or a sequence motif as an indicator of its functional importance and thus vulnerability in case of mutation. In this work, we put forward the hypothesis that in addition to conservation, co-evolution of residues in a protein influences the likelihood of a residue to be functionally important and thus associated with disease. While the basic idea of a relation between co-evolution and functional sites has been explored before, we have conducted the first systematic and comprehensive analysis of point mutations causing disease in humans with respect to correlated mutations. We included 14,211 distinct positions with known disease-causing point mutations in 1,153 human proteins in our analysis. Our data show that (1) correlated positions are significantly more likely to be disease-associated than expected by chance, and that (2) this signal cannot be explained by conservation patterns of individual sequence positions. Although correlated residues have primarily been used to predict contact sites, our data are in agreement with previous observations that (3) many such correlations do not relate to physical contacts between amino acid residues. Access to our analysis results are provided at http://webclu.bio.wzw.tum.de/~pagel/supplements/correlated-positions/. PMID:20862353

  8. Interactions in the solid state. II: Interaction of sodium bicarbonate with substituted benzoic acids in the presence of moisture

    SciTech Connect

    Wright, J.L.; Carstensen, J.T.

    1986-06-01

    The interaction of an organic acid with sodium bicarbonate in water produces an effervescent reaction. The reaction products are carbon dioxide, water, and the sodium salt of the acid. The kinetic rate-determining step for this reaction is the dehydration of carbonic acid. The solid-solid interaction with known amounts of moisture was followed by quantitatively determining carbon dioxide evolution as a function of time. The aqueous solubilities, diffusion coefficients, dissociation constants, and solid-solid interaction rates of six different substituted benzoic acids were determined. Using a model based on diffusion of the organic acid through the aqueous layer coupled with chemical reaction, predicted rates and levels of carbon dioxide production were compared with experimental results. Included in the model were the effects of the reaction products on the solution properties of the reactants. It was found that high concentrations of substituted sodium benzoate were generated very quickly and affected the solubility of the reactants, diffusion coefficient of the acid, and the carbonic acid dehydration rate constant. Moisture content was found to have a profound influence on the interaction rate. Water provides a medium for diffusion of the reacting species as well as the reaction solvent.

  9. Theoretical Analysis of Kinetic Isotope Effects on Proton Transfer Reactions between Substituted α-Methoxystyrenes and Substituted Acetic Acids

    PubMed Central

    Wong, Kin Yiu; Richard, John P.; Gao, Jiali

    2009-01-01

    Primary kinetic isotope effects (KIEs) on a series of carboxylic acid-catalyzed protonation reactions of aryl-substituted α-methoxystyrenes (X-1) to form oxocarbenium ions have been computed using the Kleinert variational second-order perturbation theory (KP2) in the framework of Feynman path integrals (PI) along with the potential energy surface obtained at the B3LYP/6-31+G(d,p) level. Good agreement with the experimental data was obtained, demonstrating that this novel computational approach for computing KIEs of organic reactions is a viable alternative to the traditional method employing Bigeleisen equation and harmonic vibrational frequencies. Although tunneling makes relative small contributions to the lowering of the free energy barriers for the carboxylic acid catalyzed protonation reaction, it is necessary to include tunneling contributions to obtain quantitative estimates of the KIEs. Consideration of anharmonicity can further improve the calculated KIEs for the protonation of substituted α-methoxystyrenes by chloroacetic acid, but for the reactions of the parent and 4-NO2 substituted α-methoxystyrene with substituted carboxylic acids, the correction of anharmonicity overestimates the computed KIEs for strong acid catalysts. In agreement with experimental findings, the largest KIEs are found in nearly ergoneutral reactions, ΔGo ≈ 0, where the transition structures are nearly symmetric and the reaction barriers are relatively low. Furthermore, the optimized transition structures are strongly dependent on the free energy for the formation of the carbocation intermediate, i.e., the driving force ΔGo, along with a good correlation of Hammond shift in the transition state structure. PMID:19754046

  10. Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene

    PubMed Central

    Erixon, Per; Oxelman, Bengt

    2008-01-01

    Background Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare. Methodology/Principle Findings We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family) and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family). Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying) selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs. Conclusions/Significance We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the controversial issue

  11. 40 CFR 721.10448 - Acetic acid, hydroxy- methoxy-, methyl ester, reaction products with substituted alkylamine...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ester, reaction products with substituted alkylamine (generic). 721.10448 Section 721.10448 Protection... NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10448 Acetic acid, hydroxy- methoxy-, methyl ester, reaction products with substituted alkylamine (generic)....

  12. 40 CFR 721.10448 - Acetic acid, hydroxy- methoxy-, methylester, reaction products with substituted alkylamine...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...-, methylester, reaction products with substituted alkylamine (generic). 721.10448 Section 721.10448 Protection... NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.10448 Acetic acid, hydroxy- methoxy-, methylester, reaction products with substituted alkylamine (generic)....

  13. 40 CFR 721.10690 - Benzenedicarboxylic acid, polymer with substituted alkanediol, dodecanedioic acid, 1,2-ethanediol...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzenedicarboxylic acid, polymer with substituted alkanediol, dodecanedioic acid, 1,2-ethanediol, alkanedioic acid, alkanediol,.alpha.-hydro-.omega.-hydroxypoly , 1,3-isobenzofurandione, methylene diphenyl diisocyanate, 2-oxepanone, 2,2'-oxybis and polymethylene polyphenylene...

  14. 40 CFR 721.7785 - Substituted alkyl aminomethylene polyphosphonic acid, salt (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... polyphosphonic acid, salt (generic). 721.7785 Section 721.7785 Protection of Environment ENVIRONMENTAL PROTECTION... acid, salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted alkylamino methylene polyphosphonic acid,...

  15. 40 CFR 721.7785 - Substituted alkyl aminomethylene polyphosphonic acid, salt (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... polyphosphonic acid, salt (generic). 721.7785 Section 721.7785 Protection of Environment ENVIRONMENTAL PROTECTION... acid, salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted alkylamino methylene polyphosphonic acid,...

  16. 40 CFR 721.10045 - Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (generic). 721.10045... Substances § 721.10045 Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid... heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (PMN P-02-737)...

  17. 40 CFR 721.10045 - Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (generic). 721.10045... Substances § 721.10045 Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid... heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (PMN P-02-737)...

  18. 40 CFR 721.10045 - Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (generic). 721.10045... Substances § 721.10045 Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid... heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (PMN P-02-737)...

  19. 40 CFR 721.10045 - Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (generic). 721.10045... Substances § 721.10045 Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid... heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (PMN P-02-737)...

  20. 40 CFR 721.7785 - Substituted alkyl aminomethylene polyphosphonic acid, salt (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polyphosphonic acid, salt (generic). 721.7785 Section 721.7785 Protection of Environment ENVIRONMENTAL PROTECTION... acid, salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted alkylamino methylene polyphosphonic acid,...

  1. 40 CFR 721.7785 - Substituted alkyl aminomethylene polyphosphonic acid, salt (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... polyphosphonic acid, salt (generic). 721.7785 Section 721.7785 Protection of Environment ENVIRONMENTAL PROTECTION... acid, salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted alkylamino methylene polyphosphonic acid,...

  2. 40 CFR 721.7785 - Substituted alkyl aminomethylene polyphosphonic acid, salt (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... polyphosphonic acid, salt (generic). 721.7785 Section 721.7785 Protection of Environment ENVIRONMENTAL PROTECTION... acid, salt (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as a substituted alkylamino methylene polyphosphonic acid,...

  3. Single amino acid substitutions influencing the folding pathway of the phage P22 tail spike endorhamnosidase.

    PubMed

    Yu, M H; King, J

    1984-11-01

    Temperature-sensitive mutations in the gene for the thermostable tail spike of phage P22 interyFere with the folding and subunit association pathway at the restrictive temperature but not with the activity or stability of the protein once matured. The local sites of these mutations and the mutant amino acid substitutions have been determined by DNA sequencing. Of 11 temperature-sensitive folding mutations, 3 were replacements of glycine residues by polar residues, and three were replacements of threonine residues by residues unable to form a side-chain H-bond. There were no proline replacements. Two of the temperature-sensitive sites in which threonine residues were replaced by isoleucine residues were homologous. These sequences probably maintain the correct local folding pathway at higher temperatures. The temperature-sensitive amino acid substitutions appear to destabilize a thermolabile intermediate in the wild-type folding pathway or to increase the rate of a competing off-pathway reaction. PMID:6387707

  4. Specificity of Aspartate Aminotransferases from Leguminous Plants for 4-Substituted Glutamic Acids 1

    PubMed Central

    Winter, Harry C.; Dekker, Eugene E.

    1989-01-01

    Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4-methyleneglutarate was least effective (lowest relative Vm/Km) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase. PMID:16666674

  5. Correlated evolution of nucleotide substitution rates and allelic variation in Mhc-DRB lineages of primates

    PubMed Central

    Garamszegi, László Z; de Groot, Natasja G; Bontrop, Ronald E

    2009-01-01

    Background The major histocompatibility complex (MHC) is a key model of genetic polymorphism. Selection pressure by pathogens or other microevolutionary forces may result in a high rate of non-synonymous substitutions at the codons specifying the contact residues of the antigen binding sites (ABS), and the maintenance of extreme MHC allelic variation at the population/species level. Therefore, selection forces favouring MHC variability for any reason should cause a correlated evolution between substitution rates and allelic polymorphism. To investigate this prediction, we characterised nucleotide substitution rates and allelic polymorphism (i.e. the number of alleles detected in relation to the number of animals screened) of several Mhc class II DRB lineages in 46 primate species, and tested for a correlation between them. Results First, we demonstrate that species-specific and lineage-specific evolutionary constraints favour species- and lineage-dependent substitution rate at the codons specifying the ABS contact residues (i.e. certain species and lineages can be characterised by high substitution rate, while others have low rate). Second, we show that although the degree of the non-synonymous substitution rate at the ABS contact residues was systematically higher than the degree of the synonymous substitution rate, these estimates were strongly correlated when we controlled for species-specific and lineage-specific effects, and also for the fact that different studies relied on different sample size. Such relationships between substitution rates of different types could even be extended to the non-contact residues of the molecule. Third, we provide statistical evidence that increased substitution rate along a MHC gene may lead to allelic variation, as a high substitution rate can be observed in those lineages in which many alleles are maintained. Fourth, we show that the detected patterns were independent of phylogenetic constraints. When we used phylogenetic

  6. Genes Translocated into the Plastid Inverted Repeat Show Decelerated Substitution Rates and Elevated GC Content.

    PubMed

    Li, Fay-Wei; Kuo, Li-Yaung; Pryer, Kathleen M; Rothfels, Carl J

    2016-01-01

    Plant chloroplast genomes (plastomes) are characterized by an inverted repeat (IR) region and two larger single copy (SC) regions. Patterns of molecular evolution in the IR and SC regions differ, most notably by a reduced rate of nucleotide substitution in the IR compared to the SC region. In addition, the organization and structure of plastomes is fluid, and rearrangements through time have repeatedly shuffled genes into and out of the IR, providing recurrent natural experiments on how chloroplast genome structure can impact rates and patterns of molecular evolution. Here we examine four loci (psbA, ycf2, rps7, and rps12 exon 2-3) that were translocated from the SC into the IR during fern evolution. We use a model-based method, within a phylogenetic context, to test for substitution rate shifts. All four loci show a significant, 2- to 3-fold deceleration in their substitution rate following translocation into the IR, a phenomenon not observed in any other, nontranslocated plastid genes. Also, we show that after translocation, the GC content of the third codon position and of the noncoding regions is significantly increased, implying that gene conversion within the IR is GC-biased. Taken together, our results suggest that the IR region not only reduces substitution rates, but also impacts nucleotide composition. This finding highlights a potential vulnerability of correlating substitution rate heterogeneity with organismal life history traits without knowledge of the underlying genome structure. PMID:27401175

  7. Genes Translocated into the Plastid Inverted Repeat Show Decelerated Substitution Rates and Elevated GC Content

    PubMed Central

    Li, Fay-Wei; Kuo, Li-Yaung; Pryer, Kathleen M.; Rothfels, Carl J.

    2016-01-01

    Plant chloroplast genomes (plastomes) are characterized by an inverted repeat (IR) region and two larger single copy (SC) regions. Patterns of molecular evolution in the IR and SC regions differ, most notably by a reduced rate of nucleotide substitution in the IR compared to the SC region. In addition, the organization and structure of plastomes is fluid, and rearrangements through time have repeatedly shuffled genes into and out of the IR, providing recurrent natural experiments on how chloroplast genome structure can impact rates and patterns of molecular evolution. Here we examine four loci (psbA, ycf2, rps7, and rps12 exon 2–3) that were translocated from the SC into the IR during fern evolution. We use a model-based method, within a phylogenetic context, to test for substitution rate shifts. All four loci show a significant, 2- to 3-fold deceleration in their substitution rate following translocation into the IR, a phenomenon not observed in any other, nontranslocated plastid genes. Also, we show that after translocation, the GC content of the third codon position and of the noncoding regions is significantly increased, implying that gene conversion within the IR is GC-biased. Taken together, our results suggest that the IR region not only reduces substitution rates, but also impacts nucleotide composition. This finding highlights a potential vulnerability of correlating substitution rate heterogeneity with organismal life history traits without knowledge of the underlying genome structure. PMID:27401175

  8. Trends in Hanging and Firearm Suicide Rates in Australia: Substitution of Method?

    ERIC Educational Resources Information Center

    De Leo, Diego; Dwyer, Jonathan; Firman, David; Neulinger, Kerryn

    2003-01-01

    This study examined the increase in the rate of suicide by hanging and an apparently simultaneous decrease in the rate of suicide by firearm as hypothetical evidence that Australian males have substituted one method of suicide for another. Individual suicide method choice may be related to independent changes in the social acceptability of each…

  9. 40 CFR 721.7770 - Alkylphenoxypoly(oxyethylene) sulfuric acid ester, substituted amine salt.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Significant New Uses for Specific Chemical Substances § 721.7770 Alkylphenoxypoly(oxyethylene) sulfuric acid...) The chemical substance identified as alkyl phenoxypoly(oxyethylene) sulfuric acid ester, substituted... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alkylphenoxypoly(oxyethylene)...

  10. 40 CFR 721.7770 - Alkylphenoxypoly(oxyethylene) sulfuric acid ester, substituted amine salt.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Significant New Uses for Specific Chemical Substances § 721.7770 Alkylphenoxypoly(oxyethylene) sulfuric acid...) The chemical substance identified as alkyl phenoxypoly(oxyethylene) sulfuric acid ester, substituted... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alkylphenoxypoly(oxyethylene)...

  11. 40 CFR 721.7770 - Alkylphenoxypoly(oxyethylene) sulfuric acid ester, substituted amine salt.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Significant New Uses for Specific Chemical Substances § 721.7770 Alkylphenoxypoly(oxyethylene) sulfuric acid...) The chemical substance identified as alkyl phenoxypoly(oxyethylene) sulfuric acid ester, substituted... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alkylphenoxypoly(oxyethylene)...

  12. 40 CFR 721.7770 - Alkylphenoxypoly(oxyethylene) sulfuric acid ester, substituted amine salt.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Significant New Uses for Specific Chemical Substances § 721.7770 Alkylphenoxypoly(oxyethylene) sulfuric acid...) The chemical substance identified as alkyl phenoxypoly(oxyethylene) sulfuric acid ester, substituted... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Alkylphenoxypoly(oxyethylene)...

  13. 40 CFR 721.7770 - Alkylphenoxypoly(oxyethylene) sulfuric acid ester, substituted amine salt.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Significant New Uses for Specific Chemical Substances § 721.7770 Alkylphenoxypoly(oxyethylene) sulfuric acid...) The chemical substance identified as alkyl phenoxypoly(oxyethylene) sulfuric acid ester, substituted... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alkylphenoxypoly(oxyethylene)...

  14. Estimating Divergence Times and Substitution Rates in Rhizobia.

    PubMed

    Chriki-Adeeb, Rim; Chriki, Ali

    2016-01-01

    Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth-death, birth-death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes. PMID:27168719

  15. Estimating Divergence Times and Substitution Rates in Rhizobia

    PubMed Central

    Chriki-Adeeb, Rim; Chriki, Ali

    2016-01-01

    Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth–death, birth–death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes. PMID:27168719

  16. Analysis of amino acid substitutions in AraC variants that respond to triacetic acid lactone.

    PubMed

    Frei, Christopher S; Wang, Zhiqing; Qian, Shuai; Deutsch, Samuel; Sutter, Markus; Cirino, Patrick C

    2016-04-01

    The Escherichia coli regulatory protein AraC regulates expression of ara genes in response to l-arabinose. In efforts to develop genetically encoded molecular reporters, we previously engineered an AraC variant that responds to the compound triacetic acid lactone (TAL). This variant (named "AraC-TAL1") was isolated by screening a library of AraC variants, in which five amino acid positions in the ligand-binding pocket were simultaneously randomized. Screening was carried out through multiple rounds of alternating positive and negative fluorescence-activated cell sorting. Here we show that changing the screening protocol results in the identification of different TAL-responsive variants (nine new variants). Individual substituted residues within these variants were found to primarily act cooperatively toward the gene expression response. Finally, X-ray diffraction was used to solve the crystal structure of the apo AraC-TAL1 ligand-binding domain. The resolved crystal structure confirms that this variant takes on a structure nearly identical to the apo wild-type AraC ligand-binding domain (root-mean-square deviation 0.93 Å), suggesting that AraC-TAL1 behaves similar to wild-type with regard to ligand recognition and gene regulation. Our results provide amino acid sequence-function data sets for training and validating AraC modeling studies, and contribute to our understanding of how to design new biosensors based on AraC. PMID:26749125

  17. The microwave spectrum of isotopically substituted hypochlorous acid: Determination of the molecular structure

    NASA Astrophysics Data System (ADS)

    Anderson, W. Darlene; Gerry, M. C. L.; Davis, R. Wellington

    1986-01-01

    Microwave spectra have been measured for four isotopically substituted species of hypochlorous acid (D 16O 35Cl, D 16O 37Cl, H 18O 35Cl, H 18O 37Cl). Both a- and b-type transitions have been analyzed for rotational, centrifugal distortion, and Cl nuclear quadrupole coupling constants. The distortion constants, together with vibrational wavenumbers, have been used to evaluate a valence harmonic force field. Effective, substitution, ground state average, and estimated equilibrium structures are presented.

  18. Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods.

    PubMed

    Wende, Frida J; Gohil, Suresh; Mojarradi, Hotan; Gerfaud, Thibaud; Nord, Lars I; Karlsson, Anders; Boiteau, Jean-Guy; Kenne, Anne Helander; Sandström, Corine

    2016-01-20

    In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required. NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at ΔGlcA-OH2 and GlcNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end GlcNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end GlcNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers. PMID:26572480

  19. 40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical...

  20. 40 CFR 721.321 - Substituted acrylamides and acrylic acid copolymer (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.321 Substituted acrylamides and acrylic acid copolymer (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical...

  1. 40 CFR 721.530 - Substituted aliphatic acid halide (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... substance and significant new uses subject to reporting. (1) The chemical substance substituted aliphatic acid halide (PMN P-84-491) is subject to reporting under this section for the significant new uses... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses...

  2. 40 CFR 721.530 - Substituted aliphatic acid halide (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... substance and significant new uses subject to reporting. (1) The chemical substance substituted aliphatic acid halide (PMN P-84-491) is subject to reporting under this section for the significant new uses... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses...

  3. Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

    PubMed

    Hellenbrand, Tim; Höfner, Georg; Wein, Thomas; Wanner, Klaus T

    2016-05-01

    In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified. PMID:27039250

  4. Efficiency of substitution of 2-ketoisocaproic acid and 2-ketoisovaleric acid in the diet of normal and uremic growing rats.

    PubMed

    Laouari, D; Kamoun, P P; Rocchiccioli, F; Dodu, C; Kleinknecht, C; Broyer, M

    1986-12-01

    Effects of various intakes of the ketoanalogues of leucine (KICA) and valine (KIVA) on growth, nitrogen, and urea excretion were examined and compared to those of an optimal intake (A) of the corresponding amino acids. Diet KICA and KIVA contents varied from 1 to 4 times A. In controls, growth was significantly reduced with equimolar substitution, corrected with twice A, and unchanged at higher levels. Doubling KICA corrected growth except with substantial anorexia. In uremic rats fed KIVA, growth was corrected at twice A. Low-KICA diets reduced plasma-leucine level; higher KICA diets normalized plasma leucine and revealed branched-chain amino acid (BCAA) antagonism. Changes in 2-ketoacids were unrelated to those of BCAA. In uremia, KICA decreased plasma and urinary urea without changing nitrogen retention. Ketoacid substitution for amino acids was 50% efficient in normal rats and not altered by uremia. BCKAs, specifically KICA, could modify urea metabolism. PMID:3788832

  5. 10-Boronic acid substituted camptothecin as prodrug of SN-38.

    PubMed

    Wang, Lei; Xie, Shao; Ma, Longjun; Chen, Yi; Lu, Wei

    2016-06-30

    Malignant tumor cells have been found to have high levels of reactive oxygen species such as hydrogen peroxide (H2O2), supporting the hypothesis that a prodrug could be activated by intracellular H2O2 and lead to a potential antitumor therapy. In this study, the 7-ethyl-10-boronic acid camptothecin (B1) was synthesized for the first time as prodrug of SN-38, by linking a cleavable aryl carbon-boron bond to the SN-38. Prodrug B1 selectively activated by H2O2, converted rapidly to the active form SN-38 under favorable oxidative conditions in cancer cells with elevated levels of H2O2. The cell survival assay showed that prodrug B1 was equally or more effective in inhibiting the growth of six different cancer cells, as compared to SN-38. Unexpectedly, prodrug B1 displayed even more potent Topo I inhibitory activity than SN-38, suggesting that it was not only a prodrug of SN-38 but also a typical Topo I inhibitor. Prodrug B1 also demonstrated a significant antitumor activity at 2.0 mg/kg in a xenograft model using human brain star glioblastoma cell lines U87MG. PMID:27060760

  6. The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates

    PubMed Central

    2012-01-01

    Background Understanding the causes underlying heterogeneity of molecular evolutionary rates among lineages is a long-standing and central question in evolutionary biology. Although several earlier studies showed that modern frogs (Neobatrachia) experienced an acceleration of mitochondrial gene substitution rates compared to non-neobatrachian relatives, no further characterization of this phenomenon was attempted. To gain new insights on this topic, we sequenced the complete mitochondrial genomes and nine nuclear loci of one pelobatoid (Pelodytes punctatus) and five neobatrachians, Heleophryne regis (Heleophrynidae), Lechriodus melanopyga (Limnodynastidae), Calyptocephalella gayi (Calyptocephalellidae), Telmatobius bolivianus (Ceratophryidae), and Sooglossus thomasseti (Sooglossidae). These represent major clades not included in previous mitogenomic analyses, and most of them are remarkably species-poor compared to other neobatrachians. Results We reconstructed a fully resolved and robust phylogeny of extant frogs based on the new mitochondrial and nuclear sequence data, and dated major cladogenetic events. The reconstructed tree recovered Heleophryne as sister group to all other neobatrachians, the Australasian Lechriodus and the South American Calyptocephalella formed a clade that was the sister group to Nobleobatrachia, and the Seychellois Sooglossus was recovered as the sister group of Ranoides. We used relative-rate tests and direct comparison of branch lengths from mitochondrial and nuclear-based trees to demonstrate that both mitochondrial and nuclear evolutionary rates are significantly higher in all neobatrachians compared to their non-neobatrachian relatives, and that such rate acceleration started at the origin of Neobatrachia. Conclusions Through the analysis of the selection coefficient (ω) in different branches of the tree, we found compelling evidence of relaxation of purifying selection in neobatrachians, which could (at least in part) explain the

  7. Natural derivatives of diphenolic acid as substitutes for bisphenol-A

    NASA Astrophysics Data System (ADS)

    Ertl, Johanna; Cerri, Elisa; Rizzuto, Matteo; Caretti, Daniele

    2014-05-01

    Diphenolic acid had been originally used in the first epoxy resins and was later on forgotten as it was substituted by the cheaper bisphenol A. But in the recent years major health concerns have been raised as bisphenol A has a pseudo-hormonal effect on the body, playing the role of estrogen it can cause a severe impact on the organism, especially in males. Moreover it is produced from acetone and phenol, both from fossil, and thus limited resources. On the contrary, diphenolic acid is synthesized from levulinic acid and phenol. Levulinic acid being directly produced by hydrolysis of biomass. By substituting the fossil phenol with natural phenols from lignin or plant extraction we are able to synthesize a fully renewable substitute for bisphenol A. The reactions to yield an epoxy resin have been examined and the reactivity with epichlorohydrin is satisfying. Moreover, some of the derivatives of diphenolic acid have interesting curing properties and preliminary results show excellent properties of the cured resin, including thermal stability and pencil hardness.

  8. Competition between the hydrolysis and deamination of cytidine and its 5-substituted derivatives in aqueous acid.

    PubMed Central

    Lönnberg, H; Käppi, R

    1985-01-01

    The monocations of a few 5-substituted cytidines have been shown to undergo competitive deamination to the corresponding uridines and hydrolysis to 5-substituted cytosines and D-ribose. The first-order rate constants measured at different temperatures indicate that the proportion of the hydrolysis is considerably increased with the increasing temperature. Electron-withdrawal by a polar substituent at C5 appears to facilitate the hydrolysis to a larger extent that the deamination. The ionic strength has practically no influence on the rate of either reaction. PMID:4000961

  9. Microheterogeneity of antithrombin III: effect of single amino acid substitutions and relationship with functional abnormalities.

    PubMed

    De Stefano, V; Leone, G; Mastrangelo, S; Lane, D A; Girolami, A; de Moerloose, P; Sas, G; Abildgaard, U; Blajchman, M; Rodeghiero, F

    1994-02-01

    Microheterogeneity of antithrombin III (AT-III) was investigated by crossed immunoelectrofocusing (CIEF) on eleven molecular variants. A normal pattern was found in five variants while two different abnormal CIEF patterns were found in the other four and two variants, respectively. Point mutations causing a major pI change (exceeding 4.0) of the amino acid substituted lead to alterations in the overall microheterogeneity. The variants thus substituted share a first type of abnormal CIEF pattern with alterations throughout the pH range, regardless of the location of the mutation (reactive site and adjacent regions or heparin binding region). Minor amino acid pI changes in these regions do not alter the AT-III overall microheterogeneity, whatever the resulting functional defect. However, if the mutation is placed in the region around positions 404 or 429, then even minor changes of the amino acid pI seem able to alter the overall charge, leading to a second type of abnormal CIEF pattern with the main alteration at pH 4.8-4.6. Neuraminidase treatment leads to disappearance of microheterogeneity except for the variants with the Arg393 to Cys substitution. Addition of thrombin induces CIEF modifications specifically related to the functional defect. A normal formation of thrombin-antithrombin complexes induces a shift towards the more acid pH range, whereas in the variants substituted at the reactive site the CIEF pattern is substantially unaffected by thrombin; variants substituted at positions 382-384 show a maximal thrombin-induced increase of the isoforms at pI 4.8-4.6. Therefore mutant antithrombins with different functional abnormalities but sharing a common CIEF pattern were well distinguished.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8180341

  10. Radiation chemistry of salicylic and methyl substituted salicylic acids: Models for the radiation chemistry of pharmaceutical compounds

    NASA Astrophysics Data System (ADS)

    Ayatollahi, Shakiba; Kalnina, Daina; Song, Weihua; Turks, Maris; Cooper, William J.

    2013-11-01

    Salicylic acid and its derivatives are components of many medications and moieties found in numerous pharmaceutical compounds. They have been used as models for various pharmaceutical compounds in pharmacological studies, for the treatment of pharmaceuticals and personal care products (PPCPs), and, reactions with natural organic matter (NOM). In this study, the radiation chemistry of benzoic acid, salicylic acid and four methyl substituted salicylic acids (MSA) is reported. The absolute bimolecular reaction rate constants for hydroxyl radical reaction with benzoic and salicylic acids as well as 3-methyl-, 4-methyl-, 5-methyl-, and 6-methyl-salicylic acid were determined (5.86±0.54)×109, (1.07±0.07)×1010, (7.48±0.17)×109, (7.31±0.29)×109, (5.47±0.25)×109, (6.94±0.10)×109 (M-1 s-1), respectively. The hydrated electron reaction rate constants were measured (3.02±0.10)×109, (8.98±0.27)×109, (5.39±0.21)×109, (4.33±0.17)×109, (4.72±0.15)×109, (1.42±0.02)×109 (M-1 s-1), respectively. The transient absorption spectra for the six model compounds were examined and their role as model compounds for the radiation chemistry of pharmaceuticals investigated.

  11. Estimating a nucleotide substitution rate for maize from polymorphism at a major domestication locus.

    PubMed

    Clark, Richard M; Tavaré, Simon; Doebley, John

    2005-11-01

    To estimate a rate for single nucleotide substitutions for maize (Zea mays ssp. mays), we have taken advantage of data from genetic and archaeological studies of the domestication of maize from its wild ancestor, teosinte (Z. mays ssp. parviglumis). Genetic studies have shown that the teosinte branched1 (tb1) gene was a major target of human selection during maize domestication, and sequence diversity in the intergenic region 5' to the tb1-coding sequence is extraordinarily low. We show that polymorphism in this region is consistent with new mutation following fixation for a small number of tb1 haplotypes during domestication. Archeological studies suggest that maize was domesticated approximately 6,250-10,000 years ago and subsequently the size of the maize population is thought to have expanded rapidly. Using the observed number of mutations within the region of selection at tb1, the approximate age of maize domestication, and approximations for the maize genealogy, we have derived estimates for the nucleotide substitution rate for the tb1 intergenic region. Using two approaches, one of which is a coalescent approach, we obtain rate estimates of approximately 2.9 x 10(-8) and 3.3 x 10(-8) substitutions per site per year. We also show that the pattern of polymorphism in the tb1 intergenic region appears to have been strongly affected by the mutagenic effect of DNA methylation. Excluding target sites of symmetric DNA methylation (CG and CNG sites) from analysis, the mutation rate estimates are reduced by approximately 50%-60%, while the rates for CG and CNG sites are nearly an order of magnitude higher. We use rate estimates from the tb1 region to estimate the timing of expansion of transposable elements in the maize genome and suggest that this expansion occurred primarily within the last million years. PMID:16079248

  12. Are Convergent and Parallel Amino Acid Substitutions in Protein Evolution More Prevalent Than Neutral Expectations?

    PubMed Central

    Zou, Zhengting; Zhang, Jianzhi

    2015-01-01

    Convergent and parallel amino acid substitutions in protein evolution, collectively referred to as molecular convergence here, have small probabilities under neutral evolution. For this reason, molecular convergence is commonly viewed as evidence for similar adaptations of different species. The surge in the number of reports of molecular convergence in the last decade raises the intriguing question of whether molecular convergence occurs substantially more frequently than expected under neutral evolution. We here address this question using all one-to-one orthologous proteins encoded by the genomes of 12 fruit fly species and those encoded by 17 mammals. We found that the expected amount of molecular convergence varies greatly depending on the specific neutral substitution model assumed at each amino acid site and that the observed amount of molecular convergence is explainable by neutral models incorporating site-specific information of acceptable amino acids. Interestingly, the total number of convergent and parallel substitutions between two lineages, relative to the neutral expectation, decreases with the genetic distance between the two lineages, regardless of the model used in computing the neutral expectation. We hypothesize that this trend results from differences in the amino acids acceptable at a given site among different clades of a phylogeny, due to prevalent epistasis, and provide simulation as well as empirical evidence for this hypothesis. Together, our study finds no genomic evidence for higher-than-neutral levels of molecular convergence, but suggests the presence of abundant epistasis that decreases the likelihood of molecular convergence between distantly related lineages. PMID:25862140

  13. The probability of speciation on an interaction network with unequal substitution rates.

    PubMed

    Olofsson, Peter; Livingstone, Kevin; Humphreys, Joshua; Steinman, Douglas

    2016-08-01

    Speciation is characterized by the development of reproductive isolating barriers between diverging groups. A seminal paper of a mathematical model of speciation was published by Orr (1995), extended by Livingstone et al. (2012) to incorporate interaction networks. Here, we further develop the model to take into account the possibility of different substitution rates for network nodes of different connectivity. Mathematically, this amounts to sampling nodes from an undirected graph where the inclusion probability for a given node depends on its degree (number of connecting edges). We establish formulas for the rate of speciation and identify a crucial parameter that is a measure of the deviation from simple random sampling. PMID:27177943

  14. Omega-3 Fatty Acid Formulations in Cardiovascular Disease: Dietary Supplements are Not Substitutes for Prescription Products.

    PubMed

    Fialkow, Jonathan

    2016-08-01

    Omega-3 fatty acid products are available as prescription formulations (icosapent ethyl, omega-3-acid ethyl esters, omega-3-acid ethyl esters A, omega-3-carboxylic acids) and dietary supplements (predominantly fish oils). Most dietary supplements and all but one prescription formulation contain mixtures of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Products containing both EPA and DHA may raise low-density lipoprotein cholesterol (LDL-C). In clinical trials, the EPA-only prescription product, icosapent ethyl, did not raise LDL-C compared with placebo. To correct a common misconception, it is important to note that omega-3 fatty acid dietary supplements are not US FDA-approved over-the-counter drugs and are not required to demonstrate safety and efficacy prior to marketing. Conversely, prescription products are supported by extensive clinical safety and efficacy investigations required for FDA approval and have active and ongoing safety monitoring programs. While omega-3 fatty acid dietary supplements may have a place in the supplementation of diet, they generally contain lower levels of EPA and DHA than prescription products and are not approved or intended to treat disease. Perhaps due to the lack of regulation of dietary supplements, EPA and DHA levels may vary widely within and between brands, and products may also contain unwanted cholesterol or fats or potentially harmful components, including toxins and oxidized fatty acids. Accordingly, omega-3 fatty acid dietary supplements should not be substituted for prescription products. Similarly, prescription products containing DHA and EPA should not be substituted for the EPA-only prescription product, as DHA may raise LDL-C and thereby complicate the management of patients with dyslipidemia. PMID:27138439

  15. Computational study of the rate constants and free energies of intramolecular radical addition to substituted anilines

    PubMed Central

    Seddiqzai, Meriam; Dahmen, Tobias; Sure, Rebecca

    2013-01-01

    Summary The intramolecular radical addition to aniline derivatives was investigated by DFT calculations. The computational methods were benchmarked by comparing the calculated values of the rate constant for the 5-exo cyclization of the hexenyl radical with the experimental values. The dispersion-corrected PW6B95-D3 functional provided very good results with deviations for the free activation barrier compared to the experimental values of only about 0.5 kcal mol−1 and was therefore employed in further calculations. Corrections for intramolecular London dispersion and solvation effects in the quantum chemical treatment are essential to obtain consistent and accurate theoretical data. For the investigated radical addition reaction it turned out that the polarity of the molecules is important and that a combination of electrophilic radicals with preferably nucleophilic arenes results in the highest rate constants. This is opposite to the Minisci reaction where the radical acts as nucleophile and the arene as electrophile. The substitution at the N-atom of the aniline is crucial. Methyl substitution leads to slower addition than phenyl substitution. Carbamates as substituents are suitable only when the radical center is not too electrophilic. No correlations between free reaction barriers and energies (ΔG ‡ and ΔG R) are found. Addition reactions leading to indanes or dihydrobenzofurans are too slow to be useful synthetically. PMID:24062821

  16. Computational study of the rate constants and free energies of intramolecular radical addition to substituted anilines.

    PubMed

    Gansäuer, Andreas; Seddiqzai, Meriam; Dahmen, Tobias; Sure, Rebecca; Grimme, Stefan

    2013-01-01

    The intramolecular radical addition to aniline derivatives was investigated by DFT calculations. The computational methods were benchmarked by comparing the calculated values of the rate constant for the 5-exo cyclization of the hexenyl radical with the experimental values. The dispersion-corrected PW6B95-D3 functional provided very good results with deviations for the free activation barrier compared to the experimental values of only about 0.5 kcal mol(-1) and was therefore employed in further calculations. Corrections for intramolecular London dispersion and solvation effects in the quantum chemical treatment are essential to obtain consistent and accurate theoretical data. For the investigated radical addition reaction it turned out that the polarity of the molecules is important and that a combination of electrophilic radicals with preferably nucleophilic arenes results in the highest rate constants. This is opposite to the Minisci reaction where the radical acts as nucleophile and the arene as electrophile. The substitution at the N-atom of the aniline is crucial. Methyl substitution leads to slower addition than phenyl substitution. Carbamates as substituents are suitable only when the radical center is not too electrophilic. No correlations between free reaction barriers and energies (ΔG (‡) and ΔG R) are found. Addition reactions leading to indanes or dihydrobenzofurans are too slow to be useful synthetically. PMID:24062821

  17. Substitution of amino acids in helix F of bacteriorhodopsin: Effects on the photochemical cycle

    SciTech Connect

    Ahl, P.L.; Stern, L.J.; Mogi, T.; Khorana, H.G.; Rothschild, K.J. )

    1989-12-26

    The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.

  18. Synthesis, characterization, and subcellular localization studies of amino acid-substituted porphyrinic pigments

    NASA Astrophysics Data System (ADS)

    van Diggelen, Lisa; Khin, Hnin; Conner, Kip; Shao, Jenny; Sweezy, Margaretta; Jung, Anna H.; Isaac, Meden; Simonis, Ursula

    2009-06-01

    Stopping cancer in its path occurs when photosensitizers (PSs) induce apoptotic cell death after their exposure to light and the subsequent formation of reactive oxygen species. In pursuit of our hypothesis that mitochondrial localizing PSs will enhance the efficacy of the photosensitizing process in photodynamic therapy, since they provoke cell death by inducing apoptosis, we synthesized and characterized tetraphenylporphyrins (TPPs) that are substituted at the paraphenyl positions by two amino acids and two fluoro or hydroxyl groups, respectively. They were prepared according to the Lindsey-modified Adler-Longo methodology using trifluoromethanesulfonylchloride (CF3SO2Cl) as a catalyst instead of trifluoroacetic acid. The use of CF3SO2Cl yielded cleaner products in significantly higher yields. During the synthesis, not only the yields and work-up procedure of the TPPs were improved by using CF3SO2Cl as a catalyst, but also a better means of synthesizing the precursor dipyrromethanes was tested by using indium(III) chloride. Column chromatography, HPLC, and NMR spectroscopy were used to separate and characterize the di-amino acid-dihydroxy, or difluoro-substituted porphyrins and to ascertain their purity before subcellular localization studies were carried out. Studies using androgen-sensitive human prostate adenocarcinoma cells LNCaP revealed that certain amino acid substituted porphyrins that are positively charged in the slightly acidic medium of cancer cells are very useful in shedding light on the targets of TPPs in subcellular organelles of cancer cells. Although some of these compounds have properties of promising photosensitizers by revealing increased water solubility, acidic properties, and innate ability to provoke cell death by apoptosis, the cell killing efficacy of these TPPs is low. This correlates with their subcellular localization. The di-amino acid, di-hydroxy substituted TPPs localize mainly to the lysosomes, whereas the di-fluoro-substituted

  19. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding

    SciTech Connect

    Kaye, F.J.; Gerster, J.L. Uniformed Services Univ. of Health Sciences, Bethesda, MD ); Kratzke, R.A. ); Horowitz, J.M. )

    1990-09-01

    The authors have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.

  20. Lewis acid tuned facial stereodivergent HDA reactions using beta-substituted N-vinyloxazolidinones.

    PubMed

    Gohier, Frédéric; Bouhadjera, Keltoum; Faye, Djibril; Gaulon, Catherine; Maisonneuve, Vincent; Dujardin, Gilles; Dhal, Robert

    2007-01-18

    The [4 + 2] acido-catalyzed heterocycloaddition between new beta-substituted N-vinyl-1,3-oxazolidin-2-ones (with R' = Me, Ar, CH2 Ar) and beta,gamma-unsaturated alpha-ketoesters (R = Ar) afforded heteroadducts with high levels of endo and facial selectivities. A complete reversal of facial differentiation was achieved by varying the Lewis acid, leading to the stereoselective formation of either endo-alpha or endo-beta adducts. [reaction: see text]. PMID:17217267

  1. An amino acid substitution in the pyruvate dehydrogenase E1{alpha} gene, affecting mitochondrial import of the precursor protein

    SciTech Connect

    Takakubo, F.; Thorburn, D.R.; Dahl, H.H.M.

    1995-10-01

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1{alpha} deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1{alpha}immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1{alpha} targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1{alpha} sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease. 58 refs., 5 figs., 1 tab.

  2. Substituent Effects on the Hydrolysis of p-Substituted Benzonitriles in Sulfuric Acid Solutions at (25.0± 0.1) °C

    NASA Astrophysics Data System (ADS)

    Abbas, Khamis A.

    2008-09-01

    The rate constants of the hydrolysis of p-substituted benzonitriles with sulfuric acid solutions (18.2 M to 10 M) have been determined spectrophotometrically at (25.1±0.1) °C. It was found that the catalytic activity of sulfuric acid was strongly inhibited by water. The logarithms of the observed rate constants were correlated with different substituent inductive (localized) and resonance (delocalized) constants. The results of the correlation studies indicated that the rate-determining step of the hydrolysis of benzonitriles in 18.2 M sulfuric acid was the addition of a nucleophile, and the hydrolysis was clearly enhanced by the electron-withdrawing inductive effect, while the rate-determining step of the hydrolysis of p-substituted benzonitriles in 10.0 M sulfuric acid was most probably the protonation of benzonitriles, and the rate constants increased by both electron-donating resonance and inductive effects. A mixture of the two mechanisms most probably occurred in 15.3 to 17.0 M sulfuric acid. HSO4 - rather thanwater most probably acted as nucleophile in the hydrolysis of benzonitriles especially at high concentrations of sulfuric acid solutions.

  3. Vapor Phase Dehydration of Glycerol to Acrolein Over SBA-15 Supported Vanadium Substituted Phosphomolybdic Acid Catalyst.

    PubMed

    Viswanadham, Balaga; Srikanth, Amirineni; Kumar, Vanama Pavan; Chary, Komandur V R

    2015-07-01

    Vapor phase dehydration of glycerol to acrolein was investigated over heteropolyacid (HPA) catalysts containing vanadium substituted phosphomolybdic acid (H4PMo11VO40) supported on mesoporous SBA-15. A series of HPA catalysts with HPA loadings varying from 10-50 wt% were prepared by impregnation method on SBA-15 support. The catalysts were characterized by X-ray diffraction, Raman spectroscopy, Fourier Transform infrared spectroscopy, temperature-programmed desorption of NH3, pyridine adsorbed FT-IR spectroscopy, scanning electron microscopy, pore size distribution and specific surface area measurements. The nature of acidic sites was examined by pyridine adsorbed FT-IR spectroscopy. XRD results suggest that the active phase containing HPA was highly dispersed at lower loadings on the support. FT-IR and Raman spectra results confirm that the presence of primary Keggin ion structure of HPA on the support and it was not affected during the preparation of catalysts. Pore size distribution results reveal that all the samples show unimodel pore size distribution with well depicted mesoporous structure. NH3-TPD results suggest that the acidity of catalysts increased with increase of HPA loading. The findings of acidity measurements by FT-IR spectra of pyridine adsorption reveals that the catalysts consist both the Brønsted and Lewis acidic sites and the amount of Brønsted acidic sites are increasing with HPA loading. SBA-15 supported vanadium substituted phosphomolybdic acid catalysts are found to be highly active during the dehydration reaction and exhibited 100% conversion of glycerol (10 wt% of glycerol) and the acrolein selectivity was appreciably changed with HPA active phase loading. The catalytic functionalities during glycerol dehydration are well correlated with surface acidity of the catalysts. PMID:26373149

  4. Massive difference in synonymous substitution rates among mitochondrial, plastid, and nuclear genes of Phaeocystis algae.

    PubMed

    Smith, David Roy; Arrigo, Kevin R; Alderkamp, Anne-Carlijn; Allen, Andrew E

    2014-02-01

    We are just beginning to understand how mutation rates differ among mitochondrial, plastid, and nuclear genomes. In most seed plants the mitochondrial mutation rate is estimated to be lower than those of the plastid and nucleus, whereas in the red alga Porphyra the opposite is true, and in certain green algae all three genomes appear to have similar rates of mutation. Relative rate statistics of organelle vs nuclear genes, however, are lacking for lineages that acquired their plastids through secondary endosymbiosis, but recent organelle DNA analyses suggest that they may differ drastically from what is observed in lineages with primary plastids, such as green plants and red algae. Here, by measuring synonymous nucleotide substitutions, we approximate the relative mutation rates within the haptophyte genus Phaeocystis, which has a red-algal-derived, secondary plastid. Synonymous-site divergence data indicate that for Phaeocystis antarctica and P. globosa the mitochondrial mutation rate is 10 and 3 times that of the plastid and nucleus, respectively. This differs drastically from relative rate estimates for primary-plastid-bearing lineages and presents a much more dynamic view of organelle vs nuclear mutation rates across the eukaryotic domain. PMID:24216019

  5. Fabrication of calcium phosphate–calcium sulfate injectable bone substitute using hydroxy-propyl-methyl-cellulose and citric acid

    PubMed Central

    Thai, Van Viet

    2010-01-01

    In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan, and hydroxyl propyl methyl cellulose (HPMC) as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate dihydrate (DCPD) and calcium sulfate dehydrate (CSD, CaSO4·2H2O) powders as the solid phase, were fabricated. Two groups were classified based on the percent of citric acid in the liquid phase (20, 40 wt%). In each groups, the HPMC percentage was 0, 2, and 4 wt%. An increase in compressive strength due to changes in morphology was confirmed by scanning electron microscopy images. A good conversion rate of HAp at 20% citric acid was observed in the XRD profiles. In addition, HPMC was not obviously affected by apatite formation. However, both HPMC and citric acid increased the compressive strength of IBS. The maximum compressive strength for IBS was with 40% citric acid and 4% HPMC after 14 days of incubation in 100% humidity at 37°C. PMID:20333539

  6. Fabrication of calcium phosphate-calcium sulfate injectable bone substitute using hydroxy-propyl-methyl-cellulose and citric acid.

    PubMed

    Thai, Van Viet; Lee, Byong-Taek

    2010-06-01

    In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan, and hydroxyl propyl methyl cellulose (HPMC) as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate dihydrate (DCPD) and calcium sulfate dehydrate (CSD, CaSO4 x 2H2O) powders as the solid phase, were fabricated. Two groups were classified based on the percent of citric acid in the liquid phase (20, 40 wt%). In each groups, the HPMC percentage was 0, 2, and 4 wt%. An increase in compressive strength due to changes in morphology was confirmed by scanning electron microscopy images. A good conversion rate of HAp at 20% citric acid was observed in the XRD profiles. In addition, HPMC was not obviously affected by apatite formation. However, both HPMC and citric acid increased the compressive strength of IBS. The maximum compressive strength for IBS was with 40% citric acid and 4% HPMC after 14 days of incubation in 100% humidity at 37 degrees C. PMID:20333539

  7. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  8. Structural consequences of amino acid substitutions causing Tay-Sachs disease.

    PubMed

    Ohno, Kazuki; Saito, Seiji; Sugawara, Kanako; Sakuraba, Hitoshi

    2008-08-01

    To determine the structural changes in the alpha-subunit of beta-hexosaminidase due to amino acid substitutions causing Tay-Sachs disease, we built structural models of mutant alpha-subunits resulting from 33 missense mutations (24 infantile and 9 late-onset), and analyzed the influence of each amino acid replacement on the structure by calculating the number of atoms affected and determining the solvent-accessible surface area of the corresponding amino acid residue in the wild-type alpha-subunit. In the infantile Tay-Sachs group, the number of atoms influenced by a mutation was generally larger than that in the late-onset Tay-Sachs group in both the main chain and the side chain, and residues associated with the mutations found in the infantile Tay-Sachs group tended to be less solvent-accessible than those in the late-onset Tay-Sachs group. Furthermore, color imaging determined the distribution and degree of the structural changes caused by representative amino acid substitutions, and that there were also differences between the infantile and late-onset Tay-Sachs disease groups. Structural study is useful for elucidating the basis of Tay-Sachs disease. PMID:18490185

  9. Computer-assisted automatic synthesis II. Development of a fully automated apparatus for preparing substituted N–(carboxyalkyl)amino acids

    PubMed Central

    Hayashi, Nobuyoshi; Sugawara, Tohru; Shintani, Motoaki; Kato, Shinji

    1989-01-01

    A versatile automated apparatus, equipped with an artificial intelligence has been developed which may be used to prepare and isolate a wide variety of compounds. The prediction of the optimum reaction conditions and the reaction control in real time, are accomplished using novel kinetic equations and substituent effects in an artificial intelligence software which has already reported [1]. This paper deals with the design and construction of the fully automated system, and its application to the synthesis of a substituted N-(carboxyalkyl)amino acid. The apparatus is composed of units for perfoming various tasks, e.g. reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes including washing and drying of the apparatus after each synthetic run were automatically performed from the mixing of the reactants to the isolation of the products as powders with purities of greater than 98%. The automated apparatus has been able to run for 24 hours per day, and the average rate of synthesis of substituted N-(carboxyalkyl)amino acids has been three compounds daily. The apparatus is extremely valuable for synthesizing many derivatives of one particular compound structure. Even if the chemical yields are low under the optimum conditions, it is still possible to obtain a sufficient amount of the desired product by repetition of the reaction. Moreover it was possible to greatly reduce the manual involvement of the many syntheses which are a necessary part of pharmaceutical research. PMID:18924679

  10. Computer-assisted automatic synthesis II. Development of a fully automated apparatus for preparing substituted N-(carboxyalkyl)amino acids.

    PubMed

    Hayashi, N; Sugawara, T; Shintani, M; Kato, S

    1989-01-01

    A versatile automated apparatus, equipped with an artificial intelligence has been developed which may be used to prepare and isolate a wide variety of compounds. The prediction of the optimum reaction conditions and the reaction control in real time, are accomplished using novel kinetic equations and substituent effects in an artificial intelligence software which has already reported [1]. This paper deals with the design and construction of the fully automated system, and its application to the synthesis of a substituted N-(carboxyalkyl)amino acid. The apparatus is composed of units for perfoming various tasks, e.g. reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes including washing and drying of the apparatus after each synthetic run were automatically performed from the mixing of the reactants to the isolation of the products as powders with purities of greater than 98%. The automated apparatus has been able to run for 24 hours per day, and the average rate of synthesis of substituted N-(carboxyalkyl)amino acids has been three compounds daily. The apparatus is extremely valuable for synthesizing many derivatives of one particular compound structure. Even if the chemical yields are low under the optimum conditions, it is still possible to obtain a sufficient amount of the desired product by repetition of the reaction. Moreover it was possible to greatly reduce the manual involvement of the many syntheses which are a necessary part of pharmaceutical research. PMID:18924679

  11. Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.

    PubMed Central

    Van de Peer, Y; Rensing, S A; Maier, U G; De Wachter, R

    1996-01-01

    Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA. PMID:8755544

  12. Impact of a single base pair substitution on the charge transfer rate along short DNA hairpins

    PubMed Central

    Renaud, Nicolas; Berlin, Yuri A.; Ratner, Mark A.

    2013-01-01

    Numerical studies of hole migration along short DNA hairpins were performed with a particular emphasis on the variations of the rate and quantum yield of the charge separation process with the location of a single guanine:cytosine (G:C) base pair. Our calculations show that the hole arrival rate increases as the position of the guanine:cytosine base pair shifts from the beginning to the end of the sequence. Although these results are in agreement with recent experimental findings, the mechanism governing the charge migration along these sequences is revisited here. Instead of the phenomenological two-step hopping mechanism via the guanine base, the charge propagation occurs through a delocalization of the hole density along the base pair stack. Furthermore, the variations of the charge transfer with the position of the guanine base are explained by the impact of the base pair substitutions on the delocalized conduction channels. PMID:23980166

  13. The Synthesis and Characterization of Substituted Polyaniline Hollow Spheres doped with a Polymeric Acid

    NASA Astrophysics Data System (ADS)

    Sui, Jing; Zhang, Lijuan; Travas-Sejdic, Jadranka; Kilmartin, Paul A.

    2009-07-01

    Self-assembled poly(o-toluidine) (POT) and poly(o-anisidine) (POA) hollow spheres were prepared by oxidative polymerization using ammonium persulfate as the oxidant in the presence of 5% by weight of a polymeric acid, poly(methyl vinyl ether-alt-maleic acid) (PMVEA). The substituent at the ortho position had a significant effect on the size of the hollow nanospheres as determined by SEM and TEM. The nanospheres were of a very uniform size in the presence of the polymeric acid, with average diameters of 338±25 nm for POT and 210±20 nm for POA. The POT and POA hollow spheres were also characterized by FTIR and UV-Vis spectroscopy, which confirmed the chemical identity of the substituted polyanilines.

  14. Aggregation of dialkyl-substituted diphosphonic acids and its effect on metal ion extraction.

    SciTech Connect

    Chiarizia, R.; Barrans, R. E., Jr.; Ferraro, J. R. Herlinger, A. W.; McAlister, D. R.

    1999-10-22

    Solvent extraction reagents containing the diphosphonic acid group exhibit an extraordinary affinity for tri-, tetra- and hexavalent actinides. Their use has been considered for actinide separation and pre-concentration procedures. Solvent extraction data obtained with P,P{prime}-di(2-ethylhexyl) methane-, ethane- and butanediphosphonic acids exhibit features that are difficult to explain without Knowledge of the aggregation state of the extractants. Information about the aggregation of the dialkyl-substituted diphosphonic acids in aromatic diluents has been obtained using the complementary techniques of vapor pressure osmometry (VPO), small angle neutron scattering (SANS), infrared spectroscopy and molecular mechanics. The results from these techniques provide an understanding of the aggregation behavior of these extractants that is fully compatible with the solvent extraction data. The most important results and their relevance to solvent extraction are reviewed in this paper.

  15. Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation.

    PubMed

    van Beek, S; Priest, F G

    2000-12-01

    Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation. PMID:11097909

  16. Decarboxylation of Substituted Cinnamic Acids by Lactic Acid Bacteria Isolated during Malt Whisky Fermentation

    PubMed Central

    van Beek, Sylvie; Priest, Fergus G.

    2000-01-01

    Seven strains of Lactobacillus isolated from malt whisky fermentations and representing Lactobacillus brevis, L. crispatus, L. fermentum, L. hilgardii, L. paracasei, L. pentosus, and L. plantarum contained genes for hydroxycinnamic acid (p-coumaric acid) decarboxylase. With the exception of L. hilgardii, these bacteria decarboxylated p-coumaric acid and/or ferulic acid, with the production of 4-vinylphenol and/or 4-vinylguaiacol, respectively, although the relative activities on the two substrates varied between strains. The addition of p-coumaric acid or ferulic acid to cultures of L. pentosus in MRS broth induced hydroxycinnamic acid decarboxylase mRNA within 5 min, and the gene was also induced by the indigenous components of malt wort. In a simulated distillery fermentation, a mixed culture of L. crispatus and L. pentosus in the presence of Saccharomyces cerevisiae decarboxylated added p-coumaric acid more rapidly than the yeast alone but had little activity on added ferulic acid. Moreover, we were able to demonstrate the induction of hydroxycinnamic acid decarboxylase mRNA under these conditions. However, in fermentations with no additional hydroxycinnamic acid, the bacteria lowered the final concentration of 4-vinylphenol in the fermented wort compared to the level seen in a pure-yeast fermentation. It seems likely that the combined activities of bacteria and yeast decarboxylate p-coumaric acid and then reduce 4-vinylphenol to 4-ethylphenol more effectively than either microorganism alone in pure cultures. Although we have shown that lactobacilli participate in the metabolism of phenolic compounds during malt whisky fermentations, the net result is a reduction in the concentrations of 4-vinylphenol and 4-vinylguaiacol prior to distillation. PMID:11097909

  17. Structure-Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates.

    PubMed

    Mascarin, Alba; Valverde, Ibai E; Mindt, Thomas L

    2016-01-01

    Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8-13) have been studied as potential imaging probes and therapeutics for NT-1-receptor-positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8-13) for radiotracer development, we herein report a side-by-side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [(177)Lu]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled examples studied, analogues of NT(8-13) containing a short hydrophilic tetraethylene glycol (PEG4 ) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro. PMID:26593062

  18. Amino acid esters substituted phosphorylated emtricitabine and didanosine derivatives as antiviral and anticancer agents.

    PubMed

    Sekhar, Kuruva Chandra; Janardhan, Avilala; Kumar, Yellapu Nanda; Narasimha, Golla; Raju, Chamarthi Naga; Ghosh, S K

    2014-07-01

    Owing to the promising antiviral activity of amino acid ester-substituted phosphorylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabine and didanosine substituted with bioactive amino acid esters at P-atom were synthesized. Initially, molecular docking studies were screened to predict their molecular interactions with hemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of human papillomavirus. The title compounds were screened for their antiviral ability against Newcastle disease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and 9c exposed well mode of interactions with HN protein and also exhibited potential growth of NDV inhibition. The remaining compounds exhibited better growth of NDV inhibition than their parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitro anticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and 100 μg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activity than that of the remaining title compounds with IC50 values of 40 and 60 μg/mL, respectively. The present in silico and in ovo antiviral and in vitro anticancer results of the title compounds are suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives, especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control and management of viral diseases with cancerous condition. PMID:24789416

  19. Synergistic Acid-Catalyzed Synthesis of N-Aryl-Substituted Azacycles from Anilines and Cyclic Ethers.

    PubMed

    Zhang, Zhenbei; Miao, Chengxia; Xia, Chungu; Sun, Wei

    2016-04-01

    A metal-free and efficient approach to N-aryl-substituted azacycles from arylamines and cyclic ethers is described. In this synthesis, the synergistic effect between Lewis and Brønsted acids is crucial to the ring-opening of cyclic ethers and the subsequent cyclization. The use of B(C6F5)3 enabled the formation of frustrated Lewis pairs (FLPs) from the reactants, and the resulting FLPs allowed ready access to the N-arylazacycles in moderate to good yields via further cyclization. Water is the sole waste resulting from the reaction, thereby making it an environmentally benign process. PMID:26962879

  20. Reversal of the surface charge asymmetry in purple membrane due to single amino acid substitutions.

    PubMed Central

    Hsu, K C; Rayfield, G W; Needleman, R

    1996-01-01

    Twenty-seven mutant bacteriorhodopsin's were screened to determine the PKa for reversal of the permanent electric dipole moment. The photoelectric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a function of pH. The pK(a) for the dipole reversal of wild-type bacteriorhodopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacteriorhodopsin. Two of these mutants, L93T and L93W, involve a neutral amino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry. We suggest that these changes to the pK(a) are due to altered protein folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior. PMID:9172760

  1. Amino acid substitutions in hepatitis C virus core region predict hepatocarcinogenesis following eradication of HCV RNA by antiviral therapy.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Hirakawa, Miharu; Kawamura, Yusuke; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2011-06-01

    Substitution of amino acid (aa) 70 and/or 91 in the core region of HCV genotype 1b (HCV-1b) is an important predictor of hepatocarcinogenesis, but its impact on the development of hepatocellular carcinoma (HCC) following eradication of HCV RNA by antiviral therapy is not clear. 1,273 patients with HCV-related chronic liver disease, with sustained virological response, defined as negative HCV RNA at 24 weeks after cessation of interferon monotherapy or interferon plus ribavirin combination therapy, were included in a follow-up study to evaluate the impact of aa substitution in the core region on hepatocarcinogenesis. Twenty six patients developed HCC during the follow-up. The cumulative rates of new HCC were 3.2%, 4.8%, and 8.6% at the end of 5, 10, and 15 years, respectively. The rates in patients infected with HCV-1b/Gln70(His70) [glutamine (histidine) at aa 70] were significantly higher than in patients infected with HCV-1b/Arg70 (arginine at aa 70) (P = 0.007; log-rank test) and HCV-2a/2b (P < 0.001; log-rank test). The rates in patients infected with HCV-1b/Arg70 were not significantly higher than in those infected with HCV-2a/2b (P = 0.617; log-rank test). Multivariate analysis identified HCV-1b/Gln70(His70) (HR 10.5, P < 0.001), advanced fibrosis (HR 9.03, P = 0.002), and old age (HR 3.09, P = 0.066) as determinants of hepatocarcinogenesis. In conclusion, aa substitution in the core region of HCV-1b at the start of antiviral therapy is an important predictor of HCC following eradication of HCV RNA. This study emphasizes the importance of detection of aa substitutions in the core region before antiviral therapy. PMID:21503914

  2. Synthesis, Characterization, and Antifungal Activity of Phenylpyrrole-Substituted Tetramic Acids Bearing Carbonates.

    PubMed

    Xu, Wen-Qin; Chen, Min; Wang, Kun-Yao; Ren, Zheng-Jiao; Lu, Ai-Min; Yang, Chun-Long

    2016-01-01

    For the aim of discovering new fungicide, a series of phenylpyrrole-substituted tetramic acid derivatives bearing carbonates 6a-q were designed and synthesized via 4-(2,4-dioxopyrrolidin-3-ylidene)-4-(phenylamino)butanoic acids 4a-k and the cyclized products 1',3,4,5'-tetrahydro-[2,3'-bipyrrolylidene]-2',4',5(1H)-triones 5a-k. The compounds were characterized using IR, ¹H- and (13)C-NMR spectroscopy, mass spectrometry (EI-MS), and elemental analysis. The structure of 6b was confirmed by X-ray diffraction crystallography. The title compounds 6a-q were bioassayed in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea and Rhizoctonia solani at a concentration of 100 μg/mL, respectively. Most compounds displayed good inhibitory activity. PMID:27007370

  3. Calcite crystal growth rate inhibition by polycarboxylic acids

    USGS Publications Warehouse

    Reddy, M.M.; Hoch, A.R.

    2001-01-01

    Calcite crystal growth rates measured in the presence of several polycarboxyclic acids show that tetrahydrofurantetracarboxylic acid (THFTCA) and cyclopentanetetracarboxylic acid (CPTCA) are effective growth rate inhibitors at low solution concentrations (0.01 to 1 mg/L). In contrast, linear polycarbocylic acids (citric acid and tricarballylic acid) had no inhibiting effect on calcite growth rates at concentrations up to 10 mg/L. Calcite crystal growth rate inhibition by cyclic polycarboxyclic acids appears to involve blockage of crystal growth sites on the mineral surface by several carboxylate groups. Growth morphology varied for growth in the absence and in the presence of both THFTCA and CPTCA. More effective growth rate reduction by CPTCA relative to THFTCA suggests that inhibitor carboxylate stereochemical orientation controls calcite surface interaction with carboxylate inhibitors. ?? 20O1 Academic Press.

  4. 40 CFR 721.10381 - Cyclic carboxylic acid, polymer with dihydroxy dialkyl ether, hydroxy substituted alkane and...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... polyester (generic). 721.10381 Section 721.10381 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY..., hydroxy substituted alkane and carboxylic acid anhydride, methacrylate terminated polyester (generic). (a... carboxylic acid anhydride, methacrylate terminated polyester (PMN P-10-290) is subject to reporting...

  5. 40 CFR 721.10381 - Cyclic carboxylic acid, polymer with dihydroxy dialkyl ether, hydroxy substituted alkane and...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... polyester (generic). 721.10381 Section 721.10381 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY..., hydroxy substituted alkane and carboxylic acid anhydride, methacrylate terminated polyester (generic). (a... carboxylic acid anhydride, methacrylate terminated polyester (PMN P-10-290) is subject to reporting...

  6. 40 CFR 721.10381 - Cyclic carboxylic acid, polymer with dihydroxy dialkyl ether, hydroxy substituted alkane and...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... polyester (generic). 721.10381 Section 721.10381 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY..., hydroxy substituted alkane and carboxylic acid anhydride, methacrylate terminated polyester (generic). (a... carboxylic acid anhydride, methacrylate terminated polyester (PMN P-10-290) is subject to reporting...

  7. 40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6181 Fatty acid, reaction product with substituted oxirane, formaldehyde... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Fatty acid, reaction product...

  8. 40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6181 Fatty acid, reaction product with substituted oxirane, formaldehyde... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Fatty acid, reaction product...

  9. 40 CFR 721.6181 - Fatty acid, reaction product with substituted oxirane, formaldehyde-phenol polymer glycidyl ether...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.6181 Fatty acid, reaction product with substituted oxirane, formaldehyde... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Fatty acid, reaction product...

  10. Controlled Rate Freezing to Regulate the Structure of Collagen-GAG Scaffolds in Engineered Skin Substitutes

    PubMed Central

    Lloyd, Christopher; Besse, John; Boyce, Steven

    2014-01-01

    Controlled rate freezing (CRF) of biopolymer scaffolds may increase reproducibility of microstructure compared to analog processes. Freezing of collagen-glycosaminoglycan (CG) scaffolds by CRF with liquid nitrogen at chamber cooling rates of −80°C, −40°C, −20°C, or −10°C/min, was compared to submersion in 95% ethanol at −55°C. Cooling rates of −80°C/min or −40°C/min generated scaffolds with pore areas and pore fractions that were comparable to scaffolds frozen in ethanol. Test and control scaffolds were populated with human dermal fibroblasts and keratinocytes to generate engineered skin substitutes (ESS), and evaluated for surface hydration and mitochondrial metabolism. ESS with scaffolds frozen by CRF at −80°C/min or −40°C/min were comparable to, or better than ESS with control scaffolds (p<0.05). These results demonstrate that fabrication of CG scaffolds by CRF offers advantages of digital programming, as well as greater reproducibility, safety and simplicity than submersion in chilled ethanol without compromise of biological properties required for biomedical applications. PMID:25132427

  11. Evolutionary dynamics of the plastid inverted repeat: the effects of expansion, contraction, and loss on substitution rates.

    PubMed

    Zhu, Andan; Guo, Wenhu; Gupta, Sakshi; Fan, Weishu; Mower, Jeffrey P

    2016-03-01

    Rates of nucleotide substitution were previously shown to be several times slower in the plastid inverted repeat (IR) compared with single-copy (SC) regions, suggesting that the IR provides enhanced copy-correction activity. To examine the generality of this synonymous rate dependence on the IR, we compared plastomes from 69 pairs of closely related species representing 52 families of angiosperms, gymnosperms, and ferns. We explored the breadth of IR boundary shifts in land plants and demonstrate that synonymous substitution rates are, on average, 3.7 times slower in IR genes than in SC genes. In addition, genes moved from the SC into the IR exhibit lower synonymous rates consistent with other IR genes, while genes moved from the IR into the SC exhibit higher rates consistent with other SC genes. Surprisingly, however, several plastid genes from Pelargonium, Plantago, and Silene have highly accelerated synonymous rates despite their IR localization. Together, these results provide strong evidence that the duplicative nature of the IR reduces the substitution rate within this region. The anomalously fast-evolving genes in Pelargonium, Plantago, and Silene indicate localized hypermutation, potentially induced by a higher level of error-prone double-strand break repair in these regions, which generates substitutional rate variation. PMID:26574731

  12. Physicochemical and acid gelation properties of commercial UHT-treated plant-based milk substitutes and lactose free bovine milk.

    PubMed

    Mäkinen, Outi E; Uniacke-Lowe, Thérèse; O'Mahony, James A; Arendt, Elke K

    2015-02-01

    Physicochemical and acid gelation properties of UHT-treated commercial soy, oat, quinoa, rice and lactose-free bovine milks were studied. The separation profiles were determined using a LUMiSizer dispersion analyser. Soy, rice and quinoa milks formed both cream and sediment layers, while oat milk sedimented but did not cream. Bovine milk was very stable to separation while all plant milks separated at varying rates; rice and oat milks being the most unstable products. Particle sizes in plant-based milk substitutes, expressed as volume mean diameters (d4.3), ranged from 0.55μm (soy) to 2.08μm (quinoa) while the average size in bovine milk was 0.52μm. Particles of plant-based milk substitutes were significantly more polydisperse compared to those of bovine milk. Upon acidification with glucono-δ-lactone (GDL), bovine, soy and quinoa milks formed structured gels with maximum storage moduli of 262, 187 and 105Pa, respectively while oat and rice milks did not gel. In addition to soy products currently on the market, quinoa may have potential in dairy-type food applications. PMID:25172757

  13. Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase

    PubMed Central

    Buček, Aleš; Matoušková, Petra; Vogel, Heiko; Šebesta, Petr; Jahn, Ullrich; Weißflog, Jerrit; Svatoš, Aleš; Pichová, Iva

    2015-01-01

    For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds. PMID:26417103

  14. Evolution of moth sex pheromone composition by a single amino acid substitution in a fatty acid desaturase.

    PubMed

    Buček, Aleš; Matoušková, Petra; Vogel, Heiko; Šebesta, Petr; Jahn, Ullrich; Weißflog, Jerrit; Svatoš, Aleš; Pichová, Iva

    2015-10-13

    For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds. PMID:26417103

  15. Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

    SciTech Connect

    Schmidheini, T.; Moesch, H.U.; Braus, G. ); Evans, J.N.S. )

    1990-04-17

    Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of M{sub r} 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by {sup 1}H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the wild-type chorismate mutase show positive cooperativity toward the substrate with a Hill coefficient of 1.71 and a (S){sub 0.5} value of 4.0 mM. In the presence of the activator tryptophan, the cooperativity is lost. The enzyme has an (S){sub 0.5} value of 1.2 mM in the presence of 10 {mu}M tryptophan and an increased (S){sub 0.5} value of 8.6 mM in the presence of 300 {mu}M tyrosine. In the mutant enzyme, a loss of the cooperativity was observed, and (S){sub 0.5} was reduced to 1.0 mM. This enzyme is therefore locked in the activated state by a single amino acid substitution.

  16. Comparative mitochondrial genomics of snakes: extraordinary substitution rate dynamics and functionality of the duplicate control region

    PubMed Central

    Jiang, Zhi J; Castoe, Todd A; Austin, Christopher C; Burbrink, Frank T; Herron, Matthew D; McGuire, Jimmy A; Parkinson, Christopher L; Pollock, David D

    2007-01-01

    Background The mitochondrial genomes of snakes are characterized by an overall evolutionary rate that appears to be one of the most accelerated among vertebrates. They also possess other unusual features, including short tRNAs and other genes, and a duplicated control region that has been stably maintained since it originated more than 70 million years ago. Here, we provide a detailed analysis of evolutionary dynamics in snake mitochondrial genomes to better understand the basis of these extreme characteristics, and to explore the relationship between mitochondrial genome molecular evolution, genome architecture, and molecular function. We sequenced complete mitochondrial genomes from Slowinski's corn snake (Pantherophis slowinskii) and two cottonmouths (Agkistrodon piscivorus) to complement previously existing mitochondrial genomes, and to provide an improved comparative view of how genome architecture affects molecular evolution at contrasting levels of divergence. Results We present a Bayesian genetic approach that suggests that the duplicated control region can function as an additional origin of heavy strand replication. The two control regions also appear to have different intra-specific versus inter-specific evolutionary dynamics that may be associated with complex modes of concerted evolution. We find that different genomic regions have experienced substantial accelerated evolution along early branches in snakes, with different genes having experienced dramatic accelerations along specific branches. Some of these accelerations appear to coincide with, or subsequent to, the shortening of various mitochondrial genes and the duplication of the control region and flanking tRNAs. Conclusion Fluctuations in the strength and pattern of selection during snake evolution have had widely varying gene-specific effects on substitution rates, and these rate accelerations may have been functionally related to unusual changes in genomic architecture. The among-lineage and

  17. IUPAC-NIST Solubility Data Series. 99. Solubility of Benzoic Acid and Substituted Benzoic Acids in Both Neat Organic Solvents and Organic Solvent Mixtures

    NASA Astrophysics Data System (ADS)

    Acree, William E.

    2013-09-01

    Solubility data are compiled and reviewed for benzoic acid and 63 substituted benzoic acids dissolved in neat organic solvents and well-defined binary and ternary organic solvent mixtures. The compiled solubility data were retrieved from the published chemical and pharmaceutical literature covering the period from 1900 to the beginning of 2013.

  18. Effects of ion substitution on bile acid-dependent and -independent bile formation by rat liver.

    PubMed Central

    Van Dyke, R W; Stephens, J E; Scharschmidt, B F

    1982-01-01

    To characterize the transport mechanisms responsible for formation of canalicular bile, we have examined the effects of ion substitution on bile acid-dependent and bile acid-independent bile formation by the isolated perfused rat liver. Complete replacement of perfusate sodium with choline and lithium abolished taurocholate-induced choleresis and reduced biliary taurocholate output by greater than 70%. Partial replacement of perfusate sodium (25 of 128 mM) by choline reduced bile acid-independent bile formation by 30% and replacement of the remaining sodium (103 mM) by choline reduced bile acid-independent bile formation by an additional 64%. In contrast, replacement of the remaining sodium (103 mM) by lithium reduced bile acid-independent bile formation by only an additional 20%, while complete replacement of sodium (128 mM) by lithium reduced bile formation by only 17%, and lithium replaced sodium as the predominant biliary cation. Replacement of perfusate bicarbonate by Tricine, a zwitterionic amino acid buffer, decreased bile acid-independent bile formation by greater than or equal to 50% and decreased biliary bicarbonate output by approximately 60%, regardless of the accompanying cation. In separate experiments, replacement of sodium by lithium essentially abolished Na,K-ATPase activity measured either as ouabain-suppressible ATP hydrolysis in rat liver or kidney homogenates, or as ouabain-suppressible 86Rb uptake by cultured rat hepatocytes. These studies indicate that bile acid(taurocholate)-dependent bile formation by rat liver exhibits a specific requirement for sodium, a finding probably attributable to the role(s) of sodium in hepatic sodium-coupled taurocholate uptake and/or in maintenance of Na,K-ATPase activity. The surprising finding that bile acid-independent bile formation was substantially unaltered by complete replacement of sodium with the permeant cation lithium does not appear to be explained by Na,K-ATPase-mediated lithium transport. Although

  19. Linear free energy relationship rate constants and basicities of N-substituted phenyl glycines in positronium-glycine complex formation

    NASA Astrophysics Data System (ADS)

    Chen, Rongti; Liang, Jiachang; Du, Youming; Cao, Chun; Yin, Dinzhen; Wang, Shuying; Zhang, Tianbao

    1987-06-01

    Complex formation between positronium and glycine derivatives in solution is discussed and the complex reaction rate constants obtained by means of a positron annihilation lifetime spectrometer with BaF 2 detectors. Rate constants mainly depend on the conjugation effect at the benzene ring and the induction effect of the substituents at the phenyl. There is a linear free energy relationship between rate constants and basicities of N-substituted phenyl glycines in orthopositronium-glycine complex formation.

  20. An amino acid substitution (Gly853-->Glu) in the collagen alpha 1(II) chain produces hypochondrogenesis.

    PubMed

    Bogaert, R; Tiller, G E; Weis, M A; Gruber, H E; Rimoin, D L; Cohn, D H; Eyre, D R

    1992-11-01

    The spondyloepiphyseal dysplasia subclassification of bone dysplasias includes achondrogenesis, hypochondrogenesis, and spondyloepiphyseal dysplasia congenita. The phenotypic expression of these disorders ranges from mild to perinatal lethal forms. We report the detection and partial characterization of a defect in type II collagen in a perinatal lethal form of hypochondrogenesis. Electrophoresis in sodium dodecyl sulfate-polyacrylamide of CB peptides (where CB represents cyanogen bromide) from type II collagen of the diseased cartilage showed a doublet band for peptide alpha 1(II)CB10 and evidence for post-translational overmodification of the major peptides (CB8, CB10, and CB11) seen as a retarded electrophoretic mobility. Peptide CB10 was digested by endoproteinase Asp-N; and on reverse-phase high pressure liquid chromatography, fragments of abnormal mobility were noted. Sequence analysis of a unique peptide D12 revealed a single amino acid substitution (Gly-->Glu) at position 853 of the triple helical domain. This was confirmed by sequence analysis of amplified COL2A1 cDNA, which revealed a single nucleotide substitution (GGA-->GAA) in 5 of 10 clones. Electron micrographs of the diseased cartilage showed a sparse extracellular matrix and chondrocytes containing dilated rough endoplasmic reticulum, which suggested impaired assembly and secretion of the mutant protein. This case further documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mutations in COL2A1. PMID:1429602

  1. Synthesis, characterization, antifungal evaluation and 3D-QSAR study of phenylhydrazine substituted tetronic acid derivatives.

    PubMed

    Hu, Ying; Wang, Junjun; Lu, Aimin; Yang, Chunlong

    2014-08-15

    A series of 3-(1-(2-(substituted phenyl)hydrazinyl)alkylidene)furan-2,4(3H,5H)-diones were designed and prepared using two synthetic routes. Their structures were confirmed by FT-IR, (1)H NMR, (13)C NMR, MS, elemental analysis and single-crystal X-ray diffraction. Their bioactivity was evaluated against Botrytis cinerea in vitro. Most target compounds exhibited remarkable antifungal activity. Two compounds 7f and 7h were highly effective and their EC50 values were 0.241 μg/mL and 0.167 μg/mL, respectively, close to that of the control drug procymidone. 3D-QSAR studies of CoMFA and CoMSIA were carried out. Models with good predictive ability were generated with the cross validated q(2) values for CoMFA and CoMSIA being 0.565 and 0.823. Conventional r(2) values were 0.983 and 0.945, respectively. The results provided a practical tool for guiding the design and synthesis of novel and more potent tetronic acid derivatives containing substituted phenylhydrazine moiety. PMID:25042337

  2. An Amino Acid Substitution (L925V) Associated with Resistance to Pyrethroids in Varroa destructor

    PubMed Central

    González-Cabrera, Joel; Davies, T. G. Emyr; Field, Linda M.; Kennedy, Peter J.; Williamson, Martin S.

    2013-01-01

    The Varroa mite, Varroa destructor, is an important pest of honeybees and has played a prominent role in the decline in bee colony numbers over recent years. Although pyrethroids such as tau-fluvalinate and flumethrin can be highly effective in removing the mites from hives, their intensive use has led to many reports of resistance. To investigate the mechanism of resistance in UK Varroa samples, the transmembrane domain regions of the V. destructor voltage-gated sodium channel (the main target site for pyrethroids) were PCR amplified and sequenced from pyrethroid treated/untreated mites collected at several locations in Central/Southern England. A novel amino acid substitution, L925V, was identified that maps to a known hot spot for resistance within the domain IIS5 helix of the channel protein; a region that has also been proposed to form part of the pyrethroid binding site. Using a high throughput diagnostic assay capable of detecting the mutation in individual mites, the L925V substitution was found to correlate well with resistance, being present in all mites that had survived tau-fluvalinate treatment but in only 8 % of control, untreated samples. The potential for using this assay to detect and manage resistance in Varroa-infected hives is discussed. PMID:24367572

  3. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

    PubMed

    Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

    2014-09-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

  4. Amino acid substitutions in the hepatitis C virus core region of genotype 1b affect very early viral dynamics during treatment with telaprevir, peginterferon, and ribavirin.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Hirakawa, Miharu; Kawamura, Yusuke; Yatsuji, Hiromi; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2010-04-01

    Substitution of amino acid (aa) 70 and 91 in the core region of hepatitis C virus (HCV) genotype 1b can predict the response to pegylated interferon (PEG-IFN)/ribavirin combination therapy, but its impact on triple therapy of telaprevir/PEG-IFN/ribavirin is not clear. The aims of this study were to investigate the rate of HCV RNA loss following 12-week triple therapy, and determine the effect of aa substitutions on very early (within 48 hr) viral dynamics. Sixty-seven patients infected with HCV genotype 1b (HCV-1b) and high viral load who received 12-week triple therapy were studied. RNA loss could be achieved in 2%, 34%, 80%, 92%, 95%, 94%, and 90% of the patients after 1, 2, 4, 6, 8, 10, and 12 weeks of triple therapy, respectively. After 24-hr treatment, the proportion of patients with Arg70 and Leu91 substitutions with > or = 3.0 log fall in HCV RNA was significantly higher than those with < 3.0 log fall (P = 0.008). However, the aa substitution patterns in the core region did not influence the fall in HCV RNA after 48-hr treatment. Multivariate analysis identified substitutions of aa 70 and 91 (P = 0.014) and level of viremia at baseline (> or = 7.0 log IU/ml; P = 0.085) as independent parameters that determined the > or = 3.0 log fall in HCV RNA level after 24-hr triple therapy. It is concluded that 12-week triple therapy achieved high rates of loss of HCV RNA in Japanese patients infected with HCV-1b and high viral load, and that the aa substitution pattern in the core region seems to influence very early viral dynamics. PMID:20166188

  5. Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction

    PubMed Central

    Teng, Shaolei; Wang, Liangjiang; Srivastava, Anand K.; Schwartz, Charles E.; Alexov, Emil

    2012-01-01

    A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases. PMID:21297231

  6. Cu(II) /TEMPO-promoted one-pot synthesis of highly substituted pyrimidines from amino acid esters.

    PubMed

    Zhou, Nini; Xie, Tao; Li, Zhongle; Xie, Zhixiang

    2014-12-22

    A novel, Cu(OAc)2/TEMPO promoted one-step approach for the preparation of fully substituted pyrimidines from readily available amino acid esters has been described. In this reaction, the amino acid esters act as the only N-C sources for the construction of corresponding pyrimidines. The mechanism of this process includes oxidative dehydrogenation, the generation of an imine radical, and a formal [3+3] cycloaddition. This methodology proves to be a high atom-economic and straightforward strategy for the synthesis of pyrimidines and diverse substrates which are substituted by various functional groups have been afforded in moderate to good yield. PMID:25377658

  7. Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase

    PubMed Central

    Iyobe, Shizuko; Kusadokoro, Haruko; Ozaki, Junko; Matsumura, Naoki; Minami, Shinzaburo; Haruta, Shin; Sawai, Tetsuo; O'Hara, Koji

    2000-01-01

    In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene blaIMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene blaIMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of blaIMP-3 was identical to that of blaIMP-1, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from blaIMP-3 and blaIMP-1 by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the blaIMP-3 gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene blaIMP-3 is an ancestor of blaIMP-1. PMID:10898670

  8. Acid mine drainage simulated leaching behavior of goethite and cobalt substituted goethite

    NASA Astrophysics Data System (ADS)

    Penprase, S. B.; Kimball, B. E.

    2015-12-01

    Though most modern day mining aims to eliminate the seepage of acid mine drainage (AMD) to the local watershed, historical mines regularly receive little to no remediation, and often release acidic, metal-rich drainage and particles to the environment. Treatment of AMD often includes neutralizing pH to facilitate the precipitation of Fe-oxides and dissolved trace metals, thereby forming Trace Metal Substituted (TMS) forms of known minerals, such as goethite (α-FeOOH). The stability of TMS precipitates is not fully understood. As a result, we conducted a 20 day leach experiment using laboratory synthesized pure (Gt) and cobalt-substituted (CoGt) goethites with a dilute ultrapure HCl solution (pH = 3.61) at T = 23.3±2.5ºC. Leached solids were characterized using X-ray diffraction (XRD) and scanning electron microscopy paired with energy dispersive spectroscopy (SEM-EDS). Leach solutions were sampled for pH and conductivity, and dissolved chemistry was determined with Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Preliminary results indicate Gt and CoGt filtered leach solutions experienced constant pH (Gt = 3.9 ± 0.1, CoGt = 6.8 ± 0.2) and conductivity (Gt = 69 ± 6.6 μS/cm, CoGt = 81 ± 16 μS/cm) for t = 0-20 days. Micro-focused XRD results indicate that leached solids did not change in mineralogy throughout the experiment, and SEM images show minor disintegration along mineral grain edges, but little overall change in shape. Preliminary ICP-MS results show lower dissolved Fe concentrations for CoGt (1.1 ± 1.1 ppb) compared to Gt (17 ± 8.9 ppb) over time. Dissolved Co concentrations ranged from 560 - 830 ppb and increased over time. Compared to leaching of pure Gt, leaching of CoGt generated significantly higher pH, slightly higher conductivity, and significantly less dissolved Fe. During the CoGt leach, Co was preferentially leached over Fe. The differences in leaching behavior between pure and TMS goethite in the laboratory have implications for

  9. Anisotropic scattering rate in Fe-substituted Bi2Sr2Ca(Cu1-xFex)2O8+δ

    DOE PAGESBeta

    Naamneh, M.; Lubashevsky, Y.; Lahoud, E.; Gu, G.; Kanigel, A.

    2015-05-27

    We measured the electronic structure of Fe substituted Bi2212 using Angle Resolved Photoemission Spectroscopy (ARPES). We find that the substitution does not change the momentum dependence of the superconducting gap but induces a very anisotropic enhancement of the scattering rate. A comparison of the effect of Fe substitution to that of Zn substitution suggests that the Fe reduces Tc so effectively because it supresses very strongly the coherence weight around the anti-nodes.

  10. Slow and Fast Evolving Endosymbiont Lineages: Positive Correlation between the Rates of Synonymous and Non-Synonymous Substitution

    PubMed Central

    Silva, Francisco J.; Santos-Garcia, Diego

    2015-01-01

    The availability of complete genome sequences of bacterial endosymbionts with strict vertical transmission to the host progeny opens the possibility to estimate molecular evolutionary rates in different lineages and understand the main biological mechanisms influencing these rates. We have compared the rates of evolution for non-synonymous and synonymous substitutions in nine bacterial endosymbiont lineages, belonging to four clades (Baumannia, Blochmannia, Portiera, and Sulcia). The main results are the observation of a positive correlation between both rates with differences among lineages of up to three orders of magnitude and that the substitution rates decrease over long endosymbioses. To explain these results we propose three mechanisms. The first, variations in the efficiencies of DNA replication and DNA repair systems, is unable to explain most of the observed differences. The second, variations in the generation time among bacterial lineages, would be based on the accumulation of fewer DNA replication errors per unit time in organisms with longer generation times. The third, a potential control of the endosymbiont DNA replication and repair systems through the transfer of nuclear-encoded proteins, could explain the lower rates in long-term obligate endosymbionts. Because the preservation of the genomic integrity of the harbored obligate endosymbiont would be advantageous for the insect host, biological mechanisms producing a general reduction in the rates of nucleotide substitution per unit of time would be a target for natural selection. PMID:26617602

  11. Time Clustered Sampling Can Inflate the Inferred Substitution Rate in Foot-And-Mouth Disease Virus Analyses

    PubMed Central

    Pedersen, Casper-Emil T.; Frandsen, Peter; Wekesa, Sabenzia N.; Heller, Rasmus; Sangula, Abraham K.; Wadsworth, Jemma; Knowles, Nick J.; Muwanika, Vincent B.; Siegismund, Hans R.

    2015-01-01

    With the emergence of analytical software for the inference of viral evolution, a number of studies have focused on estimating important parameters such as the substitution rate and the time to the most recent common ancestor (tMRCA) for rapidly evolving viruses. Coupled with an increasing abundance of sequence data sampled under widely different schemes, an effort to keep results consistent and comparable is needed. This study emphasizes commonly disregarded problems in the inference of evolutionary rates in viral sequence data when sampling is unevenly distributed on a temporal scale through a study of the foot-and-mouth (FMD) disease virus serotypes SAT 1 and SAT 2. Our study shows that clustered temporal sampling in phylogenetic analyses of FMD viruses will strongly bias the inferences of substitution rates and tMRCA because the inferred rates in such data sets reflect a rate closer to the mutation rate rather than the substitution rate. Estimating evolutionary parameters from viral sequences should be performed with due consideration of the differences in short-term and longer-term evolutionary processes occurring within sets of temporally sampled viruses, and studies should carefully consider how samples are combined. PMID:26630483

  12. Amino-acid substitution in alpha-spectrin commonly coinherited with nondominant hereditary spherocytosis.

    PubMed

    Tse, W T; Gallagher, P G; Jenkins, P B; Wang, Y; Benoit, L; Speicher, D; Winkelmann, J C; Agre, P; Forget, B G; Marchesi, S L

    1997-03-01

    Nondominant hereditary spherocytosis (ndHS) is a disorder characterized in some patients by severe hemolytic anemia and marked deficiency of erythrocyte spectrin. This report describes the identification of a variant spectrin chain, alpha-spectrin Bughill or alpha(BH), that is associated with this disorder in a number of patients. Tryptic maps of spectrin from affected individuals revealed an acidic shift in isoelectric point of the alphaII domain peptides at 46 kD and 35 kD. A point mutation at codon 970 of the alpha-spectrin gene (GCT-->GAT), that changes the encoded amino acid from an alanine to an aspartic acid, was identified in genomic DNA of affected patients. The alpha(BH) variant was present in 8 patients with ndHS from five different kindreds but was absent in 4 patients from two other kindreds. The 8 ndHS patients with the alpha(BH) variant appeared to be homozygous for the alpha(BH) variant by analysis of peptide maps of limited tryptic digests of erythrocyte spectrin. However, following genomic DNA analysis, only 2 of these patients were true homozygotes, whereas 6 were found to be doubly heterozygous for the alpha(BH) allele and a second, presumably abnormal, alpha-spectrin gene. These results suggest that, in these 6 patients, the second alpha-spectrin allele is in fact associated with one or more genetic defect(s), causing decreased accumulation of alpha-spectrin. The pattern of transmission of the alpha(BH) allele in certain families suggests that the alpha(BH) amino-acid substitution is not itself responsible for ndHS but is more likely a polymorphic variant that, in some but not all cases, is in linkage disequilibrium with another uncharacterized alpha-spectrin gene defect that itself is a cause of ndHS. PMID:9067503

  13. Role of a single amino acid substitution of VP3 H142D for increased acid resistance of foot-and-mouth disease virus serotype A.

    PubMed

    Biswal, Jitendra K; Das, Biswajit; Sharma, Gaurav K; Khulape, Sagar A; Pattnaik, Bramhadev

    2016-04-01

    Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their dissociation into pentamers at pH value below 6.5. After the uptake of FMDV by receptor-mediated endocytosis, the acid-dependent dissociation process is required for the release of FMDV genome inside endosomes. Nevertheless, dissociation of FMDV particles in mildly acidic conditions renders the inactivated FMD vaccine less effective. To improve the acid stability of inactivated FMD vaccine during the manufacturing process, a serotype A IND 40/2000 (in-use vaccine strain) mutant with increased resistance to acid inactivation was generated through reverse genetics approach. Based upon the earlier reports, the crucial amino acid residue, H142 of VP3 capsid protein was substituted separately to various amino acid residues Arg (R), Phe (F), Ala (A), and Asp (D) on the full-genome length cDNA clone. While the H142 → R or H142 → F or H142 → A substitutions resulted in non-infectious FMDV, H142 → D mutation on VP3 protein (H3142D) resulted in the generation of mutant virus with enhanced resistance to acid-induced inactivation. In addition, H3142D substitution did not alter the replication ability and antigenicity of mutant as compared to the parental virus. However, the virus competition experiments revealed that the H3142D substitution conferred a loss of fitness for the mutant virus. Results from this study demonstrate that the H3142D substitution is the molecular determinant of acid-resistant phenotype in FMDV serotype A. PMID:26873406

  14. Refocusing of B-cell responses following a single amino acid substitution in an antigen

    PubMed Central

    Chiesa, Marta Dalla; Martensen, Pia M; Simmons, Cameron; Porakishvili, Nino; Justesen, Just; Dougan, Gordon; Roitt, Ivan M; Delves, Peter J; Lund, Torben

    2001-01-01

    Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) β-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGβ, a mutated hCGβ-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGβ shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules. PMID:11412304

  15. N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids as inhibitors of ureases.

    PubMed

    Berlicki, Lukasz; Bochno, Marta; Grabowiecka, Agnieszka; Białas, Arkadiusz; Kosikowska, Paulina; Kafarski, Paweł

    2012-05-01

    Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (Ki=13±0.8 and 0.62±0.09 μM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique. PMID:21559954

  16. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  17. 40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Reaction products of secondary alkyl... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9220 Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

  18. 40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Reaction products of secondary alkyl... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9220 Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

  19. 40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Reaction products of secondary alkyl... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9220 Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

  20. 40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Reaction products of secondary alkyl... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9220 Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

  1. 40 CFR 721.9220 - Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Reaction products of secondary alkyl... SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9220 Reaction products of secondary alkyl amines with a substituted benzenesulfonic acid and sulfuric...

  2. Highly regio- and diastereoselective synthesis of CF3-substituted lactones via photoredox-catalyzed carbolactonization of alkenoic acids.

    PubMed

    Yasu, Yusuke; Arai, Yusuke; Tomita, Ren; Koike, Takashi; Akita, Munetaka

    2014-02-01

    Trifluoromethylative lactonization of both terminal and internal alkenoic acids by photoredox catalysis has been developed. The use of a Ru photocatalyst and Umemoto's reagent as a CF3 source is key in the present carbolactonization. This is the first example of a highly endo- and diastereoselective synthesis of CF3-substituted five-, six-, and seven-membered ring lactones from internal alkenoic acids. PMID:24422891

  3. A1/A2-Diamino-Substituted Pillar[5]arene-Based Acid-Base-Responsive Host-Guest System.

    PubMed

    Hu, Wei-Bo; Hu, Wen-Jing; Zhao, Xiao-Li; Liu, Yahu A; Li, Jiu-Sheng; Jiang, Biao; Wen, Ke

    2016-05-01

    An acid-base-responsive supramolecular host-guest system based on a planarly chiral A1/A2-diamino-substituted pillar[5]arene (1)/imidazolium ion recognition motif was created. The pillar[4]arene[1]diaminobenzene 1 can bring an electron-deficient imidazolium cation into its cylindrically shaped cavity under neutral or basic conditions and release it under acidic conditions. PMID:27088317

  4. Synthesis of substituted benzooxaborinin-1-ols via palladium-catalysed cyclisation of alkenyl- and alkynyl-boronic acids.

    PubMed

    Benhamou, Laure; Walker, Daniel W; Bučar, Dejan-Krešimir; Aliev, Abil E; Sheppard, Tom D

    2016-09-14

    Two new palladium-catalysed reactions have been developed for the synthesis of stable 4-substituted benzooxaborinin-1-ols. A palladium-catalysed cyclisation of ortho-alkenylbenzene boronic acids can be used to access 4-chlorobenzooxaborinin-1-ols via a Wacker-type oxidation and chlorination. Alternatively, ortho-alkynylbenzene boronic acids undergo a palladium-catalysed oxyallylation reaction to provide 4-allylbenzooxaborinin-1-ols. PMID:27506186

  5. Relative Reaction Rates of Sulfamic Acid and Hydroxylamine with Nitric Acid

    SciTech Connect

    Karraker, D.G.

    2001-03-28

    This report describes a study of comparative reaction rates where the reductant is in excess, as in the 1B bank in the Purex process. The results of this work apply to planned plant tests to partially substitute HAN for the ferrous sulfamate reductant in the Purex 1B bank.

  6. Mutational Biases Drive Elevated Rates of Substitution at Regulatory Sites across Cancer Types.

    PubMed

    Kaiser, Vera B; Taylor, Martin S; Semple, Colin A

    2016-08-01

    Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection. PMID:27490693

  7. Mutational Biases Drive Elevated Rates of Substitution at Regulatory Sites across Cancer Types

    PubMed Central

    Semple, Colin A.

    2016-01-01

    Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection. PMID:27490693

  8. ESTIMATION OF CARBOXYLIC ACID ESTER HYDROLYSIS RATE CONSTANTS

    EPA Science Inventory

    SPARC chemical reactivity models were extended to calculate hydrolysis rate constants for carboxylic acid esters from molecular structure. The energy differences between the initial state and the transition state for a molecule of interest are factored into internal and external...

  9. Heart Rate Response and Lactic Acid Concentration in Squash Players.

    ERIC Educational Resources Information Center

    Beaudin, Paula; And Others

    1978-01-01

    It was concluded that playing squash is an activity that results in heart rate responses of sufficient intensity to elicit aerobic training effects without producing high lactic acid concentration in the blood. (MM)

  10. Vapor pressures of substituted polycarboxylic acids are much lower than previously reported

    NASA Astrophysics Data System (ADS)

    Huisman, A. J.; Krieger, U. K.; Zuend, A.; Marcolli, C.; Peter, T.

    2013-07-01

    The partitioning of compounds between the aerosol and gas phase is a primary focus in the study of the formation and fate of secondary organic aerosol. We present measurements of the vapor pressure of 2-methylmalonic (isosuccinic) acid, 2-hydroxymalonic (tartronic) acid, 2-methylglutaric acid, 3-hydroxy-3-carboxy-glutaric (citric) acid and DL-2,3-dihydroxysuccinic (DL-tartaric) acid, which were obtained from the evaporation rate of supersaturated liquid particles levitated in an electrodynamic balance. Our measurements indicate that the pure component liquid vapor pressures at 298.15 K for tartronic, citric and tartaric acids are much lower than the same quantity that was derived from solid state measurements in the only other room temperature measurement of these materials (made by Booth et al., 2010). This strongly suggests that empirical correction terms in a recent vapor pressure estimation model to account for the inexplicably high vapor pressures of these and similar compounds should be revisited, and that due caution should be used when the estimated vapor pressures of these and similar compounds are used as inputs for other studies.

  11. Vapor pressures of substituted polycarboxylic acids are much lower than previously reported

    NASA Astrophysics Data System (ADS)

    Huisman, A. J.; Krieger, U. K.; Zuend, A.; Marcolli, C.; Peter, T.

    2013-01-01

    The partitioning of compounds between the aerosol and gas phase is a primary focus in the study of the formation and fate of secondary organic aerosol. We present measurements of the vapor pressure of 2-Methylmalonic (isosuccinic) acid, 2-Hydroxymalonic (tartronic) acid, 2-Methylglutaric acid, 3-Hydroxy-3-carboxy-glutaric (citric) acid and 2,3-Dihydroxysuccinic (tartaric) acid which were obtained from the evaporation rate of supersaturated liquid particles levitated in an electrodynamic balance. Our measurements indicate that the pure component liquid vapor pressures at 298.15 K for tartronic, citric and tartaric acids are much lower than the same quantity which was derived from solid state measurements in the only other room temperature measurement of these materials (made by Booth et al., 2010). This strongly suggests that empirical correction terms in vapor pressure estimation models to account for the inexplicably high vapor pressures of these and similar compounds should be revisited, and that due caution should be used when the estimated vapor pressures of these and similar compounds are used as inputs for other studies.

  12. Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids.

    PubMed

    Ratnayake, Nishanka Dilini; Theisen, Chelsea; Walter, Tyler; Walker, Kevin D

    2016-01-10

    Biologically-active β-peptides and pharmaceuticals that contain key β-amino acids are emerging as target therapeutics; thus, synthetic strategies to make substituted, enantiopure β-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼ 35) engineered to express a Pantoea agglomerans phenylalanine aminomutase (PaPAM) biocatalyst. In either 5 mL, 100mL, or 1L of M9 minimal medium containing α-phenylalanine (20mM), the cells produced ∼ 1.4 mg mL(-1) of β-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted α-arylalanines to their (S)-β-aryl-β-amino acids in vivo and were not toxic to cells at mid- to late-stage growth. The β-aryl-β-amino acids made ranged from 0.043 mg (p-nitro-β-phenylalanine, 4% converted yield) to 1.2mg (m-bromo-β-phenylalanine, 96% converted yield) over 6h in 5 mL. The substituted β-amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building blocks, or as bioisosteres in drug design. PMID:26528624

  13. High rates of substitution of the native catfish Clarias batrachus by Clarias gariepinus in India.

    PubMed

    Khedkar, Gulab D; Tiknaik, Anita D; Shinde, Rushidkumar N; Kalyankar, Amol D; Ron, Tetsuzan Benny; Haymer, David

    2016-01-01

    The clariid catfish, Clarias batrachus commonly known as Magur, has declined drastically from natural habitats in India during the last decade. This fish is highly preferred fish by Indian consumers and has high market demand. As a result traders often substitute C. batrachus with a morphologically similar but supposedly banned exotic catfish, C. gariepinus, in India. This study uses rigorous morphological comparisons confirmed by DNA barcode analysis to examine the level of substitution of C. batracus by C. gariepinus in India. Our results indicate that up to 99% (in many cases) of the market samples sold as Magur or C. batrachus were in fact C. gariepinus. PMID:24708138

  14. Microwave-Assisted Syntheses of Amino Acid Ester Substituted Benzoic Acid Amides: Potential Inhibitors of Human CD81-Receptor HCV-E2 Interaction.

    PubMed

    Holzer, Marcel; Ziegler, Sigrid; Kronenberger, Bernd; Klein, Christian D; Hartmann, Rolf W

    2008-01-01

    Results from our group showed benzyl salicylate to be a moderate inhibitor of the CD81-LEL-HCV-E2 interaction. To increase the biological activity, heterocyclic substituted benzoic acids were coupled to amino acid esters via microwave assisted DCC-reaction. The prepared compounds were tested for their inhibitory potency by means of a fluorescence labeled antibody assay system using HUH7.5 cells. PMID:19662141

  15. High-Temperature Decomposition of Brønsted Acid Sites in Gallium-Substituted Zeolites

    SciTech Connect

    K Al-majnouni; N Hould; W Lonergan; D Vlachos; R Lobo

    2011-12-31

    The dehydroxylation of Broensted acid sites (BAS) in Ga-substituted zeolites was investigated at temperatures up to 850 C using X-ray absorption spectroscopy (XAS), Fourier transform infrared spectroscopy (FTIR), and mass spectrometry-temperature programmed desorption (MS-TPD). X-ray absorption near-edge spectroscopy (XANES) revealed that the majority of gallium has tetrahedral coordination even after complete dehydroxylation. The interatomic gallium-oxygen distance and gallium coordination number determined by extended X-ray absorption fine structure (EXAFS) are consistent with gallium in tetrahedral coordination at low T (< 550 C). Upon heating Ga-Beta and Ga-ZSM5 to 850 C, analysis of the EXAFS showed that 70 and 80% of the gallium was still in tetrahedral coordination. The remainder of the gallium was found to be in octahedral coordination. No trigonal Ga atoms were observed. FTIR measurements carried out at similar temperatures show that the intensity of the OH vibration due to BAS has been eliminated. MS-TPD revealed that hydrogen in addition to water evolved from the samples during dehydroxylation. This shows that dehydrogenation in addition to dehydration is a mechanism that contributes to BAS decomposition. Dehydrogenation was further confirmed by exposing the sample to hydrogen to regenerate some of the BAS as monitored by FTIR and MS-TPD.

  16. Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

    PubMed Central

    Stavridi, E. S.; Chehab, N. H.; Caruso, L. C.; Halazonetis, T. D.

    1999-01-01

    The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain. PMID:10493578

  17. Preparation of a graphene oxide/silica composite modified with nitro-substituted tris(indolyl)methane as a solid-phase extraction sorbent for the extraction of organic acids.

    PubMed

    Wang, Na; Yu, Hui; Shao, Shijun

    2016-05-01

    This paper describes the use of graphene oxide/silica modified with nitro-substituted tris(indolyl)methane as a solid-phase extraction sorbent for the determination of organic acids. The resultant graphene oxide/silica modified with nitro-substituted tris(indolyl)methane was characterized by FTIR spectroscopy and adsorption experiments. Solid-phase extraction parameters such as sorbent type, sample solution pH, sample loading rate, eluent salt concentration, eluent methanol concentration, elution rate, sample loading, and elution volume were optimized. The method showed good precision, accuracy, sensitivity, and linear response for organic acids analysis over a concentration range of 1-100 μg/L for benzoic acid, p-methoxybenzoic acid, and salicylic acid and 5-100 μg/L for the remaining organic acids (cinnamic acid, p-chlorobenzoic acid, and p-bromobenzoic acid) with coefficients of determination (r(2) ) of higher than 0.9957. Limits of detection from 0.50 to 1.0 μg/L for six organic acids were achieved. The developed method was successfully applied to determine organic acids in real samples. PMID:26969351

  18. Syntheses of 2-substituted 1-amino-4-bromoanthraquinones (bromaminic acid analogues) – precursors for dyes and drugs

    PubMed Central

    Malik, Enas M

    2015-01-01

    Summary Anthraquinone (AQ) derivatives play a prominent role in medicine and also in textile industry. Bromaminic acid (1-amino-4-bromoanthraquinone-2-sulfonic acid) is an important precursor for obtaining dyes as well as biologically active compounds through the replacement of the C4-bromo substituent with different (ar)alkylamino residues. Here we report methods for the synthesis of bromaminic acid analogues bearing different substituents at the 2-position of the anthraquinone core. 1-Aminoanthraquinone was converted to its 2-hydroxymethyl-substituted derivative which, under different reaction conditions, yielded the corresponding carbaldehyde, carboxylic acid, and nitrile derivatives. The latter was further reacted to obtain 1-amino-2-tetrazolylanthraquinone. Subsequent bromination using bromine in DMF led to the corresponding bromaminic acid derivatives in excellent isolated yields (>90%) and high purities. Alternatively, 1-amino-4-bromo-2-hydroxymethylanthraquinone could be directly converted to the desired 2-substituted bromaminic acid analogues in high yields (85–100%). We additionally report the preparation of bromaminic acid sodium salt and 1-amino-2,4-dibromoanthraquinone directly from 1-aminoanthraquinone in excellent yields (94–100%) and high purities. The synthesized brominated AQs are valuable precursors for the preparation of AQ drugs and dyes. PMID:26734081

  19. Electronic Effects of 11β Substituted 17β-Estradiol Derivatives and Instrumental Effects on the Relative Gas Phase Acidity

    NASA Astrophysics Data System (ADS)

    Bourgoin-Voillard, Sandrine; Fournier, Françoise; Afonso, Carlos; Zins, Emilie-Laure; Jacquot, Yves; Pèpe, Claude; Leclercq, Guy; Tabet, Jean-Claude

    2012-12-01

    Numerous studies have highlighted the role of the proton donor characteristics of the phenol group of 17β-estradiol (E2) in its association with the estrogen receptor alpha (ERα). Since the substitutions at position C(11) have been reported to modulate this association, we hypothesized that such substitutions may modify the phenol acidity. Hence, phenol gas-phase acidity of nine C(11)-substituted E2-derivatives were evaluated using the extended Cooks' kinetic method, which is a method widely used to determine thermochemical properties by mass spectrometry. To enhance accuracy in data collection we recorded data from several instruments, including quadrupole ion trap, triple quadrupole, and hybrid QqTOF. Indeed, we report for the first time the use of the QqTOF instrument to provide a novel means to improve data accuracy by giving access to an intermediate effective temperature range. All experimental gas-phase acidity values were supported by theoretical calculations. Our results confirmed the ability of distant substituents at C(11) to modulate the phenol acidity through electrostatic interactions, electron withdrawing inductive effects, and mesomeric effects. However, no relationship was found between the phenol gas-phase acidity of investigated steroids and their binding affinity for ERα assessed in solution. Thus, our results highlight that the intrinsic properties of the hormone do not influence sufficiently the stabilization of the hormone/ERα complex. It is more likely that such stabilization would be more related to factors depending on the environment within the binding pocket such as hydrophobic, steric as well as direct intermolecular electrostatic effects between ERα residues and the substituted steroidal estrogens.

  20. ACUTE TOXICITY OF SELECTED SUBSTITUTED PHENOLS, BENZENES AND BENZOIC ACID ESTERS TO FATHEAD MINNOWS 'PIMEPHALES PROMELAS'

    EPA Science Inventory

    Flow-through acute toxicity tests were conducted with 24 organic compounds using fathead minnows Pimephales promelas as test organisms. The tested toxicants consisted of 11 substituted phenols, four substituted benzenes and nine esters. The 96-h LC50 values determined for these c...

  1. Spectroscopic and Computational Investigation of the H155A Variant of Cysteine Dioxygenase: Geometric and Electronic Consequences of a Third-Sphere Amino Acid Substitution.

    PubMed

    Blaesi, Elizabeth J; Fox, Brian G; Brunold, Thomas C

    2015-05-12

    Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of l-cysteine (Cys) to cysteinesulfinic acid. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a rate of Cys oxidation ∼100-fold decreased relative to that of wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155 → Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe(II)CDO active site. An analysis of these data within the framework of density functional theory calculations reveals that Cys-bound H155A Fe(II)CDO possesses a six-coordinate Fe(II) center, differing from the analogous WT CDO species in the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased level of conformational freedom of the cysteine-tyrosine cross-link in the absence of the H155 imidazole ring. Notably, the nitrosyl adduct of Cys-bound Fe(II)CDO [which mimics the (O2/Cys)-CDO intermediate] is essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO catalysis is to discourage the binding of a water molecule to the Cys-bound Fe(II)CDO active site. PMID:25897562

  2. Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs.

    PubMed

    Nojiri, Masutoshi; Hibi, Makoto; Shizawa, Hiroaki; Horinouchi, Nobuyuki; Yasohara, Yoshihiko; Takahashi, Satomi; Ogawa, Jun

    2015-12-01

    The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1% enantiomeric excess (e.e.) and 97.5% e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9% e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75% sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics. PMID:26205522

  3. Enantioconvergent Nucleophilic Substitution Reaction of Racemic Alkyne-Dicobalt Complex (Nicholas Reaction) Catalyzed by Chiral Brønsted Acid.

    PubMed

    Terada, Masahiro; Ota, Yusuke; Li, Feng; Toda, Yasunori; Kondoh, Azusa

    2016-08-31

    Catalytic enantioselective syntheses enable a practical approach to enantioenriched molecules. While most of these syntheses have been accomplished by reaction at the prochiral sp(2)-hybridized carbon atom, little attention has been paid to enantioselective nucleophilic substitution at the sp(3)-hybridized carbon atom. In particular, substitution at the chiral sp(3)-hybridized carbon atom of racemic electrophiles has been rarely exploited. To establish an unprecedented enantioselective substitution reaction of racemic electrophiles, enantioconvergent Nicholas reaction of an alkyne-dicobalt complex derived from racemic propargylic alcohol was developed using a chiral phosphoric acid catalyst. In the present enantioconvergent process, both enantiomers of the racemic alcohol were transformed efficiently to a variety of thioethers with high enantioselectivity. The key to achieving success is dynamic kinetic asymmetric transformation (DYKAT) of enantiomeric cationic intermediates generated via dehydroxylation of the starting racemic alcohol under the influence of the chiral phosphoric acid catalyst. The present fascinating DYKAT involves the efficient racemization of these enantiomeric intermediates and effective resolution of these enantiomers through utilization of the chiral conjugate base of the phosphoric acid. PMID:27490239

  4. Computer-assisted automated synthesis. III. Synthesis of substituted N-(carboxyalkyl) amino-acid tert-butyl ester derivatives.

    PubMed

    Hayashi, N; Sugawara, T; Kato, S

    1991-01-01

    A versatile automated synthesis apparatus, equipped with a chemical artificial intelligence, was developed to prepare and isolate a wide variety of compounds. The apparatus was to the synthesis of substituted N-(carboxyalkyl)amino-acids. The apparatus [1,2] is composed of units for performing various tasks,for example reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes, including washing and drying of the apparatus after each synthetic run, were automatically performed from the mixing of the reactants to the isolation of the products as powders or crystals. The reaction of an amino-acid tertbutyl ester acetic acid salt with a 2-keto acid sodium salt produces an unstable intermediate, Schiff base, which is reduced with sodum cyanoborohydride to give a substituted N-(carboxyalkyl)aminoacid tert-butyl ester sodium salt. The equilibrium and the consecutive reactions were controlled by adding sodium cyanoborohydride using the artificial intelligence software, which contained novel kinetic equations [3] and substituent effects [4].Substitued N-(carboxyalkyl)amino-acid tert-butyl esters, 90 derivatives, were automatically synthesized using the computerassisted automated synthesis apparatus. The syntheses were performed unattended 24 hours a day, except for supplying the raw materials, reagents and solvents. The apparatus is extremely valuable for synthesizing many derivatives of a particular compound. The configurations of the products were determined by circular dichroism measurements. PMID:18924904

  5. Computer-assisted automated synthesis. III. Synthesis of substituted N-(carboxyalkyl) amino-acid tert-butyl ester derivatives

    PubMed Central

    Hayashi, Nobuyoshi; Sugawara, Tohru; Kato, Shinji

    1991-01-01

    A versatile automated synthesis apparatus, equipped with a chemical artificial intelligence, was developed to prepare and isolate a wide variety of compounds. The apparatus was to the synthesis of substituted N-(carboxyalkyl)amino-acids. The apparatus [1,2] is composed of units for performing various tasks,for example reagent supply, reaction, purification and separation, each linked to a control system. All synthetic processes, including washing and drying of the apparatus after each synthetic run, were automatically performed from the mixing of the reactants to the isolation of the products as powders or crystals. The reaction of an amino-acid tertbutyl ester acetic acid salt with a 2-keto acid sodium salt produces an unstable intermediate, Schiff base, which is reduced with sodum cyanoborohydride to give a substituted N-(carboxyalkyl)aminoacid tert-butyl ester sodium salt. The equilibrium and the consecutive reactions were controlled by adding sodium cyanoborohydride using the artificial intelligence software, which contained novel kinetic equations [3] and substituent effects [4]. Substitued N-(carboxyalkyl)amino-acid tert-butyl esters, 90 derivatives, were automatically synthesized using the computerassisted automated synthesis apparatus. The syntheses were performed unattended 24 hours a day, except for supplying the raw materials, reagents and solvents. The apparatus is extremely valuable for synthesizing many derivatives of a particular compound. The configurations of the products were determined by circular dichroism measurements. PMID:18924904

  6. A New Hierarchy of Phylogenetic Models Consistent with Heterogeneous Substitution Rates

    PubMed Central

    Woodhams, Michael D.; Fernández-Sánchez, Jesús; Sumner, Jeremy G.

    2015-01-01

    When the process underlying DNA substitutions varies across evolutionary history, some standard Markov models underlying phylogenetic methods are mathematically inconsistent. The most prominent example is the general time-reversible model (GTR) together with some, but not all, of its submodels. To rectify this deficiency, nonhomogeneous Lie Markov models have been identified as the class of models that are consistent in the face of a changing process of DNA substitutions regardless of taxon sampling. Some well-known models in popular use are within this class, but are either overly simplistic (e.g., the Kimura two-parameter model) or overly complex (the general Markov model). On a diverse set of biological data sets, we test a hierarchy of Lie Markov models spanning the full range of parameter richness. Compared against the benchmark of the ever-popular GTR model, we find that as a whole the Lie Markov models perform well, with the best performing models having 8–10 parameters and the ability to recognize the distinction between purines and pyrimidines. PMID:25858352

  7. Long-term care and hospital utilisation by older people: an analysis of substitution rates.

    PubMed

    Forder, Julien

    2009-11-01

    Older people are intensive users of hospital and long-term care services. This paper explores the extent to which these services are substitutes. A small area analysis was used with both care home and (tariff cost-weighted) hospital utilisation for older people aggregated to electoral wards in England.Health and social-care structural equations were specified using a theoretical model. The estimation accounted for the skewed and censored nature of the data. For health utilisation, both a fixed effects instrumental variables GMM model and a generalised estimating equations (GEE) model were fitted, the later on a log dependent variable with predicted values of social care utilisation used to account for endogeneity (bootstrapping was used to derive standard errors). In addition to a GMM model, the social-care estimation used both two-part and tobit models (also with predicted health utilisation and bootstrapping).The results indicate that for each additional pound1 spent on care homes, hospital expenditure falls by pound0.35. Also, pound1 additional hospital spend corresponds to just over pound0.35 reduction on care home spend. With these cost substitution effects offsetting, a transfer of resources to care homes is efficient if the resultant outcome gain is greater than the outcome loss from reduced hospital use. PMID:19206085

  8. Synthesis of [.sup.13C] and [.sup.2H] substituted methacrylic acid, [.sup.13C] and [.sup.2H] substituted methyl methacrylate and/or related compounds

    DOEpatents

    Alvarez, Marc A.; Martinez, Rodolfo A.; Unkefer, Clifford J.

    2008-01-22

    The present invention is directed to labeled compounds of the formulae ##STR00001## wherein Q is selected from the group consisting of --S--, --S(.dbd.O)--, and --S(.dbd.O).sub.2--, Z is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group selected from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each independently selected from the group consisting of a C.sub.1-C.sub.4 lower alkyl, an aryl, and an alkoxy group, and X is selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl group, and a fully-deuterated C.sub.1-C.sub.4 lower alkyl group. The present invention is also directed to a process of preparing labeled compounds, e.g., process of preparing [.sup.13C]methacrylic acid by reacting a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13CH.sub.2)-- aryl sulfone precursor with .sup.13CHI to form a (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate, and, reacting the (CH.sub.3CH.sub.2O--.sup.13C(O)--.sup.13C(.sup.13CH.sub.3).sub.2)-- aryl sulfone intermediate with sodium hydroxide, followed by acid to form [.sup.13C]methacrylic acid. The present invention is further directed to a process of preparing [.sup.2H.sub.8]methyl methacrylate by reacting a (HOOC--C(C.sup.2H.sub.3).sub.2-- aryl sulfinyl intermediate with CD.sub.3I to form a (.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate, and heating the(.sup.2H.sub.3COOC--C(C.sup.2H.sub.3).sub.2)-- aryl sulfinyl intermediate at temperatures and for time sufficient to form [.sup.2H.sub.8]methyl methacrylate.

  9. Degradation rates of glycerol polyesters at acidic and basic conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyesters prepared from glycerol with mixtures of adipic and citric acids were evaluated in the laboratory to estimate degradation rates over a range of pH conditions. These renewable polymers provide a market for glycerol that is generated during biodiesel production. The polyesters were prepared...

  10. Substitution of a single amino acid in the 2b protein of Pea early-browning virus affects nematode transmission.

    PubMed

    Vellios, Evangelos; Brown, Derek J F; MacFarlane, Stuart A

    2002-07-01

    The 2b protein of Pea early-browning virus (PEBV) is required for transmission of the virus by nematodes. Comparison of the 2b proteins of highly transmissible (TpA56) and poorly transmissible (SP5) isolates of PEBV identified two amino acid substitutions (G90S and G177R) that might be responsible for the poor transmission of isolate SP5. Hybrid viruses were created in which the TpA56 2b protein carried SP5-specific substitutions at residue 90 or 177, and in which the SP5 2b protein carried TpA56-specific substitutions at these positions. Transmission tests showed that the G177R substitution is sufficient to prevent nematode transmission of the virus. Examination of the 2b proteins from PEBV and other tobraviruses predicted the presence of a coiled-coil domain in the central region of the protein. This structural element is important for the association of interacting proteins and, thus, might mediate interaction of the 2b protein with the virus coat protein or with the vector nematode. PMID:12075098

  11. Synthesis and structure--activity relationships of substituted cinnamic acids and amide analogues: a new class of herbicides.

    PubMed

    Vishnoi, Shipra; Agrawal, Vikash; Kasana, Virendra K

    2009-04-22

    In the present investigation, substituted cinnamic acids (3-hydroxy, 4-hydroxy, 2-nitro, 3-nitro, 4-nitro, 3-chloro, and 4-methoxy) and their amide analogues with four different types of substituted anilines have been synthesized. The synthesized compounds have been screened for their germination inhibition activity on radish (Raphanus sativus L. var. Japanese White) seeds at 50, 100, and 200 ppm concentrations, and the activity was compared with standard herbicide, metribuzin formulation (sencor). Significant activity was exhibited by all of the compounds. It was observed that with the increase in concentration of the test solution, the activity also increased. All of the compounds showed more than 70% inhibition at 100 ppm concentration except 4-hydroxy cinnamanilide. The compound, 2-chloro (4'-hydroxy) cinnamanilide was the best among the tested compounds, and it was found to be at par with the standard, metribuzin at all concentrations. Thus, it can be concluded that substituted cinnamic acids and their amide analogues may be developed as potential herbicides. PMID:19368353

  12. Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes.

    PubMed

    Yuan, Yuan; Wang, Zhouyong; Jiang, Chao; Wang, Xumin; Huang, Luqi

    2014-01-25

    Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species. PMID:23085319

  13. Effect of chemical substitutions on photo-switching properties of 3-hydroxy-picolinic acid studied by ab initio methods

    NASA Astrophysics Data System (ADS)

    Rode, Michał F.; Sobolewski, Andrzej L.

    2014-02-01

    Effect of chemical substitutions to the molecular structure of 3-hydroxy-picolinic acid on photo-switching properties of the system operating on excited-state intramolecular double proton transfer (d-ESIPT) process [M. F. Rode and A. L. Sobolewski, Chem. Phys. 409, 41 (2012)] was studied with the aid of electronic structure theory methods. It was shown that simultaneous application of electron-donating and electron-withdrawing substitutions at certain positions of the molecular frame increases the height of the S0-state tautomerization barrier (ensuring thermal stability of isomers) and facilitates a barrierless access to the S1/S0 conical intersection from the Franck-Condon region of the S1 potential-energy surface. Results of study point to the conclusion that the most challenging issue for practical design of a fast molecular photoswitch based on d-ESIPT phenomenon are to ensure a selectivity of optical excitation of a given tautomeric form of the system.

  14. Nucleophilic Aromatic Substitution.

    ERIC Educational Resources Information Center

    Avila, Walter B.; And Others

    1990-01-01

    Described is a microscale organic chemistry experiment which demonstrates one feasible route in preparing ortho-substituted benzoic acids and provides an example of nucleophilic aromatic substitution chemistry. Experimental procedures and instructor notes for this activity are provided. (CW)

  15. The green-absorbing Drosophila Rh6 visual pigment contains a blue-shifting amino acid substitution that is conserved in vertebrates.

    PubMed

    Salcedo, Ernesto; Farrell, David M; Zheng, Lijun; Phistry, Meridee; Bagg, Eve E; Britt, Steven G

    2009-02-27

    The molecular mechanisms that regulate invertebrate visual pigment absorption are poorly understood. Through sequence analysis and functional investigation of vertebrate visual pigments, numerous amino acid substitutions important for this adaptive process have been identified. Here we describe a serine/alanine (S/A) substitution in long wavelength-absorbing Drosophila visual pigments that occurs at a site corresponding to Ala-292 in bovine rhodopsin. This S/A substitution accounts for a 10-17-nm absorption shift in visual pigments of this class. Additionally, we demonstrate that substitution of a cysteine at the same site, as occurs in the blue-absorbing Rh5 pigment, accounts for a 4-nm shift. Substitutions at this site are the first spectrally significant amino acid changes to be identified for invertebrate pigments sensitive to visible light and are the first evidence of a conserved tuning mechanism in vertebrate and invertebrate pigments of this class. PMID:19126545

  16. Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats, and nucleotide substitution rates.

    PubMed

    Weng, Mao-Lun; Blazier, John C; Govindu, Madhumita; Jansen, Robert K

    2014-03-01

    Geraniaceae plastid genomes are highly rearranged, and each of the four genera already sequenced in the family has a distinct genome organization. This study reports plastid genome sequences of six additional species, Francoa sonchifolia, Melianthus villosus, and Viviania marifolia from Geraniales, and Pelargonium alternans, California macrophylla, and Hypseocharis bilobata from Geraniaceae. These genome sequences, combined with previously published species, provide sufficient taxon sampling to reconstruct the ancestral plastid genome organization of Geraniaceae and the rearrangements unique to each genus. The ancestral plastid genome of Geraniaceae has a 4 kb inversion and a reduced, Pelargonium-like small single copy region. Our ancestral genome reconstruction suggests that a few minor rearrangements occurred in the stem branch of Geraniaceae followed by independent rearrangements in each genus. The genomic comparison demonstrates that a series of inverted repeat boundary shifts and inversions played a major role in shaping genome organization in the family. The distribution of repeats is strongly associated with breakpoints in the rearranged genomes, and the proportion and the number of large repeats (>20 bp and >60 bp) are significantly correlated with the degree of genome rearrangements. Increases in the degree of plastid genome rearrangements are correlated with the acceleration in nonsynonymous substitution rates (dN) but not with synonymous substitution rates (dS). Possible mechanisms that might contribute to this correlation, including DNA repair system and selection, are discussed. PMID:24336877

  17. The complete mitochondrial genome of the black surfperch, Embiotoca jacksoni: Selection and substitution rates among surfperches (Embiotocidae).

    PubMed

    Longo, Gary C; O'Connell, Brendan; Green, Richard E; Bernardi, Giacomo

    2016-08-01

    The complete 16,515bp nucleotide sequence of the mitochondrial genome was determined for the black surfperch, Embiotoca jacksoni (Perciformes: Embiotocidae). The black surfperch mitochondrial genome contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and the non-coding control region (D-loop), the gene order of which is identical to that observed in most vertebrates. The protein-coding gene sequences of E. jacksoni mitochondrial DNA were compared with two other embiotocid surfperches with available complete mitochondrial genomes, Cymatogaster aggregata and Ditrema temminckii. Across all mitochondrial protein-coding genes in surfperches the weighted average substitution rate was 2.079% per My and average dN/dS ratios for each protein-coding gene ranged from 0.016 in CytB to 0.608 in ND3. Substitution rates and dN/dS ratios were relatively high for ATP8 compared to other protein-coding genes. Although most protein-coding genes showed signals of purifying selection, we found evidence for positive selection in ND3 in E. jacksoni. PMID:26994927

  18. Genotype–phenotype associations: substitution models to detect evolutionary associations between phenotypic variables and genotypic evolutionary rate

    PubMed Central

    O'Connor, Timothy D.; Mundy, Nicholas I.

    2009-01-01

    Motivation: Mapping between genotype and phenotype is one of the primary goals of evolutionary genetics but one that has received little attention at the interspecies level. Recent developments in phylogenetics and statistical modelling have typically been used to examine molecular and phenotypic evolution separately. We have used this background to develop phylogenetic substitution models to test for associations between evolutionary rate of genotype and phenotype. We do this by creating hybrid rate matrices between genotype and phenotype. Results: Simulation results show our models to be accurate in detecting genotype–phenotype associations and robust for various factors that typically affect maximum likelihood methods, such as number of taxa, level of relevant signal, proportion of sites affected and length of evolutionary divergence. Further, simulations show that our method is robust to homogeneity assumptions. We apply the models to datasets of male reproductive system genes in relation to mating systems of primates. We show that evolution of semenogelin II is significantly associated with mating systems whereas two negative control genes (cytochrome b and peptidase inhibitor 3) show no significant association. This provides the first hybrid substitution model of which we are aware to directly test the association between genotype and phenotype using a phylogenetic framework. Availability: Perl and HYPHY scripts are available upon request from the authors. Contact: to252@cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19478022

  19. 40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Agency, at 40 CFR 747.195, as published in the Federal Register of June 14, 1984. A copy of the... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted... leads to formation of a substance known to cause cancer. This product is designed to be used...

  20. 40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Agency, at 40 CFR 747.195, as published in the Federal Register of June 14, 1984. A copy of the... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted... leads to formation of a substance known to cause cancer. This product is designed to be used...

  1. 40 CFR 747.195 - Triethanolamine salt of a substituted organic acid.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Agency, at 40 CFR 747.195, as published in the Federal Register of June 14, 1984. A copy of the... of a substance known to cause cancer in laboratory animals. The triethanolamine salt of a substituted... leads to formation of a substance known to cause cancer. This product is designed to be used...

  2. In vitro and in vivo antitumor effects of novel actinomycin D analogs with amino acid substituted in the cyclic depsipeptides.

    PubMed

    Zhang, Bang-zhi; Wang, Kai-rong; Yan, Jie-xi; Zhang, Wei; Song, Jing-jing; Ni, Jing-man; Wang, Rui

    2010-04-01

    The actinomycin D (AMD) analogs in which the D-valine residues (the second amino acid residue in the cyclic depsipeptide of AMD) and the N-methyl-L-valine residues (the fifth amino acid residue in the cyclic depsipeptide of AMD) were replaced with D-Phe or l- and D-forms N-methylvalines, N-methylisoleucine, N-methylleucine, N-methylphenylalanine, N-methylalanine, and sarcosine were synthesized. The antimicrobial activity and cytotoxic activities of these compounds in vitro were investigated. The results showed that most D-valine substituted analogs had much lower antimicrobial activity and cytotoxic activities in vitro than AMD itself, but three N-methyl-L-valine substituted analogs had comparable or even more remarkable cytotoxic activities in vitro than AMD. Acute toxicities and antitumor effects of the N-methyl-L-valine substituted analogs in mice were also examined. The result showed that the acute toxicity of compound 4 L-methylleucine(5)-AMD analog is comparable to AMD itself and that of compound 3(L-Methylisoleucine(5)-AMD analog) is slightly more toxic, about 1.25-fold than AMD. However, the acute toxicity of compound 5 D-methylleucine5-AMD analog is about 2-fold lower than AMD. This suggested that the N-methyl-D-amino acid replacement in the cyclic ring might play a vital role in their decreased acute toxicities, and perhaps the N-methyl-D-leucine substituent is more favorable, though there may be a slight loss of antitumor activity. This finding may be helpful for the design and development of more potent antitumor agents together with low acute toxicity, and suggests that the N-methyl-D-leucine substituent has the potential to be used as antitumor drug lead. PMID:20045716

  3. Amino acid substitutions in the thymidine kinase gene of induced acyclovir-resistant herpes simplex virus type 1

    NASA Astrophysics Data System (ADS)

    Hussin, Ainulkhir; Nor, Norefrina Shafinaz Md; Ibrahim, Nazlina

    2013-11-01

    Acyclovir (ACV) is an antiviral drug of choice in healthcare setting to treat infections caused by herpes viruses, including, but not limited to genital herpes, cold sores, shingles and chicken pox. Acyclovir resistance has emerged significantly due to extensive use and misuse of this antiviral in human, especially in immunocompromised patients. However, it remains unclear about the amino acid substitutions in thymidine (TK) gene, which specifically confer the resistance-associated mutation in herpes simplex virus. Hence, acyclovir-resistant HSV-1 was selected at high concentration (2.0 - 4.5 μg/mL), and the TK-gene was subjected to sequencing and genotypic characterization. Genotypic sequences comparison was done using HSV-1 17 (GenBank Accesion no. X14112) for resistance-associated mutation determination whereas HSV-1 KOS, HSV-1 473/08 and HSV clinical isolates sequences were used for polymorphism-associated mutation. The result showed that amino acid substitutions at the non-conserved region (UKM-1: Gln34Lys, UKM-2: Arg32Ser & UKM-5: Arg32Cys) and ATP-binding site (UKM-3: Tyr53End & UKM-4: Ile54Leu) of the TK-gene. These discoveries play an important role to extend another dimension to the evolution of acyclovir-resistant HSV-1 and suggest that selection at high ACV concentration induced ACV-resistant HSV-1 evolution. These findings also expand the knowledge on the type of mutations among acyclovir-resistant HSV-1. In conclusion, HSV-1 showed multiple strategies to exhibit acyclovir resistance, including amino acid substitutions in the TK gene.

  4. Identification by random forest method of HLA class I amino acid substitutions associated with lower survival at day 100 in unrelated donor hematopoietic cell transplantation

    PubMed Central

    Marino, Susana R.; Lin, Shang; Maiers, Martin; Haagenson, Michael; Spellman, Stephen; Klein, John P.; Binkowski, T. Andrew; Lee, Stephanie J.; van Besien, Koen

    2011-01-01

    The identification of important amino acid substitutions associated with low survival in hematopoietic cell transplantation (HCT) is hampered by the large number of observed substitutions compared to the small number of patients available for analysis. Random forest analysis is designed to address these limitations. We studied 2,107 HCT recipients with good or intermediate risk hematologic malignancies to identify HLA class I amino acid substitutions associated with reduced survival at day 100 post-transplant. Random forest analysis and traditional univariate and multivariate analyses were used. Random forest analysis identified amino acid substitutions in 33 positions that were associated with reduced 100 day survival, including HLA-A 9, 43, 62, 63, 76, 77, 95, 97, 114, 116, 152, 156, 166, and 167; HLA-B 97, 109, 116, and 156; and HLA-C 6, 9, 11, 14, 21, 66, 77, 80, 95, 97, 99, 116, 156, 163, and 173. Thirteen had been previously reported by other investigators using classical biostatistical approaches. Using the same dataset, traditional multivariate logistic regression identified only 5 amino acid substitutions associated with lower day 100 survival. Random forest analysis is a novel statistical methodology for analysis of HLA-mismatching and outcome studies, capable of identifying important amino acid substitutions missed by other methods. PMID:21441965

  5. Biphenyl-4-yl-acrylohydroxamic acids: Identification of a novel indolyl-substituted HDAC inhibitor with antitumor activity.

    PubMed

    Cincinelli, Raffaella; Zwick, Vincent; Musso, Loana; Zuco, Valentina; De Cesare, Michelandrea; Zunino, Franco; Simoes-Pires, Claudia; Nurisso, Alessandra; Giannini, Giuseppe; Cuendet, Muriel; Dallavalle, Sabrina

    2016-04-13

    Modification of the cap group of biphenylacrylohydroxamic acid-based HDAC inhibitors led to the identification of a new derivative (3) characterized by an indolyl-substituted 4-phenylcinnamic skeleton. Molecular docking was used to predict the optimal conformation in the class I HDACs active site. Compound 3 showed HDAC inhibitory activity and antiproliferative activity against a panel of tumor cell lines, in the low μM range. The compound was further tested in vitro for acetylation of histone H4 and other non-histone proteins, and in vivo in a colon carcinoma model, showing significant proapoptotic and antitumor activities. PMID:26890116

  6. Highly Amino Acid Selective Hydrolysis of Myoglobin at Aspartate Residues as Promoted by Zirconium(IV)-Substituted Polyoxometalates.

    PubMed

    Ly, Hong Giang T; Absillis, Gregory; Janssens, Rik; Proost, Paul; Parac-Vogt, Tatjana N

    2015-06-15

    SDS-PAGE/Edman degradation and HPLC MS/MS showed that zirconium(IV)-substituted Lindqvist-, Keggin-, and Wells-Dawson-type polyoxometalates (POMs) selectively hydrolyze the protein myoglobin at Asp-X peptide bonds under mildly acidic and neutral conditions. This transformation is the first example of highly sequence selective protein hydrolysis by POMs, a novel class of protein-hydrolyzing agents. The selectivity is directed by Asp residues located on the surface of the protein and is further assisted by electrostatic interactions between the negatively charged POMs and positively charged surface patches in the vicinity of the cleavage site. PMID:25950869

  7. A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

    PubMed Central

    Martín-Acebes, Miguel A.; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses. PMID:23874963

  8. The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.

    PubMed

    Alvarez-Rueda, Nidia; Fleury, Audrey; Logé, Cédric; Pagniez, Fabrice; Robert, Estelle; Morio, Florent; Le Pape, Patrice

    2016-10-01

    Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals. PMID:27143634

  9. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    PubMed Central

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  10. 40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Naphthalenedisulfonic acid, azo... Significant New Uses for Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, azo]-hydroxy... chemical substance identified generically as naphthalenedisulfonic acid, azo]-hydroxy- , metal salt (PMN...

  11. 40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Naphthalenedisulfonic acid, azo... Significant New Uses for Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, azo]-hydroxy... chemical substance identified generically as naphthalenedisulfonic acid, azo]-hydroxy- , metal salt (PMN...

  12. 40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Naphthalenedisulfonic acid, azo... Significant New Uses for Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, azo]-hydroxy... chemical substance identified generically as naphthalenedisulfonic acid, azo]-hydroxy- , metal salt (PMN...

  13. 40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Naphthalenedisulfonic acid, azo... Significant New Uses for Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, azo]-hydroxy... chemical substance identified generically as naphthalenedisulfonic acid, azo]-hydroxy- , metal salt (PMN...

  14. 40 CFR 721.10107 - Naphthalenedisulfonic acid, [amino-hydroxy-[(substituted)azo-sulfo-naphthaleneyl]azo]-hydroxy...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Naphthalenedisulfonic acid, azo... Significant New Uses for Specific Chemical Substances § 721.10107 Naphthalenedisulfonic acid, azo]-hydroxy... chemical substance identified generically as naphthalenedisulfonic acid, azo]-hydroxy- , metal salt (PMN...

  15. 40 CFR 721.9460 - Tall oil fatty acids, reaction products with polyamines, alkyl substituted.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9460 Tall oil fatty acids, reaction... reporting. (1) The chemical substance identified generically as tall oil fatty acids, reaction products with... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Tall oil fatty acids,...

  16. 40 CFR 721.9460 - Tall oil fatty acids, reaction products with polyamines, alkyl substituted.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9460 Tall oil fatty acids, reaction... reporting. (1) The chemical substance identified generically as tall oil fatty acids, reaction products with... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Tall oil fatty acids,...

  17. 40 CFR 721.9460 - Tall oil fatty acids, reaction products with polyamines, alkyl substituted.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9460 Tall oil fatty acids, reaction... reporting. (1) The chemical substance identified generically as tall oil fatty acids, reaction products with... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Tall oil fatty acids,...

  18. 40 CFR 721.9460 - Tall oil fatty acids, reaction products with polyamines, alkyl substituted.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9460 Tall oil fatty acids, reaction... reporting. (1) The chemical substance identified generically as tall oil fatty acids, reaction products with... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Tall oil fatty acids,...

  19. 40 CFR 721.9460 - Tall oil fatty acids, reaction products with polyamines, alkyl substituted.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.9460 Tall oil fatty acids, reaction... reporting. (1) The chemical substance identified generically as tall oil fatty acids, reaction products with... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Tall oil fatty acids,...

  20. Reduction Rates for Higher Americium Oxidation States in Nitric Acid

    SciTech Connect

    Grimes, Travis Shane; Mincher, Bruce Jay; Schmitt, Nicholas C

    2015-09-30

    The stability of hexavalent americium was measured using multiple americium concentrations and nitric acid concentrations after contact with the strong oxidant sodium bismuthate. Contrary to our hypotheses Am(VI) was not reduced faster at higher americium concentrations, and the reduction was only zero-order at short time scales. Attempts to model the reduction kinetics using zero order kinetic models showed Am(VI) reduction in nitric acid is more complex than the autoreduction processes reported by others in perchloric acid. The classical zero-order reduction of Am(VI) was found here only for short times on the order of a few hours. We did show that the rate of Am(V) production was less than the rate of Am(VI) reduction, indicating that some Am(VI) undergoes two electron-reduction to Am(IV). We also monitored the Am(VI) reduction in contact with the organic diluent dodecane. A direct comparison of these results with those in the absence of the organic diluent showed the reduction rates for Am(VI) were not statistically different for both systems. Additional americium oxidations conducted in the presence of Ce(IV)/Ce(III) ions showed that Am(VI) is reduced without the typical growth of Am(V) observed in the systems sans Ce ion. This was an interesting result which suggests a potential new reduction/oxidation pathway for Am in the presence of Ce; however, these results were very preliminary, and will require additional experiments to understand the mechanism by which this occurs. Overall, these studies have shown that hexavalent americium is fundamentally stable enough in nitric acid to run a separations process. However, the complicated nature of the reduction pathways based on the system components is far from being rigorously understood.

  1. Calcite growth-rate inhibition by fulvic acid and magnesium ion—Possible influence on biogenic calcite formation

    USGS Publications Warehouse

    Reddy, Michael M.

    2012-01-01

    Increases in ocean surface water dissolved carbon dioxide (CO2) concentrations retard biocalcification by reducing calcite supersaturation (Ωc). Reduced calcification rates may influence growth-rate dependent magnesium ion (Mg) incorporation into biogenic calcite modifying the use of calcifying organisms as paleoclimate proxies. Fulvic acid (FA) at biocalcification sites may further reduce calcification rates. Calcite growth-rate inhibition by FA and Mg, two common constituents of seawater and soil water involved in the formation of biogenic calcite, was measured separately and in combination under identical, highly reproducible experimental conditions. Calcite growth rates (pH=8.5 and Ωc=4.5) are reduced by FA (0.5 mg/L) to 47% and by Mg (10−4 M) to 38%, compared to control experiments containing no added growth-rate inhibitor. Humic acid (HA) is twice as effective a calcite growth-rate inhibitor as FA. Calcite growth rate in the presence of both FA (0.5 mg/L) and Mg (10−4 M) is reduced to 5% of the control rate. Mg inhibits calcite growth rates by substitution for calcium ion at the growth site. In contrast, FA inhibits calcite growth rates by binding multiple carboxylate groups on the calcite surface. FA and Mg together have an increased affinity for the calcite growth sites reducing calcite growth rates.

  2. Synthesis of a Stable Primary-Alkyl-Substituted Selenenyl Iodide and Its Hydrolytic Conversion to the Corresponding Selenenic Acid.

    PubMed

    Sase, Shohei; Kakimoto, Ryo; Kimura, Ryutaro; Goto, Kei

    2015-01-01

    A primary-alkyl-substituted selenenyl iodide was successfully synthesized through oxidative iodination of a selenol with N-iodosuccinimide by taking advantage of a cavity-shaped steric protection group. The selenenyl iodide exhibited high thermal stability and remained unchanged upon heating at 100 °C for 3 h in [D₈]toluene. The selenenyl iodide was reduced to the corresponding selenol by treatment with dithiothreitol. Hydrolysis of the selenenyl iodide under alkaline conditions afforded the corresponding selenenic acid almost quantitatively, corroborating the chemical validity of the recent proposal that hydrolysis of a selenenyl iodide to a selenenic acid is potentially involved in the catalytic mechanism of an iodothyronine deiodinase. PMID:26633336

  3. SPECTROSCOPIC AND COMPUTATIONAL INVESTIGATION OF THE H155A VARIANT OF CYSTEINE DIOXYGENASE: GEOMETRIC AND ELECTRONIC CONSEQUENCES OF A THIRD-SPHERE AMINO ACID SUBSTITUTION

    PubMed Central

    Blaesi, Elizabeth J.; Fox, Brian G.; Brunold, Thomas C.

    2015-01-01

    Cysteine dioxygenase (CDO) is a mononuclear, non-heme iron(II)-dependent enzyme that utilizes molecular oxygen to catalyze the oxidation of L-cysteine (Cys) to cysteine sulfinic acid. X-ray crystal structures of CDO have revealed that unusual structural motifs are present in both the first and the second coordination spheres of the Fe center, including a three-histidine metal binding site and a cysteine-tyrosine crosslink that is formed post-translationally. Although the kinetic consequences of various outer-sphere amino acid substitutions have previously been assessed, the effects of these substitutions on the geometric and electronic structures of the active site remained largely unexplored. In this work, we have performed a spectroscopic and computational characterization of the H155A CDO variant, which was previously shown to display a ~100-fold decreased rate of Cys oxidation relative to wild-type (WT) CDO. Magnetic circular dichroism and electron paramagnetic resonance spectroscopic data indicate that the His155→Ala substitution has a significant effect on the electronic structure of the Cys-bound Fe(II)CDO active site. An analysis of these data within the framework of quantum mechanics/molecular mechanics (QM/MM) and single-point density functional theory calculations reveals that Cys-bound H155A Fe(II)CDO possesses a six-coordinate Fe(II) center, differing from the analogous WT CDO species by the presence of an additional water ligand. The enhanced affinity of the Cys-bound Fe(II) center for a sixth ligand in the H155A CDO variant likely stems from the increased conformational freedom of the cysteine-tyrosine crosslink in the absence of the H155 imidazole ring. Despite these differences between the WT and H155A Cys-Fe(II)CDO species, the nitrosyl adducts of Cys- and Sec-bound Fe(II)CDO [which mimic the (O2/Cys)-CDO intermediate] are essentially unaffected by the H155A substitution, suggesting that the primary role played by the H155 side chain in CDO

  4. Antibacterial and anticancer activity of a series of novel peptides incorporating cyclic tetra-substituted C(α) amino acids.

    PubMed

    Hicks, Rickey P

    2016-09-15

    Eleven antimicrobial peptides (AMP) based on the incorporation of cyclic tetra substituted C(α) amino acids, as well as other unnatural amino acids were designed, synthesized and screened for in vitro activity against 18 strains of bacteria as well as 12 cancer cell lines. The AMPs discussed herein are derived from the following peptide sequence: Ac-GF(X)G(X)B(X)G(X)F(X)G(X)GB(X)BBBB-amide, X=any one of the following residues, A5c, A6c, Tic or Oic and B=any one of the following residues, Arg, Lys, Orn, Dpr or Dab. A diversity of in vitro inhibitory activity was observed for these AMPs. Several analogs exhibited single digit μM activity against drug resistant bacteria including; multiple drug resistant Mycobacterium tuberculosis, extremely drug resistant Mycobacterium tuberculosis and MRSA. The physicochemical properties of the basic amino acid residues incorporated into these AMPs seem to play a major role in defining antibacterial activity. Overall hydrophobicity seems to play a limited role in defining antibacterial activity. The ESKAPE pathogens were used to compare the activity of these AMPs to another family of synthetic AMPs incorporating the unnatural amino acids Tic and Oic. In most cases similarly substituted members of both families exhibited similar inhibitory activity against the ESKAPE pathogens. In specific cases differences in activity as high as 15 fold were observed between analogs. In addition four of these AMPs exhibited promising IC50 (<7.5μM) values against 12 different and diverse cancer cell lines. Five other AMPs exhibited promising IC50 (<7.5μM) values against selected cancer cell lines. PMID:27387357

  5. Design, synthesis and SAR studies of GABA uptake inhibitors derived from 2-substituted pyrrolidine-2-yl-acetic acids.

    PubMed

    Steffan, Tobias; Renukappa-Gutke, Thejavathi; Höfner, Georg; Wanner, Klaus T

    2015-03-15

    In this paper, we disclose the design and synthesis of a series of 2-substituted pyrrolidine-2-yl-acetic acid as core structures and the N-arylalkyl derivatives thereof as potential GABA transport inhibitors. The 2-position in the side chain of pyrrolidine-2-yl-acetic acid derivatives was substituted with alkyl, hydroxy and amino groups to modulate the activity and selectivity to mGAT1 and mGAT4 proteins. SAR studies of the compounds performed for the four mouse GABA transporter proteins (mGAT1-mGAT4) implied significant potencies and subtype selectivities for 2-hydroxy-2-pyrrolidine-2-yl-acetic acid derivatives. The racemate rac-(u)-13c exhibited the highest potency (pIC50 5.67) at and selectivity for mGAT1 in GABA uptake assays. In fact, the potency of rac-(u)-13c at hGAT-1 (pIC50 6.14) was even higher than its potency at mGAT1. These uptake results for rac-(u)-13c are in line with the binding affinities to the aforesaid proteins mGAT1 (pKi 6.99) and hGAT-1 (pKi 7.18) determined by MS Binding Assay based on NO711 as marker quantified by LC-ESI-MS-MS analysis. Interestingly, the 2-hydroxy-2-pyrrolidine-2-yl-acetic acid rac-(u)-13d containing 2-{[tris(4-methoxyphenyl)]methoxy} ethyl group at the nitrogen atom of the pyrrolidine ring showed high potency at mGAT4 and a comparatively better selectivity for this protein (>15 against mGAT3) than the well known mGAT4 uptake inhibitor (S)-SNAP-5114. PMID:25698617

  6. Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus.

    PubMed

    Schrauwen, Eefje J A; Richard, Mathilde; Burke, David F; Rimmelzwaan, Guus F; Herfst, Sander; Fouchier, Ron A M

    2016-01-01

    Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission. PMID:26792744

  7. Influence of the substitution of a grass cover by a mulch on infiltration rate

    NASA Astrophysics Data System (ADS)

    Sastre-Merlín, A.; Martínez-Pérez, S.; Bienes-Allas, R.; Molina-Navarro, E.; Martínez de Baroja, L.

    2012-04-01

    The study was carried out in an urban park of Madrid, in which it was decided to remove part of the prairie, replacing it by a mulch (pine bark). One year after this change in soil cover, infiltration tests were performed using the double ring infiltrometer (Müntz method). In each treatment the number of repetitions was 3. In the infiltration tests carried out the mulch not was withdraw, since we want to study their behavior before a rain or overhead irrigation. After one year, the infiltration rate showed ??much higher values in the prairie (18.9 mm h-1) than in the pine bark (8.4 mm h-1). Removing the prairie has meant a reduction in permeability of about 55%, which demonstrates the important role exerted by the radicular systems on infiltration. The origin is in the ability of roots to create preferred pathways circulation of the water. These pathways are of various types, and perhaps the most important are the root tubes, which are the channels that occur in the soil once the roots decompose. The finer roots create these pathways faster. These root tubes end up crumbling over time, so that is necessary to maintain the constant creation of new pipes in the soil. Under a prairie the number of root tubes that forms annually is enormous. By contrast, in absence of roots, in the surface horizon begins a process of gradual compaction, with reducing of the macroporosity and consequently impacting on the infiltration rate. The first consequence of this reduction in the infiltration rate is a poor flushing of salts from the soil of reclaimed water used in irrigation. This assertion has been corroborated by the analysis of the soil saturated paste, which shows an increasing of the electrical conductivities under the mulch. E.C. (μS cm-1) at the beginning of 2011 irrigation season (March) at different depths. Efficiency of the rains of autumn-winter by to wash soil salts. Depth (cm) PrairiePine bark 20 340 320 35 310 480 60 340 550 Therefore, the results indicate that

  8. A More Challenging Interpretative Nitration Experiment Employing Substituted Benzoic Acids and Acetanilides

    ERIC Educational Resources Information Center

    Treadwell, Edward M.; Lin, Tung-Yin

    2008-01-01

    An experiment is described involving the nitration of ortho or meta monosubstituted benzoic acids (XC[subscript 6]H[subscript 4]CO[subscript 2]H, X = Halogen, Me, OH, or OMe) and monochlorinated acetanilides with nitric acid to determine the regioselectivity of addition by [superscript 1]H NMR spectroscopy and molecular modeling. Students were…

  9. 40 CFR 721.10045 - Diazotized substituted heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (generic). 721.10045... derivative, nickel complex, alkaline salt (generic). (a) Chemical substance and significant new uses subject... heteromonocycle coupled with naphthalene sulfonic acid derivative, nickel complex, alkaline salt (PMN P-02-737)...

  10. The evolution of the RNase P- and RNase MRP-associated RNAs: phylogenetic analysis and nucleotide substitution rate.

    PubMed

    Sbisà, E; Pesole, G; Tullo, A; Saccone, C

    1996-07-01

    We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments. PMID:8660429

  11. A Single Amino Acid Substitution in 1918 Influenza Virus Hemagglutinin Changes Receptor Binding Specificity

    PubMed Central

    Glaser, Laurel; Stevens, James; Zamarin, Dmitriy; Wilson, Ian A.; García-Sastre, Adolfo; Tumpey, Terrence M.; Basler, Christopher F.; Taubenberger, Jeffery K.; Palese, Peter

    2005-01-01

    The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the α2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the α2,6 and the α2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor. PMID:16103207

  12. A role for residue 151 of LamB in bacteriophage lambda adsorption: possible steric effect of amino acid substitutions.

    PubMed

    Charbit, A; Werts, C; Michel, V; Klebba, P E; Quillardet, P; Hofnung, M

    1994-06-01

    LamB is the cell surface receptor for bacteriophage lambda. LamB missense mutations yielding resistance to lambda have been previously grouped in two classes. Class I mutants block growth of lambda with wild-type host range (lambda h+) but support growth of one-step extended-host-range mutants (lambda h). Class II mutants block lambda h but support growth of two-step extended host range mutants (lambda hh*). While Class I mutations occur at 11 different amino acid sites, in five distinct portions of LamB, all the Class II mutations analyzed previously correspond to the same G-to-D change at amino acid 151. We generated by in vitro mutagenesis four different new substitutions at site 151 (to S, V, R, and C). Two of the mutants (G-151-->V [G151V] and G151R) were of Class II, while the two others (G151S and G151C) were of Class I, demonstrating that not only the site but also the nature of the substitutions at residue 151 was critical for the phage sensitivity phenotypes. The introduction of a negatively charged, a positively charged, or an aliphatic nonpolar residue at site 151 of LamB prevented both lambda h+ and lambda h adsorption, indicating that the block is not due to a charge effect. In contrast to G151D, which was sensitive to all the lambda hh* phages, G151V and G151R conferred sensitivity to only four of the five lambda hh* phages. Thus, G151V and G151R represent a new subclass of Class II LamB mutations that is more restrictive with respect to the growth of lambda hh*. Our results agree with the hypothesis that residue 151 belongs to an accessibility gate controlling the access to the phage tight-binding site and that substitutions at this residue affect the access of the phage to the binding site in relation to the size of the substitute side chain (surface area): the most restrictive changes are G151V and G151R, followed to a lesser extent by G151D and they by G151S and G151C. PMID:8195074

  13. Susceptibilities of human immunodeficiency virus type 1 enzyme and viral variants expressing multiple resistance-engendering amino acid substitutions to reserve transcriptase inhibitors.

    PubMed Central

    Byrnes, V W; Emini, E A; Schleif, W A; Condra, J H; Schneider, C L; Long, W J; Wolfgang, J A; Graham, D J; Gotlib, L; Schlabach, A J

    1994-01-01

    To evaluate the potential that multiply resistant human immunodeficiency virus type 1 variants may arise during combination nucleoside and nonnucleoside reverse transcriptase inhibitor therapy, we constructed a series of mutant reverse transcriptase enzymes and viruses that coexpressed various combinations of resistance-associated amino acid substitutions. Substitutions at residues 100 (Leu-->Ile) and 181 (Tyr-->Cys), which mediate resistance to the nonnucleosides, suppressed resistance to 3'-azido-3'-deoxythymidine (AZT) when coexpressed with AZT-specific substitutions. However, a number of viral variants that exhibited significantly reduced susceptibilities to both classes of inhibitors were constructed. PMID:7522428

  14. Effect of single amino acid substitution on oxidative modifications of the Parkinson's disease-related protein, DJ-1.

    PubMed

    Madian, Ashraf G; Hindupur, Jagadish; Hulleman, John D; Diaz-Maldonado, Naomi; Mishra, Vartika R; Guigard, Emmanuel; Kay, Cyril M; Rochet, Jean-Christophe; Regnier, Fred E

    2012-02-01

    Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD. PMID:22104028

  15. Acid-Triggered Release via dePEGylation of Fusogenic Liposomes Mediated by Heterobifunctional Phenyl Substituted Vinyl Ethers with Tunable pH-Sensitivity

    PubMed Central

    Kim, Hee-Kwon; Van den Bossche, Jeroen; Hyun, Seok-Hee; Thompson, David H

    2012-01-01

    A new family of heterobifunctional phenyl-substituted vinyl ether (PIVE) coupling agents with tunable acid-sensitivity has been developed. The PIVE compounds are designed to hydrolyze under acidic conditions with hydrolysis rates that can be varied by rational selection of the phenyl ring substituent. These reagents were incorporated within 2-methoxypoly(ethylene glycol) PEG-conjugated 1,3-dioctadecyl-rac-glycerol lipids to produce the acid-cleavable lipopolymers mPEG-[H-PIVE]-DOG, mPEG-[F-PIVE]-DOG, mPEG-[Me-PIVE]-DOG, and mPEG-[MeO-PIVE]-DOG. These lipopolymers were hydrolyzed under acidic conditions (pH 3.5 or 4.5) at rates that were dependent on the electron donating or withdrawing character of the α-phenyl vinyl ether substituent, while remaining stable at pH 7.4. Blending of these compounds with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 10:90 mPEG-PIVE-Lipid:DOPE ratio produced stable liposomes at neutral pH; however, acidification of the solution led to dePEGylation and release of the liposomal cargo in a manner that correlated with the PIVE proton affinity. Specifically, we observed 70 % calcein release within 12 h from mPEG-[MeO-PIVE]-DOG-containing liposomes at pH 4.5, whereas only 22% calcein release was observed from mPEG-[F-PIVE]-DOG:DOPE liposomes over this same timescale and pH. These results indicate that dePEGylation following acidification is a triggering mechanism that can be rationally designed and controlled through the appropriate selection of PIVE moieties. PMID:22988941

  16. Self-assembled nanoparticles based on chondroitin sulfate-deoxycholic acid conjugates for docetaxel delivery: Effect of degree of substitution of deoxycholic acid.

    PubMed

    Liu, Mengrui; Du, Hongliang; Zhai, Guangxi

    2016-10-01

    Hydrophobically-modified polymers based on chondroitin sulfate with different degree of substitution (DS) of deoxycholic acid (DOCA) were developed for docetaxel delivery. Chondroitin sulfate-deoxycholic acid (CSAD) bioconjugates were synthesized via the linker of adipic dihydrazide by amide bond. They were characterized with spherical shape, mean diameter of around 165.2nm and negative zeta potential (-14.87 to -20.53mV). An increase of DOCA DS reduced size of nanoparticles, while increasing drug loading efficiency. Drug release in vitro showed a triphasic sustained pattern and higher accumulative drug release percentage was observed with increased DS of DOCA on polymer. Self-assemblies with higher DS also had enhanced internalization of nanoparticles and stronger cytotoxicity at the cellular level. The self-assemble nanoparticles demonstrate to be excellent targeting drug delivery systems and the desired therapeutics can be achieved via the alteration of DS. PMID:27343846

  17. In Vitro characterization of low modulus linoleic acid coated strontium-substituted hydroxyapatite containing PMMA bone cement.

    PubMed

    Lam, W M; Pan, H B; Fong, M K; Cheung, W S; Wong, K L; Li, Z Y; Luk, K D K; Chan, W K; Wong, C T; Yang, C; Lu, W W

    2011-01-01

    Poly (methyl methacrylate) (PMMA) bone cement is widely used in vertebral body augmentation procedures such as vertebroplasty and balloon kyphoplasty. Filling high modulus PMMA increases the modulus of filled verterbra, increasing the risk of fracture in the adjacent vertebra. On the other hand, in porous PMMA bone cements, wear particle generation and deterioration of mechanical performance are the major drawbacks. This study adopts a new approach by utilizing linoleic acid coated strontium substituted hydroxyapatite nanoparticle (Sr-5 HA) and linoleic acid as plasticizer reducing bone cement's modulus with minimal impact on its strength. We determined the compressive strength (UCS) and modulus (Ec), hydrophobicity, injectability, in vitro bioactivity and biocompatibility of this bone cement at different filler and linoleic acid loading. At 20 wt % Sr5-HA incorporation, UCS and Ec were reduced from 63 ± 2 MPa, 2142 ± 129 MPa to 58 ± 2 MPa, 1785 ± 64 MPa, respectively. UCS and Ec were further reduced to 49 ± 2 MPa and 774 ± 70 MPa respectively when 15 v/v of linoleic acid was incorporated. After 7 days of incubation, pre-osteoblast cells (MC3T3-E1) attached on 20 wt % Sr5-HA and 20 wt % Sr5-HA with 15 v/v of linoleic acid group were higher (3.73 ± 0.01 x 10⁴, 2.27 ± 0.02 x 10⁴) than their PMMA counterpart (1.83 ± 0.04 x 10⁴). Incorporation of Sr5-HA with linoleic acid in monomer phase is more effective in reducing the bone cement's stiffness than Sr5-HA alone. Combination of low stiffness and high mechanical strength gives the novel bone cement the potential for use in vertebroplasty cement applications. PMID:21053263

  18. Stability constants of europium complexes with a nitrogen heterocycle substituted methane-1,1-diphosphonic acid

    SciTech Connect

    Jensen, M.P.; Rickert, P.G.; Schmidt, M.A.; Nash, K.L.

    1996-06-01

    Even in moderately acidic solutions ([H{sup +}] > 0.01 M), N-piperidinomethane-1,1-diphosphonic acid (H{sub 4}PMDPA) is a strong complexant of trivalent lanthanide ions that shows enhanced complex solubility over previously studied 1,1-diphosphonic acids. The protonation constants of PMDPA in 2.0 M H/NaClO{sub 4} were determined by potentiometric and NMR titrations, and the stability constants for formation of complexes with Eu{sup 3+} were determined by solvent extraction. Difference in protonation equilibria induced by addition of the nitrogen heterocycle results in an increase in the complexation strength of PMDPA. In solutions containing 0.1 M H{sup +} and ligand concentrations greater than 0.02 M, PMDPA is the most effective 1,1-diphosphonic acid for europium complexation studied thus far.

  19. Uptake of benzoic acid and chloro-substituted benzoic acids by alcaligenes denitrificans BRI 3010 and BRI 6011

    SciTech Connect

    Miguez, C.B.; Ingram, J.M.; MacLeod, R.A.

    1995-12-01

    The mechanism of uptake of benzoic and 2,4-dichlorobenzoic acid (2,4-DCBA) by Alcaligenes denitrificans BRI 3010 and BRI 6011 and Pseudomonas sp. strain B13, three organisms capable of degrading isomers of chlorinated benzoic acids, was investigated. In all three organisms, uptake of benzoic acid was inducible. For benzoic acid uptake into BRI 3010, monophasic saturation kinetics with apparent K{sub m} and V{sub max} values of 1.4 {mu}M and 3.2 nmol/min/mg of cell dry weight, respectively, were obtained. For BRI 6011, biphasic saturation kinetics were observed, suggesting presence of two uptake systems for benzoic acid with distinct K{sub m} (0.72 and 5.3 {mu}M) and V{sub max} (3.3 and 4.6 nmol/min/mg of cell dry weight) values. BRI 3010 and BRI 6011 accumulated benzoic acid against a concentration gradient by a factor of 8 and 10, respectively. A wide range of structural analogs, at 50-fold excess concentrations, inhibited benzoic acid uptake by BRI 3010 and BRI 6011, whereas with B13, only 3-chlorobenzoic acid was an effective inhibitor. For BRI 3010 and BRI 6011, the inhibition by the structural analogs was not of a competitive nature. Uptake of benzoic acid by BRI 3010 and BRI 6011 was inhibited by KCN, by the protonophore 3,5,3`, 4`-tetrachlorosalicylanilide (TCS), and, for BRI 6011, by anaerobiosis unless nitrate was present, thus indicating that energy was required for the uptake process. Uptake of 2,4-DCBA by BRI 6011 was constitutive and saturation uptake kinetics were not observed. Uptake of 2,4-DCBA by BRI 6011 was inhibited by KCN, TCS, and anaerobiosis even if nitrate was present, but the compound was not accumulated intracellularly against a concentration gradient. Uptake of 2,4-DCBA by BRI 6011 appears to occur by passive diffusion into the cell down its concentration gradient, which is maintained by the intracellular metabolism of the compound. This process could play an important role in the degradation of xenobiotic compounds by microorganisms.

  20. Synthesis and evaluation of dual antiplatelet activity of bispidine derivatives of N-substituted pyroglutamic acids.

    PubMed

    Misra, Ankita; Anil Kumar, K S; Jain, Manish; Bajaj, Kirti; Shandilya, Shyamali; Srivastava, Smriti; Shukla, Pankaj; Barthwal, Manoj K; Dikshit, Madhu; Dikshit, Dinesh K

    2016-03-01

    N-aralkylpyroglutamides of substituted bispidine were prepared and evaluated for their ability to inhibit collagen induced platelet aggregation, both in vivo and in vitro. Some compounds showed high anti-platelet efficacy (in vitro) of which six inhibited both collagen as well as U46619 induced platelet aggregation with concentration dependent anti-platelet efficacy through dual mechanism. In particular, the compound 4j offered significant protection against collagen epinephrine induced pulmonary thromboembolism as well as ferric chloride induced arterial thrombosis, without affecting bleeding tendency in mice. Therefore, the present study suggests that the compound 4j displays a remarkable antithrombotic efficacy much better than aspirin and clopidogrel. PMID:26807542

  1. Functional impact of polar and acidic substitutions in the lactose repressor hydrophobic monomer.monomer interface with a buried lysine.

    PubMed

    Zhan, Hongli; Sun, Zhifei; Matthews, Kathleen Shive

    2009-02-17

    Despite predicted energetic penalties, the charged K84 side chains of tetrameric lactose repressor protein (LacI) are found buried within the highly hydrophobic monomer.monomer interface that includes side chains of V94 and V96. Once inducer binding has occurred, these K84 side chains move to interact with the more solvent-exposed side chains of D88 and E100'. Previous studies demonstrated that hydrophobic substitutions for K84 increased protein stability and significantly impaired the allosteric response. These results indicated that enhanced hydrophobic interactions at the monomer.monomer interface remove the energetic driving force of the buried charges, decreasing the likelihood of a robust conformational change and stabilizing the structure. We hypothesized that creating a salt bridge network with the lysine side chains by including nearby negatively charged residues might result in a similar outcome. To that end, acidic residues, D and E, and their neutral amides, N and Q, were substituted for the valines at positions 94 and 96. These variants exhibited one or more of the following functional changes: weakened inducer binding, impaired allosteric response, and diminished protein stability. For V96D and V96E, ion pair formation with K84 appears optimal, and the loss of inducer response exceeds that of the hydrophobic K84A and -L variants. However, impacts on functional properties indicate that stabilizing the buried positive charge with polar or ion pair interactions is not functionally equivalent to structural stabilization via hydrophobic enhancement. PMID:19166325

  2. A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

    PubMed

    DeJesus, E; Kieft, R; Albright, B; Stephens, N A; Hajduk, S L

    2013-01-01

    Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens. PMID:23637606

  3. A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding

    PubMed Central

    DeJesus, E.; Kieft, R.; Albright, B.; Stephens, N. A.; Hajduk, S. L.

    2013-01-01

    Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens. PMID:23637606

  4. Characterization of all possible single-nucleotide change caused amino acid substitutions in the kinase domain of Bruton tyrosine kinase.

    PubMed

    Väliaho, Jouni; Faisal, Imrul; Ortutay, Csaba; Smith, C I Edvard; Vihinen, Mauno

    2015-06-01

    Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease-causing variations among the human protein kinases, still it is just 10% of all the possible single-nucleotide substitution-caused amino acid variations (SNAVs). In the BTK kinase domain (BTK-KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease-causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty-seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms. PMID:25777788

  5. Effect of chemical substitutions on photo-switching properties of 3-hydroxy-picolinic acid studied by ab initio methods

    SciTech Connect

    Rode, Michał F. Sobolewski, Andrzej L.

    2014-02-28

    Effect of chemical substitutions to the molecular structure of 3-hydroxy-picolinic acid on photo-switching properties of the system operating on excited-state intramolecular double proton transfer (d-ESIPT) process [M. F. Rode and A. L. Sobolewski, Chem. Phys. 409, 41 (2012)] was studied with the aid of electronic structure theory methods. It was shown that simultaneous application of electron-donating and electron-withdrawing substitutions at certain positions of the molecular frame increases the height of the S{sub 0}-state tautomerization barrier (ensuring thermal stability of isomers) and facilitates a barrierless access to the S{sub 1}/S{sub 0} conical intersection from the Franck-Condon region of the S{sub 1} potential-energy surface. Results of study point to the conclusion that the most challenging issue for practical design of a fast molecular photoswitch based on d-ESIPT phenomenon are to ensure a selectivity of optical excitation of a given tautomeric form of the system.

  6. Remarkable alkaline stability of an engineered protein A as immunoglobulin affinity ligand: C domain having only one amino acid substitution

    PubMed Central

    Minakuchi, Kazunobu; Murata, Dai; Okubo, Yuji; Nakano, Yoshiyuki; Yoshida, Shinichi

    2013-01-01

    Protein A affinity chromatography is the standard purification process for the capture of therapeutic antibodies. The individual IgG-binding domains of protein A (E, D, A, B, C) have highly homologous amino acid sequences. From a previous report, it has been assumed that the C domain has superior resistance to alkaline conditions compared to the other domains. We investigated several properties of the C domain as an IgG-Fc capture ligand. Based on cleavage site analysis of a recombinant protein A using a protein sequencer, the C domain was found to be the only domain to have neither of the potential alkaline cleavage sites. Circular dichroism (CD) analysis also indicated that the C domain has good physicochemical stability. Additionally, we evaluated the amino acid substitutions at the Gly-29 position of the C domain, as the Z domain (an artificial B domain) acquired alkaline resistance through a G29A mutation. The G29A mutation proved to increase the alkaline resistance of the C domain, based on BIACORE analysis, although the improvement was significantly smaller than that observed for the B domain. Interestingly, a number of other amino acid mutations at the same position increased alkaline resistance more than did the G29A mutation. This result supports the notion that even a single mutation on the originally alkali-stable C domain would improve its alkaline stability. An engineered protein A based on this C domain is expected to show remarkable performance as an affinity ligand for immunoglobulin. PMID:23868198

  7. [Influence of the degree of substitution on the absorptivity of acidic carboxymethyl cellulose in the form of nonwoven fabric].

    PubMed

    Masteiková, Ruta; Vinklárková, Lenka; Muselík, Jan; Vetchý, David; Bernatoniene, Jurga; Sopuch, Tomáš

    2013-04-01

    Modern wound treatment is based on the creation of moist wound environment which accelerates healing. For these purposes some devices and materials may be used, including carboxymethyl cellulose (CMC). Wound dressings currently available on the market contain CMC in the form of sodium salt. CMC in the acidic form has not been used in wound healing therapy yet. Likewise, there are only a few papers describing the acidic CMC preparation and properties, which are inter alia dependent on the degree of substitution (DS). Therefore, the aim of the study was to evaluate the influence of DS on absorptivity, which is one of the main features of dressings after application on the wound. Samples with DS from 0.1 to 0.45 were examined using five media: purified water, normal saline, buffer solution with pH 7.4, physiological buffer solution with pH 7.2, and solution A. Absorptivity was evaluated using a model wound created by us. It has been found that from the viewpoint of absorptivity the optimal DS of acidic CMC in the form of nonwoven fabric lies in the range from 0.25 to 0.35. Below or above these values the absorptivity is worse. PMID:23822574

  8. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-01

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended. PMID:23796243

  9. X-ray photoelectron spectroscopy study of para-substituted benzoic acids chemisorbed to aluminum oxide thin films

    SciTech Connect

    Kreil, Justin; Ellingsworth, Edward; Szulczewski, Greg

    2013-11-15

    A series of para-substituted, halogenated (F, Cl, Br, and I) benzoic acid monolayers were prepared on the native oxide of aluminum surfaces by solution self-assembly and spin-coating techniques. The monolayers were characterized by x-ray photoelectron spectroscopy (XPS) and water contact angles. Several general trends are apparent. First, the polarity of the solvent is critical to monolayer formation. Protic polar solvents produced low coverage monolayers; in contrast, nonpolar solvents produced higher coverage monolayers. Second, solution deposition yields a higher surface coverage than spin coating. Third, the thickness of the monolayers determined from XPS suggests the plane of the aromatic ring is perpendicular to the surface with the carboxylate functional group most likely binding in a bidentate chelating geometry. Fourth, the saturation coverage (∼2.7 × 10{sup 14} molecules cm{sup −2}) is independent of the para-substituent.

  10. Divergent reactivity in palladium-catalyzed annulation with diarylamines and α,β-unsaturated acids: direct access to substituted 2-quinolinones and indoles.

    PubMed

    Kancherla, Rajesh; Naveen, Togati; Maiti, Debabrata

    2015-06-01

    A palladium-catalyzed CH activation strategy has been successfully employed for exclusive synthesis of a variety of 3-substituted indoles. A [3+3] annulation for synthesizing substituted 2-quinolinones was recently developed by reaction of α,β-unsaturated carboxylic acids with diarylamines under acidic conditions. In the present work, an analogous [3+2] annulation is achieved from the same set of starting materials under basic conditions to generate 1,3-disubstituted indoles exclusively. Mechanistic studies revealed an ortho-palladation-π-coordination-β-migratory insertion-β-hydride elimination reaction sequence to be operative under the reaction conditions. PMID:25941155

  11. Use of 8-substituted-FAD analogues to investigate the hydroxylation mechanism of the flavoprotein 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

    PubMed

    Chaiyen, Pimchai; Sucharitakul, Jeerus; Svasti, Jisnuson; Entsch, Barrie; Massey, Vincent; Ballou, David P

    2004-04-01

    2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is a flavoprotein that catalyzes the oxygenation of MHPC to form alpha-(N-acetylaminomethylene)-succinic acid. Although formally similar to the oxygenation reactions catalyzed by phenol hydroxylases, MHPCO catalyzes the oxygenation of a pyridyl derivative rather than a simple phenol. Therefore, in this study, the mechanism of the reaction was investigated by replacing the natural cofactor FAD with FAD analogues having various substituents (-Cl, -CN, -NH(2), -OCH(3)) at the C8-position of the isoalloxazine. Thermodynamic and catalytic properties of the reconstituted enzyme were investigated and found to be similar to those of the native enzyme, validating that these FAD analogues are reasonable to be used as mechanistic probes. Dissociation constants for the binding of MHPC or the substrate analogue 5-hydroxynicotinate (5HN) to the reconstituted enzymes indicate that the reconstituted enzymes bind well with ligands. Redox potential values of the reconstituted enzymes were measured and found to be more positive than the values of free FAD analogues, which correlated well with the electronic effects of the 8-substituents. Studies of the reductive half-reaction of MHPCO have shown that the rates of flavin reduction by NADH could be described as a parabolic relationship with the redox potential values of the reconstituted enzymes, which is consistent with the Marcus electron transfer theory. Studies of the oxidative half-reaction of MHPCO revealed that the rate of hydroxylation depended upon the different analogues employed. The rate constants for the hydroxylation step correlated with the calculated pK(a) values of the 8-substituted C(4a)-hydroxyflavin intermediates, which are the leaving groups in the oxygen transfer step. It was observed that the rates of hydroxylation were greater when the pK(a) values of C(4a)-hydroxyflavins were lower. Although these results are not as dramatic as those from

  12. Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

    PubMed Central

    Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

    2015-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

  13. Amino acids form strongly bound anions when substituted with superhalogen ligands

    NASA Astrophysics Data System (ADS)

    Sieradzan, Iwona; Anusiewicz, Iwona

    2013-04-01

    The properties of AA-Y- anions (where AA = cysteine, aspartic acid, lysine; Y = BF3, PF5) were investigated at the ab initio Outer Valence Green's Function (OVGF)/6-311++G(3df,3pd)//MP2/6-311++G(d,p) level of theory. It is shown that introducing a superhalogen-like substituent to an amino acid (i.e., Cys, Asp, and Lys) results in obtaining molecules that bind an excess electron relatively strongly. The electronic stabilities of such resulting daughter anions are predicted to be substantial (5.3-6.9 eV).

  14. High emission rate of sulfuric acid from Bezymianny volcano, Kamchatka

    NASA Astrophysics Data System (ADS)

    Zelenski, Michael; Taran, Yuri; Galle, Bo

    2015-09-01

    High concentrations of primary sulfuric acid (H2SO4) in fumarolic gases and high emission rate of sulfuric acid aerosol in the plume were measured at Bezymianny volcano, an active dome-growing andesitic volcano in central Kamchatka. Using direct sampling, filter pack sampling, and differential optical absorption spectroscopy measurements, we estimated an average emission of H2SO4 at 243 ± 75 t/d in addition to an average SO2 emission of 212 ± 65 t/d. The fumarolic gases of Bezymianny correspond to arc gases released by several magma bodies at different stages of degassing and contain 25-92% of entrained air. H2SO4 accounts for 6-87 mol% of the total sulfur content, 42.8 mol% on average, and SO2 is the rest. The high H2SO4 in Bezymianny fumaroles can be explained by catalytic oxidation of SO2 inside the volcanic dome. Because sulfate aerosol is impossible to measure remotely, the total sulfur content in a plume containing significant H2SO4 may be seriously underestimated.

  15. Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses.

    PubMed

    Harvey, William T; Benton, Donald J; Gregory, Victoria; Hall, James P J; Daniels, Rodney S; Bedford, Trevor; Haydon, Daniel T; Hay, Alan J; McCauley, John W; Reeve, Richard

    2016-04-01

    Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997-2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens. PMID:27057693

  16. Identification of Low- and High-Impact Hemagglutinin Amino Acid Substitutions That Drive Antigenic Drift of Influenza A(H1N1) Viruses

    PubMed Central

    Harvey, William T.; Benton, Donald J.; Gregory, Victoria; Hall, James P. J.; Daniels, Rodney S.; Bedford, Trevor; Haydon, Daniel T.; Hay, Alan J.; McCauley, John W.; Reeve, Richard

    2016-01-01

    Determining phenotype from genetic data is a fundamental challenge. Identification of emerging antigenic variants among circulating influenza viruses is critical to the vaccine virus selection process, with vaccine effectiveness maximized when constituents are antigenically similar to circulating viruses. Hemagglutination inhibition (HI) assay data are commonly used to assess influenza antigenicity. Here, sequence and 3-D structural information of hemagglutinin (HA) glycoproteins were analyzed together with corresponding HI assay data for former seasonal influenza A(H1N1) virus isolates (1997–2009) and reference viruses. The models developed identify and quantify the impact of eighteen amino acid substitutions on the antigenicity of HA, two of which were responsible for major transitions in antigenic phenotype. We used reverse genetics to demonstrate the causal effect on antigenicity for a subset of these substitutions. Information on the impact of substitutions allowed us to predict antigenic phenotypes of emerging viruses directly from HA gene sequence data and accuracy was doubled by including all substitutions causing antigenic changes over a model incorporating only the substitutions with the largest impact. The ability to quantify the phenotypic impact of specific amino acid substitutions should help refine emerging techniques that predict the evolution of virus populations from one year to the next, leading to stronger theoretical foundations for selection of candidate vaccine viruses. These techniques have great potential to be extended to other antigenically variable pathogens. PMID:27057693

  17. Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

    PubMed Central

    Franco, Paola; Pavone, Vincenzo; Mugione, Pietro; Di Carluccio, Gioconda; Masucci, Maria Teresa; Arra, Claudio; Pirozzi, Giuseppe; Stoppelli, Maria Patrizia; Carriero, Maria Vincenza

    2012-01-01

    The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPARS90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPARS90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPARS90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPARS90P. In conclusion, our findings indicate that Ser90 is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPARS90E and uPARS90P are forced into inactive and active forms, respectively

  18. Laboratory measurements of H-D substitution rates in solid methanol-dn (n=0-2) at 10 K

    NASA Astrophysics Data System (ADS)

    Nagaoka, Akihiro; Watanabe, Naoki; Kouchi, Akira

    The deuterium fractionation of interstellar methanol is investigated experimentally using the ASURA (Apparatus for SUrface Reactions in Astrophysics) system. Recent observations toward the low-mass protostars IRAS16293 found the very high D/H ratios in formaldehyde and methanol up to 0.2 and 0.4, respectively (Loinard et al. 2000; Parise et al. 2004; Aikawa et al. 2005). To date, several models have been proposed to explain D-fractionation mechanism. Pure gas-phase models are difficult to reproduce the D-fractionation, particularly, for multideuterated species, while the results of some gas-grain models can achieve the observed fractionation levels fairly well (Stantcheva & Herbst 2003). However, the gas-grain models require many assumptions regarding the grain surface reactions. Then, the experiments on the surface reaction have been highly desirable. In this context, we performed the experiments on the formation of deuterated formaldehyde and methanol on cold (10 K) interstellar grain analogues and revealed that a key route for the D-fractionation is not successive addition of H and D to CO as previously considered (e.g., Charnley, Tielens, & Rodgers 1997) but H-D substitution in solid CH3OH on icy grains (Nagaoka, Watanabe, & Kouchi 2005). We report the results of further experiments on the deuteration of CH3OH using a cold (30 K) atomic D beam. The relative rates of H-D substitution reactions; CH3OH → CH2DOH, CH2DOH → CHD2OH, CHD2OH → CD3OH, were measured. Experiments were performed using the ASURA system described previously (Watanabe et al. 2004; Nagaoka, Watanabe, & Kouchi 2005). The experimental procedure is as follows. An aluminum substrate was placed in the centre of an ultra-high vacuum chamber (10-10 Torr) and cooled to 10 K by a helium refrigerator. The solid samples of normal and deuterated methanol (CH3OH, CH2DOH, CHD2OH) were vapor-deposited on the substrate. The D atoms produced by dissociation of D2 molecules by microwave discharge were

  19. Pleiotropic effects of hemagglutinin amino acid substitutions of H5 influenza escape mutants

    SciTech Connect

    Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.; Shilov, Aleksandr A.; Krylov, Petr S.; Ilyushina, Natalia A.; Kaverin, Nikolai V.

    2013-12-15

    In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and in vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects.

  20. Single Amino Acid Substitution in the Pullulanase of Klebsiella variicola for Enhancing Thermostability and Catalytic Efficiency.

    PubMed

    Mu, Guo Cui; Nie, Yao; Mu, Xiao Qing; Xu, Yan; Xiao, Rong

    2015-07-01

    Based on conserved sites and homology modeling analysis, the residue Phe581 in the Klebsiella variicola SHN-1 pullulanase was selected as the potential thermostability-related site and its role on thermostability and activity was investigated by site-saturated mutagenesis. Compared with the wild-type pullulanase, the optimum temperature of the mutants including F581L, F581Q, F581R, F581T, F581V, and F581Y was increased from 53 to 56 °C, and correspondingly the half lives of these mutants at 55 °C were increased by 4.20, 3.70, 1.90, 7.16, 3.01, and 1.75 min, respectively. By modeling the structure of the pullulanase, formation of more hydrogen bonds by single-site substitution was supposed to be responsible for the improvement of thermostability. Of these mutants, furthermore, F581L and F581V exhibited higher values of V max and k cat/K m, compared with the wild-type enzyme. Therefore, the residue Phe581 was identified as an important site relevant to the activity and thermostability of the pullulanase of K. variicola, and by mutation at this single site, the mutated enzymes with enhanced thermostability and catalytic efficiency were achieved consequently. PMID:26018345

  1. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein.

    PubMed

    Zhang, Xinsheng; Wallace, Olivia L; Domi, Arban; Wright, Kevin J; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J; Kamali, Anatoli; Karita, Etienne; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A; Parks, Christopher L

    2015-08-01

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. PMID:25880113

  2. Pentahaloethane-based chlorofluorocarbon substitutes and halothane: Correlation of in vivo hepatic protein trifluoroacetylation and urinary trifluoroacetic acid excretion with calculated enthalpies of activation

    SciTech Connect

    Harris, J.W.; Jones, J.P.; Martin, J.L.; LaRosa, A.C.; Olson, M.J.; Pohl, L.R.; Anders, M.W. )

    1992-09-01

    The hydrochlorofluorocarbons (HCFCs) 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) and 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124) and the hydrofluorocarbon (HFC) pentafluoroethane (HFC-125) are being developed as substitutes for chlorofluorocarbons that deplete stratospheric ozone. The structural similarity of these HCFCs and HFCs to halothane, which is hepatotoxic under certain circumstances, indicates that the metabolism and cellular interactions of HCFCs and HFCs must be explored. In a previous study [Harris et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1407], similar patterns of trifluoroacetylated proteins (TFA-proteins) were detected by immunoblotting with anti-TFA-protein antibodies in livers of rats exposed to halothane or HCFC-123. The present study extends these results and demonstrates that in vivo TFA-protein formation resulting from a 6-h exposure to a 1% atmosphere of these compounds follows the trend: halothane approximately HCFC-123 much greater than HFC-124, greater than HFC-125. The calculated enthalpies of activation of halothane, HCFC-123, HCFC-124, and HFC-125 paralleled the observed rate of trifluoroacetic acid excretion in HCFC- or HFC-exposed rats. Exposure of rats to a range of HCFC-123 concentrations indicated that TFA-protein formation was saturated at an exposure concentration between 0.01% and 0.1% HCFC-123. Deuteration of HCFC-123 decreased TFA-protein formation in vivo. Urinary trifluoroacetic acid excretion by treated rats correlated with the levels of TFA-proteins found after each of these treatments.

  3. Nitrogen mineralization rates of the acidic, xeric soils of the New Jersey Pinelands: field rates

    SciTech Connect

    Poovarodom, S.; Tate, R.L. III; Bloom, R.A.

    1988-04-01

    Using the buried-bag procedure, the authors quantified nitrogen mineralization rates in the xeric, acidic Lakehurst, and Atsion sands of the New Jersey Pine Barrens. Average annual nitrogen yields in the upper 15 cm for the Lakehurst and the Atsion sands were 38.4 and 53.0 kg N/ha, corresponding to 4.5 and 2.5% of the total nitrogen, respectively. Net nitrogen mineralization in both soils exhibited distinct seasonal patterns with maxima in summer and minimum rates in the winter. Nitrification accounted for only 5% of the total N mineralized in both soils. This is consistent with the finding of low populations of autotrophic nitrifiers in these soils.

  4. Surface-active ionic liquids in micellar catalysis: impact of anion selection on reaction rates in nucleophilic substitutions.

    PubMed

    Cognigni, Alice; Gaertner, Peter; Zirbs, Ronald; Peterlik, Herwig; Prochazka, Katharina; Schröder, Christian; Bica, Katharina

    2016-05-21

    A series of surface-active ionic liquids based on the 1-dodecyl-3-methylimidazolium cation and different anions such as halides and alkylsulfates was synthesized. The aggregation behavior of these ionic liquids in water was characterized by surface tension, conductivity measurements and UV-Vis spectroscopy in order to determine the critical micelle concentration (CMC) and to provide aggregation parameters. The determination of surface activity and aggregation properties of amphiphilic ionic liquids was accompanied by SAXS studies on selected surface-active ionic liquids. The application of these surface-active ionic liquids with different anions was tested in nucleophilic substitution reactions for the degradation of organophosphorus compounds. Kinetic studies via UV-Vis spectrophotometry showed a strong acceleration of the reaction in the micellar system compared to pure water. In addition, an influence of the anion was observed, resulting in a correlation between the anion binding to the micelle and the reaction rate constants, indicating that the careful choice of the surface-active ionic liquid can considerably affect the outcome of reactions. PMID:27121134

  5. Synthesis and cytotoxicity screening of substituted isobenzofuranones designed from anacardic acids.

    PubMed

    Logrado, Lúcio P L; Santos, Camila O; Romeiro, Luiz A S; Costa, Arinice M; Ferreira, José R O; Cavalcanti, Bruno C; Manoel de Moraes, O; Costa-Lotufo, Letícia V; Pessoa, Cláudia; Dos Santos, Maria L

    2010-08-01

    This work is part of a large program, which seeks to discover new antitumor isobenfuranones designed from anacardic acids. The synthetic strategy for the construction of the title compounds takes into consideration the use of inexpensive anacardic acids (2), the major natural cashew (Anacardium occidentale) nut-shell phenolic lipid, and features one-pot construction of fused-ring aromatic gamma-lactones, phthalides. The cytotoxicity screening in different human cancer cell lines (HL-60 leukemia, SF295 glioblastoma and MDA-MB435 melanoma) by the MTT assay showed that acyclic precursor (6), and isobenfuranones (1a and 1b) are active compounds. Interestingly, 1a exhibits significant antiproliferative effect against HL-60 cells and moderate activity against SF295 and MDA-MB435 cell lines. Analysis of mechanisms involved in the cytotoxic activity showed that active compounds were leading to DNA damage, triggering apoptosis or necrosis induction. PMID:20537433

  6. A stable enol from a 6-substituted benzanthrone and its unexpected behaviour under acidic conditions

    PubMed Central

    Debeaux, Marc; Brandhorst, Kai; Jones, Peter G; Hopf, Henning; Grunenberg, Jörg; Kowalsky, Wolfgang

    2009-01-01

    Summary Treatment of benzanthrone (1) with biphenyl-2-yl lithium leads to the surprisingly stable enol 4, which is converted by dehydrogenation into the benzanthrone derivative 7. Under acidic conditions 4 isomerises to the spiro compound 11 and the bicyclo[4.3.1]decane derivative 12. Furthermore, the formation of 7 and the hydrogenated compound 13 is observed. A mechanism for the formation of the reaction products is proposed and supported by DFT calculations. PMID:19597557

  7. Anisotropic scattering rate in Fe-substituted Bi2Sr2Ca(Cu1-xFex)2O8+δ

    SciTech Connect

    Naamneh, M.; Lubashevsky, Y.; Lahoud, E.; Gu, G.; Kanigel, A.

    2015-05-27

    We measured the electronic structure of Fe substituted Bi2212 using Angle Resolved Photoemission Spectroscopy (ARPES). We find that the substitution does not change the momentum dependence of the superconducting gap but induces a very anisotropic enhancement of the scattering rate. A comparison of the effect of Fe substitution to that of Zn substitution suggests that the Fe reduces Tc so effectively because it supresses very strongly the coherence weight around the anti-nodes.

  8. Functional role of positively selected amino acid substitutions in mammalian rhodopsin evolution

    PubMed Central

    Fernández-Sampedro, Miguel A.; Invergo, Brandon M.; Ramon, Eva; Bertranpetit, Jaume; Garriga, Pere

    2016-01-01

    Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution. PMID:26865329

  9. Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature.

    PubMed

    Morio, Florent; Loge, Cedric; Besse, Bernard; Hennequin, Christophe; Le Pape, Patrice

    2010-04-01

    For several years, azole antifungal drugs have been a treatment option for potentially life-threatening Candida infections. However, azole resistance can occur through various mechanisms such as alterations in ERG11, encoding lanosterol 14alpha-demethylase (CYP51). In this study, we investigated the antifungal susceptibility to fluconazole, itraconazole, and voriconazole of 73 clinical isolates of Candida albicans. Screening for amino acid substitutions in Erg11 was performed on each of the 73 isolates. Twenty isolates displayed a marked decrease in azole susceptibility. Amino acid substitutions were detected in more than two-thirds of the strains. In all, 23 distinct substitutions were identified. Four have not been described previously, among which N136Y and Y447H are suspected to be involved in azole resistance. We suggest that the high genetic polymorphism of ERG11 must be considered in the rationale design of new azole compounds targeting lanosterol 14alpha-demethylase. A review of all Erg11 amino acid polymorphisms described to date is given. PMID:20226328

  10. The nucleotide sequence of HLA-B{sup *}2704 reveals a new amino acid substitution in exon 4 which is also present in HLA-B{sup *}2706

    SciTech Connect

    Rudwaleit, M.; Bowness, P.; Wordsworth, P.

    1996-12-31

    The HLA-B27 subtype HLA-B{sup *}2704 is virtually absent in Caucasians but common in Orientals, where it is associated with ankylosing spondylitis. The amino acid sequence of HLA-B{sup *}2704 has been established by peptide mapping and was shown to differ by two amino acids from HLA-B{sup *}2705, HLA-B{sup *}2704 is characterized by a serine for aspartic acid substitution at position 77 and glutamic acid for valine at position 152. To date, however, no nucleotide sequence confirming these changes at the DNA level has been published. 13 refs., 2 figs.

  11. Variable clinical manifestations of a glycine to glutamic acid substitution of the COL3A1 gene at residue 736

    SciTech Connect

    Pope, F.M.; Narcisi, P.; Richards, A.J.

    1994-09-01

    Glycine substitutions at the 3{prime} end of the COL3A1 gene generally produce a characteristic clinical phenotype including acrogeria and severe vascular fragility. Here we report a three generation British family in which the propositus presented with aneurysms of the groins. He, his mother, sister and elder daughter all had the external clinical phenotype of vascular EDS IV whilst another daughter and nephew were clinically normal. Cultured skin fibroblasts from the propositus and his clinically affected relatives poorly secreted normal and overmodified collagen III species. Normal components of secreted proteins predominated whilst overmodified molecules were prominent in intracellular material. Surprisingly the normal children also secreted less collagen type III than expected (though more than their clinically abnormal relatives). cDNA from bases 2671 to 3714 were amplified as four overlapping PCR fragments and analysed by DGGE. The region between 2671 and 3015 was heterozygous. Sequencing showed a mutation of glycine to glutamic acid at residue 736. This mutation created an extra Apa 1 restriction site which was suitable for family studies. These showed inheritance of the mutant gene by both vascular and non-vascular clinical phenotypes. This family therefore illustrates that replacement of glycine to glutamic acid at position 736 produces variable clinical and biochemical phenotypes ranging from easily recognizable vascular EDS IV with very poor collagen secretion to an EDS III-like picture and with less severe protein disturbance. The reasons for these differences are at present unexplained.

  12. Amino acid substitutions of cysteine residues near the amino terminus of Wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The amino-terminal half of HC-Pro of Wheat streak mosaic virus (WSMV) is required for semi-persistent transmission by the wheat curl mite (Aceria tosichella Keifer). The amino-proximal region of WSMV HC-Pro is cysteine-rich with a zinc finger-like motif. Amino acid substitutions were made in this re...

  13. 3-Component synthesis of α-substituted sulfonamides via Brønsted acid-catalyzed C(sp(3))-H bond functionalization of 2-alkylazaarenes.

    PubMed

    Beisel, T; Kirchner, J; Kaehler, T; Knauer, J; Soltani, Y; Manolikakes, G

    2016-06-28

    A Brønsted acid-catalyzed addition of 2-alkylazaarenes to in situ generated N-sulfonylimines through selective C(sp(3))-H bond functionalization has been developed. This protocol provides an atom- and step-economic approach to α-substituted sulfonamides. PMID:26868020

  14. Investigation on the reactivity of α-azidochalcones with carboxylic acids: Formation of α-amido-1,3-diketones and highly substituted 2-(trifluoromethyl)oxazoles.

    PubMed

    Rajaguru, Kandasamy; Mariappan, Arumugam; Suresh, Rajendran; Manivannan, Periasamy; Muthusubramanian, Shanmugam

    2015-01-01

    The reaction of α-azidochalcones with carboxylic acids has been investigated resulting in the formation of α-amido-1,3-diketones under microwave irradiation via in situ formation of 2H-azirine intermediates. An interesting reaction is described wherein, with trifluoroacetic acid at lower temperature, it affords highly substituted 2-(trifluoromethyl)oxazoles. These flexible transformations proceed under solvent free conditions in good to excellent yields without any catalyst. PMID:26664623

  15. Investigation on the reactivity of α-azidochalcones with carboxylic acids: Formation of α-amido-1,3-diketones and highly substituted 2-(trifluoromethyl)oxazoles

    PubMed Central

    Rajaguru, Kandasamy; Mariappan, Arumugam; Suresh, Rajendran; Manivannan, Periasamy

    2015-01-01

    Summary The reaction of α-azidochalcones with carboxylic acids has been investigated resulting in the formation of α-amido-1,3-diketones under microwave irradiation via in situ formation of 2H-azirine intermediates. An interesting reaction is described wherein, with trifluoroacetic acid at lower temperature, it affords highly substituted 2-(trifluoromethyl)oxazoles. These flexible transformations proceed under solvent free conditions in good to excellent yields without any catalyst. PMID:26664623

  16. Randomized trial of high-dose interferon-alpha-2b combined with ribavirin in patients with chronic hepatitis C: Correlation between amino acid substitutions in the core/NS5A region and virological response to interferon therapy.

    PubMed

    Mori, Nami; Imamura, Michio; Kawakami, Yoshiiku; Saneto, Hiromi; Kawaoka, Tomokazu; Takaki, Shintaro; Aikata, Hiroshi; Takahashi, Shoichi; Chayama, Kazuaki

    2009-04-01

    The aim of this study was to compare the efficacy of high-dose interferon (IFN)-alpha-2b with standard dose of IFN-alpha-2b in combination with ribavirin (RBV) for patients with chronic hepatitis C virus (HCV) infection, and to investigate the predictive factors associated with virological response. Two hundred Japanese patients with high HCV viral load (>100 KIU/ml) were randomized to 6 or 10 mega units (MU) of 24-week IFN-alpha-2b with RBV. Predictive factors were investigated; including pretreatment amino acid (aa) sequences of the core region and the IFN-sensitive determining region (ISDR). The sustained virological response rate was not different in the two groups (24% vs. 30%) but the incidence of depression was significantly higher in the 10 MU group than 6 MU group (7% vs. 0%, P = 0.02). Younger age (<60) and HCV genotype (2a/b) were significant predictors of sustained virological response. In patients infected with genotype 1b, substitutions of core aa 70 and/or 91 were predictive for non-virological response (P < 0.001), and substitutions in the ISDR was observed frequently in virological responders. Early viral kinetics study showed that serum HCV core antigen decreased more slowly in both patients with aa 70 and/or 91 substitutions in the core and with absence of substitutions in the ISDR. In conclusion, the use of a higher dose of IFN-alpha-2b in combination with RBV did not improve virological response but resulted in higher incidence of depression. Amino acid substitutions in the core and ISDR are predictive of virological response to the therapy in patients with genotype 1b and high viral load. PMID:19235866

  17. Acceleration of protein folding by four orders of magnitude through a single amino acid substitution

    PubMed Central

    Roderer, Daniel J. A.; Schärer, Martin A.; Rubini, Marina; Glockshuber, Rudi

    2015-01-01

    Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

  18. Acceleration of protein folding by four orders of magnitude through a single amino acid substitution.

    PubMed

    Roderer, Daniel J A; Schärer, Martin A; Rubini, Marina; Glockshuber, Rudi

    2015-01-01

    Cis prolyl peptide bonds are conserved structural elements in numerous protein families, although their formation is energetically unfavorable, intrinsically slow and often rate-limiting for folding. Here we investigate the reasons underlying the conservation of the cis proline that is diagnostic for the fold of thioredoxin-like thiol-disulfide oxidoreductases. We show that replacement of the conserved cis proline in thioredoxin by alanine can accelerate spontaneous folding to the native, thermodynamically most stable state by more than four orders of magnitude. However, the resulting trans alanine bond leads to small structural rearrangements around the active site that impair the function of thioredoxin as catalyst of electron transfer reactions by more than 100-fold. Our data provide evidence for the absence of a strong evolutionary pressure to achieve intrinsically fast folding rates, which is most likely a consequence of proline isomerases and molecular chaperones that guarantee high in vivo folding rates and yields. PMID:26121966

  19. Destabilization of pea lectin by substitution of a single amino acid in a surface loop.

    PubMed

    Hoedemaeker, F J; van Eijsden, R R; Díaz, C L; de Pater, B S; Kijne, J W

    1993-09-01

    Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability of Pisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val103) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (Tm) by some 10 degrees C in comparison with wild-type (wt) PSL, and the denaturation energy (delta H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe104) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugar-binding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced in Escherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 degrees C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are active in planta at ambient temperatures, but are inactive and possibly non-toxic at 37 degrees C in the intestines of mammals. PMID:8400124

  20. Probing structural features of Alzheimer's amyloid-β pores in bilayers using site-specific amino acid substitutions.

    PubMed

    Capone, Ricardo; Jang, Hyunbum; Kotler, Samuel A; Kagan, Bruce L; Nussinov, Ruth; Lal, Ratnesh

    2012-01-24

    A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-β (Aβ) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aβ channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aβ channel is an assembly of loosely associated mobile β-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aβ(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining β-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a β-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aβ-dependent ion fluxes. PMID:22242635

  1. Predicting HLA Class I Non-Permissive Amino Acid Residues Substitutions

    PubMed Central

    Binkowski, T. Andrew; Marino, Susana R.; Joachimiak, Andrzej

    2012-01-01

    Prediction of peptide binding to human leukocyte antigen (HLA) molecules is essential to a wide range of clinical entities from vaccine design to stem cell transplant compatibility. Here we present a new structure-based methodology that applies robust computational tools to model peptide-HLA (p-HLA) binding interactions. The method leverages the structural conservation observed in p-HLA complexes to significantly reduce the search space and calculate the system’s binding free energy. This approach is benchmarked against existing p-HLA complexes and the prediction performance is measured against a library of experimentally validated peptides. The effect on binding activity across a large set of high-affinity peptides is used to investigate amino acid mismatches reported as high-risk factors in hematopoietic stem cell transplantation. PMID:22905104

  2. Ascorbic acid and rates of cognitive decline in Alzheimer's disease.

    PubMed

    Bowman, Gene L; Dodge, Hiroko; Frei, Balz; Calabrese, Carlo; Oken, Barry S; Kaye, Jeffrey A; Quinn, Joseph F

    2009-01-01

    The brain maintains high levels of ascorbic acid (AA) despite a concentration gradient favoring diffusion from brain to peripheral tissues. Dietary antioxidants, including AA, appear to modify the risk of Alzheimer's disease (AD). The objective of this study was to test the hypothesis that neurodegeneration in AD is modified by brain levels of AA. Thirty-two patients with mild to moderate AD participated in a biomarker study involving standardized clinical assessments over one year. Cerebrospinal fluid (CSF) and serum were collected at baseline for AA and albumin content. Cognitive measures were collected at baseline and one year. CSF and plasma AA failed to predict cognitive decline independently, however, CSF: plasma AA ratio did. After adding CSF Albumin Index (an established marker of blood-brain barrier integrity) to the regression models the effect of CSF: plasma AA ratio as a predictor of cognitive decline was weakened. CSF: plasma AA ratio predicts rate of decline in AD. This relationship may indicate that the CSF: plasma AA ratio is an index of AA availability to the brain or may be an artifact of a relationship between blood-brain barrier impairment and neurodegeneration. PMID:19158425

  3. Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae

    PubMed Central

    Ha, Suk-Jin; Kim, Heejin; Lin, Yuping; Jang, Myoung-Uoon; Galazka, Jonathan M.; Kim, Tae-Jip; Cate, Jamie H. D.

    2013-01-01

    Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter (cdt-1) and intracellular β-glucosidase (gh1-1) genes from Neurospora crassa. Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae. However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1. The rate of cellobiose fermentation by the HXT2.4-expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher Km and 4-fold higher Vmax values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains. PMID:23263959

  4. Substitution of amino acid 70 in the hepatitis C virus core region of genotype 1b is an important predictor of elevated alpha-fetoprotein in patients without hepatocellular carcinoma.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Kawamura, Yusuke; Yatsuji, Hiromi; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2008-08-01

    Previous studies identified amino acid (aa) substitutions of the hepatitis C virus core region of genotype 1b (HCV-1b core region) and elevated serum alpha-fetoprotein (AFP) levels as predictors of poor virologic response to pegylated interferon (PEG-IFN) plus ribavirin (RBV), and also as risk factors for hepatocarcinogenesis. The present study evaluated the impact of aa substitutions of HCV-1b core region on AFP, as a surrogate marker of hepatocarcinogenesis, on AFP levels in 569 Japanese patients with HCV-1b but without HCC, and investigated the predictive factors of elevated AFP (> or =11 microg/L). High AFP levels were detected in 27.4% of the patients. The rate of hepatocarcinogenesis in a group of 109 patients who received IFN monotherapy and followed-up for 15 years, was significantly higher in patients with abnormal than normal AFP. Multivariate analysis of 569 patients identified fibrosis stage (F3,4), aspartate aminotransferase (> or =76 IU/L), substitution of aa 70 (glutamine or histidine), and platelet count (<15.0 x 10(4)/microl) as significant determinants of elevated AFP. In 49 patients with abnormal AFP levels and substitutions at aa 70 who were treated with PEG-IFN + RBV, the rate of normalization of AFP was significantly lower in non-virological responders (28.6%) than in transient (71.4%) and sustained (100%) virological responders. The results indicated that substitution of aa 70 of HCV-1b core region is an important predictor of elevated AFP in non-HCC patients, and that eradication of the mutant virus normalizes AFP. The results highlight the importance of eradication of mutant type virus of aa 70 for reducing the risk of hepatocarcinogenesis. PMID:18551609

  5. Amino Acid Substitutions of CrrB Responsible for Resistance to Colistin through CrrC in Klebsiella pneumoniae.

    PubMed

    Cheng, Yi-Hsiang; Lin, Tzu-Lung; Lin, Yi-Tsung; Wang, Jin-Town

    2016-06-01

    Colistin is a last-resort antibiotic for treatment of carbapenem-resistant Klebsiella pneumoniae A recent study indicated that missense mutations in the CrrB protein contribute to colistin resistance. In our previous study, mechanisms of colistin resistance were defined in 17 of 26 colistin-resistant K. pneumoniae clinical isolates. Of the remaining nine strains, eight were highly resistant to colistin. In the present study, crrAB sequences were determined for these eight strains. Six separate amino acid substitutions in CrrB (Q10L, Y31H, W140R, N141I, P151S, and S195N) were detected. Site-directed mutagenesis was used to generate crrB loci harboring individual missense mutations; introduction of the mutated genes into a susceptible strain, A4528, resulted in 64- to 1,024-fold increases in colistin MICs. These crrB mutants showed increased accumulation of H239_3062, H239_3059, pmrA, pmrC, and pmrH transcripts by quantitative reverse transcription (qRT)-PCR. Deletion of H239_3062 (but not that of H239_3059) in the A4528 crrB(N141I) strain attenuated resistance to colistin, and H239_3062 was accordingly named crrC Similarly, accumulation of pmrA, pmrC, and pmrH transcripts induced by crrB(N141I) was significantly attenuated upon deletion of crrC Complementation of crrC restored resistance to colistin and accumulation of pmrA, pmrC, and pmrH transcripts in a crrB(N141I) ΔcrrC strain. In conclusion, novel individual CrrB amino acid substitutions (Y31H, W140R, N141I, P151S, and S195N) were shown to be responsible for colistin resistance. We hypothesize that CrrB mutations induce CrrC expression, thereby inducing elevated expression of the pmrHFIJKLM operon and pmrC (an effect mediated via the PmrAB two-component system) and yielding increased colistin resistance. PMID:27067316

  6. Amino acid side chain induced selectivity in the hydrolysis of peptides catalyzed by a Zr(IV)-substituted Wells-Dawson type polyoxometalate.

    PubMed

    Vanhaecht, Stef; Absillis, Gregory; Parac-Vogt, Tatjana N

    2013-11-21

    In this paper the reactivity of K15H[Zr(α2-P2W17O61)2]·25H2O (1), a Zr(IV)-substituted Wells-Dawson polyoxometalate, is examined towards a series of Gly-Aa, Aa-Gly or Aa-Ser dipeptides, in which the nature and the size of the Aa amino acid side chain were varied. The rate of peptide bond hydrolysis, determined by (1)H NMR experiments, in Gly-Aa dipeptides is strongly dependent on the molecular volume and the chemical structure of the Aa side chain. When the volume of the aliphatic side chain of the Aa residue in Gly-Aa increased, a clear decrease in the hydrolysis rate was observed. Replacing one α-H in the C-terminal Gly residue of Gly-Gly by a methyl group (Gly-Ala) resulted in a 6-fold reactivity decrease, pointing towards the importance of steric factors for efficient peptide bond hydrolysis. The rate constants for peptide bond hydrolysis in Gly-Aa dipeptides at pD 5.0 and 60 °C ranged from 208.0 ± 15.6 × 10(-6) min(-1) for Gly-Ser to 5.0 ± 1.0 × 10(-6) min(-1) for Gly-Glu, reflecting the influence of the different nature of the amino acid side chains on the hydrolysis rate. Faster hydrolysis was observed for peptides containing Ser and Thr since the hydroxyl group in their side chain is able to facilitate amide bond hydrolysis by promoting an N→O acyl rearrangement. Peptides containing positively charged side chains at pD 5.0 show enhanced hydrolysis rates as a result of the secondary electrostatic interactions with the negatively charged surface of the polyoxometalate, which stabilize the peptide-polyoxometalate complex. A slow hydrolysis rate was observed for Gly-Glu, because of the preferential coordination of the carboxylate group in the side chain of Glu to Zr(IV), which prevents coordination of the peptide carbonyl group and its activation towards hydrolysis. PMID:24018583

  7. Amino acid substitution in the core protein has no impact on relapse in hepatitis C genotype 1 patients treated with peginterferon and ribavirin.

    PubMed

    Inoue, Yuko; Hiramatsu, Naoki; Oze, Tsugiko; Yakushijin, Takayuki; Mochizuki, Kiyoshi; Fukuda, Kazuto; Mita, Eiji; Haruna, Yoshimichi; Inoue, Atsuo; Imai, Yasuharu; Hosui, Atsushi; Miyagi, Takuya; Yoshida, Yuichi; Tatsumi, Tomohide; Kiso, Shinichi; Kanto, Tatsuya; Kasahara, Akinori; Takehara, Tetsuo; Hayashi, Norio

    2011-03-01

    Previous reports demonstrated that amino acid (aa) substitutions in the hepatitis C virus (HCV) core protein are predictors of non-virological responses to pegylated interferon (Peg-IFN) and ribavirin combination therapy. The aim of this study was to investigate the impact of core aa substitutions on viral kinetics during the treatment and relapse after the treatment. The 187 patients with HCV genotype 1 enrolled in this study were categorized into four groups according to core aa substitution patterns: double-wild group (n=92), Arg70/Leu91; 70-mutant group (n=42), Gln70/Leu91; 91-mutant group (n=31), Arg70/Met91; and double-mutant group (n=22), Gln70/Met91. The relationship between the core aa substitutions and the virological response was examined. Multivariate logistic regression analyses showed that substitution at aa 70 was significantly associated with a poor virological response during the first 12 weeks (decline of <1 log from baseline at week 4, <2 log at week 12), and substitution at aa 91 was significantly associated with detectable HCV RNA at week 24. With respect to relapse, only the ribavirin exposure (odds ratio (OR), 0.77; 95% confidence interval (CI), 0.60-0.98) and HCV RNA disappearance between weeks 13 and 24 (OR, 23.69; 95% CI, 5.44-103.08) were associated independently with relapse, with no correlation being found with the core aa substitutions and relapse. In conclusion, the results showed that core aa substitutions can be strong predictive factors at pretreatment of the non-response, but not for relapse, for virological responders with HCV RNA disappearance during treatment. PMID:21264862

  8. l-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs

    PubMed Central

    Frikke-Schmidt, Henriette; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2015-01-01

    Vitamin C deficiency globally affects several hundred million people and has been associated with increased morbidity and mortality in numerous studies. In this study, bioavailability of the oxidized form of vitamin C (l-dehydroascorbic acid or DHA)—commonly found in vitamin C containing food products prone to oxidation—was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences in vitamin C accumulation from ascorbate or DHA were observed except for the lowest dose of DHA (0.25 mg/ml in the drinking water), where approximately half of the tissues had slightly yet significantly less vitamin C accumulation than from the ascorbate source. As these results contradicted data from rats, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs—as in humans—is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin C and further suggest that the guinea pig is superior to other rodents in modeling human vitamin C homeostasis. PMID:26609560

  9. L-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs.

    PubMed

    Frikke-Schmidt, Henriette; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-04-01

    Vitamin C deficiency globally affects several hundred million people and has been associated with increased morbidity and mortality in numerous studies. In this study, bioavailability of the oxidized form of vitamin C (l-dehydroascorbic acid or DHA)-commonly found in vitamin C containing food products prone to oxidation-was studied. Our aim was to compare tissue accumulation of vitamin C in guinea pigs receiving different oral doses of either ascorbate or DHA. In all tissues tested (plasma, liver, spleen, lung, adrenal glands, kidney, muscle, heart, and brain), only sporadic differences in vitamin C accumulation from ascorbate or DHA were observed except for the lowest dose of DHA (0.25mg/ml in the drinking water), where approximately half of the tissues had slightly yet significantly less vitamin C accumulation than from the ascorbate source. As these results contradicted data from rats, we continued to explore the ability to recycle DHA in blood, liver and intestine in guinea pigs, rats and mice. These investigations revealed that guinea pigs have similar recycling capacity in red blood cells as observed in humans, while rats and mice do not have near the same ability to reduce DHA in erythrocytes. In liver and intestinal homogenates, guinea pigs also showed a significantly higher ability to recycle DHA compared to rats and mice. These data demonstrate that DHA in guinea pigs-as in humans-is almost as effective as ascorbate as vitamin C source when it comes to taking up and storing vitamin C and further suggest that the guinea pig is superior to other rodents in modeling human vitamin C homeostasis. PMID:26609560

  10. Coordination compounds of hafnium(IV) with some N-substituted derivatives of unsaturated hydroxamic acids

    SciTech Connect

    Stratulat, A.A.; Batyr, D.G.

    1987-05-01

    Coordination compounds of hafnium(IV) with N-o(or m)-X-phenylacryl- and methacrylhydroxamic acids with the general formula (Hf/XC/sub 6/H/sub 4/-N(O)-C(O)-R//sub 4/), where X = 4-CH/sub 3/, H, 4-Cl, 4-Br, 4-CH/sub 3/C(O), 4-CH=CH/sub 2/, 4-CH/sub 3/OC(O), 3-CH/sub 3/, 3-Cl, and 3-Br, and R = CH=CH/sub 2/ and C(CH/sub 3/)=CH/sub 2/, have been synthesized and characterized. The type of coordination of the organic ligands and the structure of the complexes have been established on the basis of the data from IR, electronic, and PMR spectra. It has been shown that the complexation process involves the replacement of the proton of the hydroxyl group of the hydroxamic grouping by a metal ion and the coordination of the oxygen atom of the carbonyl group. The coordination compounds obtained have been assigned a square-antiprismatic structure. The introduction of a methyl radical into the vinyl grouping R results in a significant increase in the strength of the complex.

  11. Antimicrobial effect of para-alkoxyphenylcarbamic acid esters containing substituted N-phenylpiperazine moiety

    PubMed Central

    Malík, Ivan; Bukovský, Marián; Andriamainty, Fils; Gališinová, Jana

    2013-01-01

    In current research, nine basic esters of para-alkoxyphenylcarbamic acid with incorporated 4-(4-fluoro-/3-trifluoromethylphenyl)piperazin-1-yl fragment, 6i–6m and 8f–8i, were screened for their in vitro antimicrobial activity against Candida albicans, Staphylococcus aureus and Escherichia coli, respectively. Taking into account the minimum inhibitory concentration assay (MIC), as the most active against given yeast was evaluated 8i (MIC = 0.20 mg/mL), the most lipophilic structure containing para-butoxy and trifluoromethyl substituents. Investigating the efficiency of the compounds bearing only a single atom of fluorine and appropriate para-alkoxy side chain against Candida albicans, the cut-off effect was observed. From evaluated homological series, the maximum of the effectiveness was noticed for the stucture 6 k (MIC = 0.39 mg/mL), containing para-propoxy group attached to phenylcarbamoyloxy fragment, beyond which the compounds ceased to be active. On the contrary, all the tested molecules were against Staphylococcus aureus and Escherichia coli (MICs > 1.00 mg/mL) practically inactive. PMID:24294237

  12. Antimicrobial effect of para-alkoxyphenylcarbamic acid esters containing substituted N-phenylpiperazine moiety.

    PubMed

    Malík, Ivan; Bukovský, Marián; Andriamainty, Fils; Gališinová, Jana

    2013-01-01

    In current research, nine basic esters of para-alkoxyphenylcarbamic acid with incorporated 4-(4-fluoro-/3-trifluoromethylphenyl)piperazin-1-yl fragment, 6i-6m and 8f-8i, were screened for their in vitro antimicrobial activity against Candida albicans, Staphylococcus aureus and Escherichia coli, respectively. Taking into account the minimum inhibitory concentration assay (MIC), as the most active against given yeast was evaluated 8i (MIC = 0.20 mg/mL), the most lipophilic structure containing para-butoxy and trifluoromethyl substituents. Investigating the efficiency of the compounds bearing only a single atom of fluorine and appropriate para-alkoxy side chain against Candida albicans, the cut-off effect was observed. From evaluated homological series, the maximum of the effectiveness was noticed for the stucture 6 k (MIC = 0.39 mg/mL), containing para-propoxy group attached to phenylcarbamoyloxy fragment, beyond which the compounds ceased to be active. On the contrary, all the tested molecules were against Staphylococcus aureus and Escherichia coli (MICs > 1.00 mg/mL) practically inactive. PMID:24294237

  13. Preparation of a nitro-substituted tris(indolyl)methane modified silica in deep eutectic solvents for solid-phase extraction of organic acids.

    PubMed

    Wang, Na; Wang, Jiamin; Liao, Yuan; Shao, Shijun

    2016-05-01

    A new sorbent for solid-phase extraction was synthesized by chemical immobilization of nitro-substituted tris(indolyl)methane on silica in new and green deep eutectic solvents. Elemental analysis results indicated that deep eutectic solvents could be an alternative to the traditional solvents in preparing nitro-substituted tris(indolyl)methane modified silica. Coupled with high performance liquid chromatography, the extraction performance of the sorbent was evaluated by using four organic acids as model analytes. The rebinding experiments results showed that the nitro-substituted tris(indolyl)methane modified silica sorbent had a good adsorption capacity towards the selected organic acids. Under the appropriate experimental conditions, good precision and wide linear ranges with coefficient of determination (R(2)) of higher than 0.9957 were obtained, and the limits of detection were in the range of 0.50-2.0μgL(-1) for the organic acids tested. The developed solid-phase extraction-high performance liquid chromatography-diode array detection (SPE-HPLC-DAD) method was successfully applied for the determination of organic acids in two drinking samples with recoveries ranging from 76.7% to 110.0% and 67.7% to 104.0% for all the selected organic acids, respectively. PMID:26946003

  14. A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis1[OPEN

    PubMed Central

    Yuan, Hui; Owsiany, Katherine; Sheeja, T.E.; Zhou, Xiangjun; Rodriguez, Caroline; Li, Yongxi; Welsch, Ralf; Chayut, Noam; Yang, Yong; Thannhauser, Theodore W.; Parthasarathy, Mandayam V.; Xu, Qiang; Deng, Xiuxin; Fei, Zhangjun; Schaffer, Ari; Katzir, Nurit; Burger, Joseph; Tadmor, Yaakov; Li, Li

    2015-01-01

    Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtORHis (R90H) or SbORHis (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtORAla (R90A) functioned similarly to AtORHis to promote carotenoid overproduction. Neither AtOR nor AtORHis greatly affected carotenogenic gene expression. AtORHis exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHis triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtORHis in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates ORHis/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying ORHis-regulated carotenoid accumulation. PMID:26224804

  15. THE ANTIMUTAGENICITY OF 2-SUBSTITUTED SELENAZOLIDINE-4-(R)-CARBOXYLIC ACIDS.

    PubMed Central

    El-Sayed, Wael M.; Hussin, Warda A; Franklin, Michael R.

    2007-01-01

    Selenium can have cancer chemopreventive activity, although the mechanism of action has not been well defined. Selenazolidine-4-(R)-carboxylic acids (SCAs) were devised as prodrugs of L-selenocysteine, to provide selenium in a form and at a concentration commensurate with cancer chemopreventive activity. In the present study, a series of selenazolidines has been evaluated in the Salmonella typhimurium TA98 tester strain and all were found to possess antimutagenic activity. There was little difference between the seven selenazolidines in their effectiveness against either benzo[a]pyrene (B[a]P) or 3,6-bis(dimethylamino)acridine (acridine orange), agents which differ in their requirement for mammalian enzyme bioactivation for mutagenicity. Antimutagenic activity against acridine orange was dependent on selenazolidine concentration, and EC50 values were in the 5 –10 μM range. At 25 μM, the concentration tested in common for the two mutagens, the selenazolidines were more effective antimutagens against acridine orange than against B[a]P, with reductions in mutant frequency ranging from 54–71% for B[a]P and 79–93% for acridine orange. Efficacy against B[a]P was not enhanced when the concentration was increased to 50 μM. The similarity in efficacy among the selenazolidines against B[a]P mutagenicity, contrasted with inter-compound differences in their ability to inhibit S9 CYP1A activity. The CYP1A Ki values ranged from a low of 63 μM (2-[2'-hydroxyphenyl]SCA) to a high of 1.1 mM (2-cyclohexylSCA), but all were above the concentration required to inhibit mutagenicity by 50%. Thus, all the SCAs possess antimutagenic activity against both B[a]P and acridine orange, the efficacy varies little between the individual selenazolidines, and for B[a]P, the efficacy is not proportional to the inhibitory effect on the mutagen bioactivating enzyme. PMID:17166761

  16. Hydrogenation of imines catalyzed by trisphosphine-substituted molybdenum and tungsten nitrosyl hydrides and co-catalytic acid.

    PubMed

    Chakraborty, Subrata; Blacque, Olivier; Fox, Thomas; Berke, Heinz

    2014-10-01

    Hydride complexes Mo,W(CO)(NO)H(mer-etp(i)p) (iPr2PCH2CH2)2PPh=etp(i)p) (2 a,b(syn), syn and anti of NO and Ph(etp(i)p) orientions) were prepared and probed in imine hydrogenations together with co-catalytic [H(Et2O)2][B(C6F5)4] (140 °C, 60 bar H2). 2 a,b(syn) were obtained via reduction of syn/anti-Mo,W(NO)Cl3(mer-etp(i)p) and syn,anti-Mo,W(NO)(CO)Cl(mer-etp(i)p). [H(Et2O)2][B(C6F5)4] in THF converted the hydrides into THF complexes syn-[Mo,W(NO)(CO)(etp(i)p)(THF)][B(C6F5)4]. Combinations of the p-substituents of aryl imines p-R(1)C6H4CH=N-p-C6H4R(2) (R(1),R(2)=H,F,Cl,OMe,α-Np) were hydrogenated to amines (maximum initial TOFs of 1960 h(-1) (2 a(syn)) and 740 h(-1) (2 b(syn)) for N-(4-methoxybenzylidene)aniline). An 'ionic hydrogenation' mechanism based on linear Hammett plots (ρ=-10.5, p-substitution on the C-side and ρ=0.86, p-substitution on the N-side), iminium intermediates, linear P(H2) dependence, and DKIE=1.38 is proposed. Heterolytic splitting of H2 followed by 'proton before hydride' transfers are the steps in the ionic mechanism where H2 ligand addition is rate limiting. PMID:25048293

  17. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    PubMed Central

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  18. Two single amino acid substitutions in the intervening region of Newcastle disease virus HN protein attenuate viral replication and pathogenicity

    PubMed Central

    Liu, Bin; Ji, Yanhong; Lin, Zhongqing; Fu, Yuguang; Muhammad Dafallah, Rihab; Zhu, Qiyun

    2015-01-01

    Among the proteins encoded by Newcastle disease virus (NDV), the attachment protein (HN) is an important determinant of virulence and pathogenicity. HN has been molecularly characterized at the protein level; however, the relationship between the molecular character of HN and the animal pathotype it causes has not been well explored. Here, we revisited the intervening region (IR) of the HN stalk and extended the known biological functions of HN. Three distinct substitutions (A89Q, P93A, and L94A) in the IR of genotype VII NDV (G7 strain) HN protein were analyzed. The A89Q and L94A mutations weakened the fusion promotion activity of HN to 44% and 41% of that of wild type, respectively, whereas P93A decreased the neuraminidase activity to 21% of the parental level. At the virus level, P93A and L94A-bearing viruses displayed impaired receptor recognition ability, neuraminidase activity, and fusion-promoting activity, all of which led to virus attenuation. In addition, the L94A-mutated virus showed a dramatic decline in replication and was attenuated in cells and in chickens. Our data demonstrate that the HN biological activities and functions modulated by these specific amino acids in the IR are associated with NDV replication and pathogenicity. PMID:26267791

  19. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.

    PubMed

    Roney, Ian J; Rudner, Adam D; Couture, Jean-François; Kærn, Mads

    2016-01-01

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount. PMID:27323850

  20. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels

    PubMed Central

    Roney, Ian J.; Rudner, Adam D.; Couture, Jean-François; Kærn, Mads

    2016-01-01

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount. PMID:27323850

  1. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    PubMed

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T G; Hendriks, Wiljan J A J; Cortés, Jesús M; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  2. Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120

    PubMed Central

    Kirchherr, Jennifer L; Hamilton, Jennifer; Lu, Xiaozhi; Gnanakaran, S; Muldoon, Mark; Daniels, Marcus; Kasongo, Webster; Chalwe, Victor; Mulenga, Chanda; Mwananyanda, Lawrence; Musonda, Rosemary M; Yuan, Xing; Montefiori, David C; Korber, Bette T; Haynes, Barton F; Gao, Feng

    2010-01-01

    Neutralizing antibodies (Nabs) are thought to play an important role in prevention and control of HIV-1 infection and should be targeted by an AIDS vaccine. It is critical to understand how HIV-1 induces Nabs by analyzing viral sequences in both tested viruses and sera. Neutralization susceptibility to antibodies in autologous and heterologous plasma was determined for multiple Envs (3–6) from each of 15 subtype C infected-individuals. Heterologous neutralization was divided into two distinct groups: plasma with strong, cross-reactive neutralization (N=9) and plasma with weak neutralization (N=6). Plasma with cross-reactive heterologous Nabs also more potently neutralized contemporaneous autologous viruses. Analysis of Env sequences in plasma from both groups revealed a three-amino acid substitution pattern in the V4 region that was associated with greater neutralization potency and breadth. Identification of such potential neutralization signatures may have important implications for the development of HIV-1 vaccines capable of inducing Nabs to subtype C HIV-1. PMID:21036380

  3. Designing Inhibitors of Cytochrome c/Cardiolipin Peroxidase Complexes: Mitochondria-Targeted Imidazole-Substituted Fatty Acids

    PubMed Central

    Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A.; Silva, K. Ishara; Huang, Zhentai; Amoscato, Andrew A.; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E.

    2014-01-01

    Mitochondria have emerged as the major regulatory platform responsible for coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (cyt) c. As this oxidation occurs within the peroxidase complex of cyt c with CL, the latter represents a promising target for the discovery and design of drugs with anti-apoptotic mechanism of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogues of stearic acid TPP-n-ISA with different positions of the attached imidazole group on the fatty acid (n=6, 8, 10, 13 and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance, and electron spin echo envelope modulation) we demonstrated that TPP-n-ISA indeed were able to potently suppress CL induced structural re-arrangements in cyt c paving the way to its peroxidase competence. TPP-n-ISA analogues preserved the low spin hexa-coordinated heme iron state in cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant anti-apoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all atom molecular dynamics simulations. Based on the experimental data and computations predictions, we identified TPP-6-ISA as a candidate drug with optimized anti-apoptotic potency. PMID:24631490

  4. Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids.

    PubMed

    Jiang, Jianfei; Bakan, Ahmet; Kapralov, Alexandr A; Silva, K Ishara; Huang, Zhentai; Amoscato, Andrew A; Peterson, James; Garapati, Venkata Krishna; Saxena, Sunil; Bayir, Hülya; Atkinson, Jeffrey; Bahar, Ivet; Kagan, Valerian E

    2014-06-01

    Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency. PMID:24631490

  5. Low Phytic Acid Barley Responses to Phosphorus Rates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low phytic acid (LPA) barley (Hordeum vulgare L.) cultivars partition phosphorus in seed tissue differently than conventional barley cultivars through a reduction in seed phytic acid (myo-inositol-1,2,3,4,5,6-hexkisphosphate) coupled with an increase in inorganic phosphorus. The response of the LPA...

  6. Synthesis of [.sup.13C] and [.sup.2H] substituted methacrylic acid, [.sup.13C] and [.sup.2H] substituted methyl methacrylate and/or related compounds

    DOEpatents

    Alvarez, Marc A.; Martinez, Rodolfo A.; Unkefer, Clifford J.

    2010-02-16

    The present invention is directed to labeled compounds of the formulae ##STR00001## wherein Q is selected from the group consisting of --S(.dbd.O)--, and --S(.dbd.O).sub.2--, Z is selected from the group consisting of 1-naphthyl, substituted 1-naphthyl, 2-naphthyl, substituted 2-naphthyl, and phenyl groups with the structure ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each independently selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl, a halogen, and an amino group selected from the group consisting of NH.sub.2, NHR and NRR' where R and R' are each independently selected from the group consisting of a C.sub.1-C.sub.4 lower alkyl, an aryl, and an alkoxy group, and X is selected from the group consisting of hydrogen, a C.sub.1-C.sub.4 lower alkyl group, and a fully-deuterated C.sub.1-C.sub.4 lower alkyl group.

  7. Requirement for Asn298 on D1 protein for oxygen evolution: analyses by exhaustive amino acid substitution in the green alga Chlamydomonas reinhardtii.

    PubMed

    Kuroda, Hiroshi; Kodama, Natsumi; Sun, Xiao-Yu; Ozawa, Shin-ichiro; Takahashi, Yuichiro

    2014-07-01

    PSII generates strong oxidants used for water oxidation. The secondary electron donor, Y(Z), is Tyr161 on PSII reaction center D1 protein and mediates electron transfer from the oxygen-evolving Mn(4)CaO(5) cluster to the primary electron donor, P680. The latest PSII crystal structure revealed the presence of a hydrogen bond network around Y(Z), which is anticipated to play important roles in the electron and proton transfer reactions. Y(Z) forms a hydrogen bond with His190 which in turn forms a hydrogen bond with Asn298 on D1 protein. Although functional roles of Y(Z) and His190 have already been characterized, little is known about the functional role of Asn298. Here we have generated 19 mutants from a green alga Chlamydomonas reinhardtii, in which the Asn298 has been substituted by each of the other 19 amino acid residues. All mutants showed significantly impaired or no photosynthetic growth. Seven mutants capable of photosynthetic growth showed oxygen-evolving activity although at a significantly reduced rate. Interestingly the oxygen-evolving activity of these mutants was markedly photosensitive. The 19 mutants accumulated PSII at variable levels and showed a light-induced electron transfer reaction from 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolindophenol (DCIP), suggesting that Asn298 is important for the function and photoprotection of the Mn(4)CaO(5) cluster. PMID:24853102

  8. Multiple amino acid substitutions involved in the adaptation of avian-origin influenza A (H10N7) virus in mice.

    PubMed

    Wu, Haibo; Peng, Xiuming; Peng, Xiaorong; Cheng, Linfang; Jin, Changzhong; Lu, Xiangyun; Xie, Tiansheng; Yao, Hangping; Wu, Nanping

    2016-04-01

    To identify substitutions that are possibly associated with the adaptation of avian-origin H10N7 virus to mammals, adaptation of the H10N7 virus in mouse lung was carried out by serial lung-to-lung passage. Genomic analysis of the mouse-adapted virus revealed amino acid changes in the PB2 (E627K), PA (T97I), and HA (G409E) proteins, and this virus was more virulent in mice than the wild-type virus. Our results suggest that these substitutions are involved in the enhancement of the replication efficiency of avian-origin H10N7 virus, resulting in severe disease in mice. Continued poultry surveillance of these substitutions in H10N7 viruses is required. PMID:26699787

  9. DNA-LCEB: a high-capacity and mutation-resistant DNA data-hiding approach by employing encryption, error correcting codes, and hybrid twofold and fourfold codon-based strategy for synonymous substitution in amino acids.

    PubMed

    Hafeez, Ibbad; Khan, Asifullah; Qadir, Abdul

    2014-11-01

    Data-hiding in deoxyribonucleic acid (DNA) sequences can be used to develop an organic memory and to track parent genes in an offspring as well as in genetically modified organism. However, the main concerns regarding data-hiding in DNA sequences are the survival of organism and successful extraction of watermark from DNA. This implies that the organism should live and reproduce without any functional disorder even in the presence of the embedded data. Consequently, performing synonymous substitution in amino acids for watermarking becomes a primary option. In this regard, a hybrid watermark embedding strategy that employs synonymous substitution in both twofold and fourfold codons of amino acids is proposed. This work thus presents a high-capacity and mutation-resistant watermarking technique, DNA-LCEB, for hiding secret information in DNA of living organisms. By employing the different types of synonymous codons of amino acids, the data storage capacity has been significantly increased. It is further observed that the proposed DNA-LCEB employing a combination of synonymous substitution, lossless compression, encryption, and Bose-Chaudary-Hocquenghem coding is secure and performs better in terms of both capacity and robustness compared to existing DNA data-hiding schemes. The proposed DNA-LCEB is tested against different mutations, including silent, miss-sense, and non-sense mutations, and provides substantial improvement in terms of mutation detection/correction rate and bits per nucleotide. A web application for DNA-LCEB is available at http://111.68.99.218/DNA-LCEB. PMID:25195035

  10. Comparisons of Pollen Substitute Diets for Honey bees: Consumprion Rates by Colonies and Effects on Brood and Adult Populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercially available pollen substitute diets for honey bees (Apis mellifera L.) were evaluated for consumption and colony growth (brood and adult populations) and compared with pollen cake and high fructose corn syrup (HFCS). Two trials were conducted; the first for 4 months during the fall and wi...

  11. Comparisons of pollen substitute diets for honey bees: consumption rates by colonies and effects on brood and adult populations.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Commercially available pollen substitute diets for honey bees (Apis mellifera L.) were evaluated for consumption and colony growth (brood and adult populations) and compared with pollen cake and high fructose corn syrup (HFCS). Two trials were conducted; the first for 3 months during the fall and w...

  12. Hotspots of Biased Nucleotide Substitutions in Human Genes

    PubMed Central

    Berglund, Jonas; Pollard, Katherine S; Webster, Matthew T

    2009-01-01

    Genes that have experienced accelerated evolutionary rates on the human lineage during recent evolution are candidates for involvement in human-specific adaptations. To determine the forces that cause increased evolutionary rates in certain genes, we analyzed alignments of 10,238 human genes to their orthologues in chimpanzee and macaque. Using a likelihood ratio test, we identified protein-coding sequences with an accelerated rate of base substitutions along the human lineage. Exons evolving at a fast rate in humans have a significant tendency to contain clusters of AT-to-GC (weak-to-strong) biased substitutions. This pattern is also observed in noncoding sequence flanking rapidly evolving exons. Accelerated exons occur in regions with elevated male recombination rates and exhibit an excess of nonsynonymous substitutions relative to the genomic average. We next analyzed genes with significantly elevated ratios of nonsynonymous to synonymous rates of base substitution (dN/dS) along the human lineage, and those with an excess of amino acid replacement substitutions relative to human polymorphism. These genes also show evidence of clusters of weak-to-strong biased substitutions. These findings indicate that a recombination-associated process, such as biased gene conversion (BGC), is driving fixation of GC alleles in the human genome. This process can lead to accelerated evolution in coding sequences and excess amino acid replacement substitutions, thereby generating significant results for tests of positive selection. PMID:19175294

  13. Ultrasound-assisted one-pot synthesis of substituted coumarins catalyzed by poly(4-vinylpyridinium) hydrogen sulfate as an efficient and reusable solid acid catalyst.

    PubMed

    Khaligh, Nader Ghaffari

    2013-07-01

    Poly(4-vinylpyridinium) hydrogen sulfate solid acid was found to be efficient catalyst for synthesis of substituted coumarins via Pechmann reaction using ultrasound irradiation at room temperature and neat condition in high yields with short reaction times. This methodology offers momentous improvements over various options for the synthesis of coumarins with regard to yield of products, simplicity in operation and green aspects by avoiding toxic catalysts and solvents. Further, the catalyst can be reused and recovered for several times. PMID:23395258

  14. Hydroxyapatite-calcium sulfate-hyaluronic acid composite encapsulated with collagenase as bone substitute for alveolar bone regeneration.

    PubMed

    Subramaniam, Sadhasivam; Fang, Yen-Hsin; Sivasubramanian, Savitha; Lin, Feng-Huei; Lin, Chun-pin

    2016-01-01

    Periodontitis is a very severe inflammatory condition of the periodontium that progressively damages the soft tissue and destroys the alveolar bone that supports the teeth. The bone loss is naturally irreversible because of limited reparability of the teeth. Advancement in tissue engineering provides an effective regeneration of osseous defects with suitable dental implants or tissue-engineered constructs. This study reports a hydroxyapatite, calcium sulfate hemihydrate and hyaluronic acid laden collagenase (HAP/CS/HA-Col) as a bone substitute for the alveolar bone regeneration. The composite material was mechanically tested and the biocompatibility was evaluated by WST-1 assay. The in vivo bone formation was assessed in rat with alveolar bone defects and the bone augmentation by the HAP/CS/HA-Col composite was confirmed by micro-CT images and histological examination. The mechanical strength of 6.69 MPa with excellent biocompatibility was obtained for the HAP/CS/HA-Col composite. The collagenase release profile had facilitated the acceleration of bone remodeling process and it was confirmed by the findings of micro-CT and H&E staining. The bone defects implanted with HAP/CS/HA composite containing 2 mg/mL type I collagenase have shown improved new bone formation with matured bone morphology in comparison with the HAP/CS/HA composite that lacks the collagenase and the porous hydroxyapatite (p-HAP) granules. The said findings demonstrated that the collagenase inclusion in HAP/CS/HA composite is a feasible approach for the alveolar bone regeneration and the same design can also be applied to other defective tissues. PMID:26454048

  15. FT-IR, Raman and DFT study of 2-amino-5-fluorobenzoic acid and its biological activity with other halogen (Cl, Br) substitution

    NASA Astrophysics Data System (ADS)

    Xavier, T. S.; Hubert, Joe I.

    2011-07-01

    The Fourier-transform Raman and infrared spectra of 2-amino-5-fluoro benzoic acid has been recorded and analyzed. The optimized geometry of the other halogen substitution (Cl, Br) have been computed with the help of density functional theory. The detailed interpretation of vibrational spectra of 2-amino-5-fluoro benzoic acid have performed in terms of potential energy distribution analysis. Natural bond orbital analysis on 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid has been carried out for various intramolecular interactions that are responsible for the stabilization of the molecule. The p Ka values of 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid are computed using MOPAC and it is related with HOMO-LUMO energy difference obtained from Gaussian 03 software. The biological activity of 2-amino-5-fluoro benzoic acid has been predicted based on these values. The inhibition activity of 2-amino-5-bromo benzoic acid with the protein tyrosine kinase 3LQ8 is simulated by using Autodock software.

  16. FT-IR, Raman and DFT study of 2-amino-5-fluorobenzoic acid and its biological activity with other halogen (Cl, Br) substitution.

    PubMed

    Xavier, T S; Joe, I Hubert

    2011-07-01

    The Fourier-transform Raman and infrared spectra of 2-amino-5-fluoro benzoic acid has been recorded and analyzed. The optimized geometry of the other halogen substitution (Cl, Br) have been computed with the help of density functional theory. The detailed interpretation of vibrational spectra of 2-amino-5-fluoro benzoic acid have performed in terms of potential energy distribution analysis. Natural bond orbital analysis on 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid has been carried out for various intramolecular interactions that are responsible for the stabilization of the molecule. The pKa values of 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid are computed using MOPAC and it is related with HOMO-LUMO energy difference obtained from Gaussian 03 software. The biological activity of 2-amino-5-fluoro benzoic acid has been predicted based on these values. The inhibition activity of 2-amino-5-bromo benzoic acid with the protein tyrosine kinase 3LQ8 is simulated by using Autodock software. PMID:21497545

  17. The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

    PubMed Central

    Buhrke, David; Velazquez Escobar, Francisco; Sauthof, Luisa; Wilkening, Svea; Herder, Nico; Tavraz, Neslihan N.; Willoweit, Mario; Keidel, Anke; Utesch, Tillmann; Mroginski, Maria-Andrea; Schmitt, Franz-Josef; Hildebrandt, Peter; Friedrich, Thomas

    2016-01-01

    Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores. PMID:27329837

  18. Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels.

    PubMed

    Guo, Kai; Wang, Xianming; Gao, Guofeng; Huang, Congxin; Elmslie, Keith S; Peterson, Blaise Z

    2010-11-01

    We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart. PMID:20720179

  19. TREATOGENIC ACTIVITY OF TRICHOLORACETIC ACID IN THE RATE

    EPA Science Inventory

    Trichloroacetic acid (TCA) is a by-product of the chlorine disinfection of water containing natural organic material. t is detectable in finished drinking water at levels comparable to the trihalomethanes (930-160 ug/L). CA is also formed in vivo after ingestion of hypochelorite ...

  20. Amino acid-based enantiomerically pure 3-substituted 1,4-benzodiazepin-2-ones: a new class of anti-ischemic agents.

    PubMed

    Mishra, Jitendra Kumar; Garg, Puja; Dohare, Preeti; Kumar, Ashutosh; Siddiqi, Mohammad Imran; Ray, Madhur; Panda, Gautam

    2007-03-01

    A series of 3-substituted 1,4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ischemic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling studies. PMID:17178221

  1. Bioresorbable bone graft substitutes of different osteoconductivities: a histologic evaluation of osteointegration of poly(propylene glycol-co-fumaric acid)-based cement implants in rats.

    PubMed

    Lewandrowski, K U; Gresser, J D; Wise, D L; Trantol, D J

    2000-04-01

    Bioresorbable bone graft substitutes may significantly reduce the disadvantages associated with autografts, allografts and other synthetic materials currently used in bone graft procedures. We investigated the biocompatibility and osteointegration of a bioresorbable bone graft substitute made from the unsaturated polyester poly(propylene-glycol-co-fumaric acid), or simply poly(propylene fumarate), PPF, which is crosslinked in the presence of soluble and insoluble calcium filler salts. Four sets of animals each having three groups of 8 were evaluated by grouting bone graft substitutes of varying compositions into 3-mm holes that were made into the anteromedial tibial metaphysis of rats. Four different formulations varying as to the type of soluble salt filler employed were used: set 1--calcium acetate, set 2--calcium gluconate, set 3--calcium propionate, and set 4--control with hydroxapatite, HA, only. Animals of each of the three sets were sacrificed in groups of 8 at postoperative week 1, 3, and 7. Histologic analysis revealed that in vivo biocompatibility and osteointegration of bone graft substitutes was optimal when calcium acetate was employed as a soluble salt filler. Other formulations demonstrated implant surface erosion and disintegration which was ultimately accompanied by an inflammatory response. This study suggested that PPF-based bone graft substitutes can be designed to provide an osteoconductive pathway by which bone will grow in faster because of its capacity to develop controlled porosities in vivo. Immediate applicability of this bone graft substitute, the porosity of which can be tailored for the reconstruction of defects of varying size and quality of the recipient bed, is to defects caused by surgical debridement of infections, previous surgery, tumor removal, trauma, implant revisions and joint fusion. Clinical implications of the relation between developing porosity, resulting osteoconduction, and bone repair in vivo are discussed. PMID

  2. Amino acid substitutions in malate dehydrogenases of piezophilic bacteria isolated from intestinal contents of deep-sea fishes retrieved from the abyssal zone.

    PubMed

    Saito, Rie; Kato, Chiaki; Nakayama, Akihiko

    2006-02-01

    To examine the occurrence in other deep-sea bacteria of two amino acid substitutions (Ala-180 and His-229) in malate dehydrogenase (MDH) found previously in the deep-sea piezophilic Moritella sp. strain 2D2, we cloned and sequenced MDH genes of deep-sea piezophilic Moritella and Shewanella strains isolated from intestinal contents of deep-sea fishes, as well as other Moritella species from deep-sea water and sediments: M. marina, M. japonica, and M. yayanosii. The piezophilic Moritella strains had a Val residue or an Ala residue at position 180 and all the Moritella strains except for one had a His residue at position 229. However, four piezophilic-strain-specific substitutions at positions 103, 111, 229, and 283 were found to be completely conserved in the MDH of the intestinal Moritella strains of deep-sea fishes, indicating the substitutions may be habitat-specific. The piezophilic Shewanella strains had a Val residue and a Gln residue at positions 180 and 229, respectively. However, the MDHs of the Shewanella strains had five piezophilic-strain-specific substitutions at positions 61, 65, 107, 161, and 202. Therefore, the enzymatic strategies for responding to deep-sea high pressure environments of the MDHs between the genera Moritella and Shewanella are potentially different. Moreover, homology modeling shows these substitutions found in the MDHs of both genera except for position 229 in the subunit interface are located on the exposed region of the MDH molecules, indicating the substitutions may be related to the hydration state of the molecules. PMID:16598154

  3. Accelerated hydrolysis of substituted cellulose for potential biofuel production: kinetic study and modeling.

    PubMed

    Mu, Bingnan; Xu, Helan; Yang, Yiqi

    2015-11-01

    In this work, kinetics of substitution accelerated cellulose hydrolysis with multiple reaction stages was investigated to lay foundation for mechanism study and molecular design of substituting compounds. High-efficiency hydrolysis of cellulose is critical for cellulose-based bioethanol production. It is known that, substitution could substantially decrease activation energy and increase reaction rate of acidic hydrolysis of glycosidic bonds in cellulose. However, reaction kinetics and mechanism of the accelerated hydrolysis were not fully revealed. In this research, it was proved that substitution therefore accelerated hydrolysis only occurred in amorphous regions of cellulose fibers, and was a process with multiple reaction stages. With molar ratio of substitution less than 1%, the overall hydrolysis rate could be increased for around 10 times. We also quantified the relationship between the hydrolysis rate of individual reaction stage and its major influences, including molar ratio of substitution, activation energy of acidic hydrolysis, pH and temperature. PMID:26253917

  4. A review of molecular-clock calibrations and substitution rates in liverworts, mosses, and hornworts, and a timeframe for a taxonomically cleaned-up genus Nothoceros.

    PubMed

    Villarreal, Juan Carlos; Renner, Susanne S

    2014-09-01

    Absolute times from calibrated DNA phylogenies can be used to infer lineage diversification, the origin of new ecological niches, or the role of long distance dispersal in shaping current distribution patterns. Molecular-clock dating of non-vascular plants, however, has lagged behind flowering plant and animal dating. Here, we review dating studies that have focused on bryophytes with several goals in mind, (i) to facilitate cross-validation by comparing rates and times obtained so far; (ii) to summarize rates that have yielded plausible results and that could be used in future studies; and (iii) to calibrate a species-level phylogeny for Nothoceros, a model for plastid genome evolution in hornworts. Including the present work, there have been 18 molecular clock studies of liverworts, mosses, or hornworts, the majority with fossil calibrations, a few with geological calibrations or dated with previously published plastid substitution rates. Over half the studies cross-validated inferred divergence times by using alternative calibration approaches. Plastid substitution rates inferred for "bryophytes" are in line with those found in angiosperm studies, implying that bryophyte clock models can be calibrated either with published substitution rates or with fossils, with the two approaches testing and cross-validating each other. Our phylogeny of Nothoceros is based on 44 accessions representing all suspected species and a matrix of six markers of nuclear, plastid, and mitochondrial DNA. The results show that Nothoceros comprises 10 species, nine in the Americas and one in New Zealand (N. giganteus), with the divergence between the New Zealand species and its Chilean sister species dated to the Miocene and therefore due to long-distance dispersal. Based on the new tree, we formally transfer two species of Megaceros into Nothoceros, resulting in the new combinations N. minarum (Nees) J.C. Villarreal and N. schizophyllus (Gottsche ex Steph.) J.C. Villarreal, and we also

  5. Water evaporation rates across hydrophobic acid monolayers at equilibrium spreading pressure.

    PubMed

    Tsuji, Minami; Nakahara, Hiromichi; Moroi, Yoshikiyo; Shibata, Osamu

    2008-02-15

    The effect of alkanoic acid [CH(3)(CH(2))(n-2)COOH; HCn] and perfluoroalkanoic acid [CF(3)(CF(2))(n-2)COOH; FCn] monolayers on the water evaporation rate was investigated by thermogravimetry tracing the decrease in amount of water with time. The evaporation rate from the surface covered by a monolayer was measured as a function of temperature and hydrophobic chain length of the acids, where the monolayer was under an equilibrium spreading pressure. From thermal behavior of the crystallized acids, their solid states are C-type in crystalline state over the temperature range from 298.2 to 323.2 K. The dry air was flowed through a furnace tube of a thermogravimetry apparatus at the flow rate of 80 mL min(-1), where the evaporation rate becomes almost constant irrespective of the flow rate. The temperature dependence of the evaporation rate was analyzed kinetically to evaluate the activation energy and thermodynamics values for the activated complex, which demonstrated that these values were almost the same for both alkanoic acids and perfluoroalkanoic acids, although the effect of perfluoroalkanoic acids on the evaporation rate was smaller than that of corresponding hydrogenated fatty acids. The difference in the evaporation rate between FCn and HCn was examined by atomic force microscopy (AFM), Brewster angle microscopy (BAM), surface potential (DeltaV) at equilibrium spreading pressure, and Langmuir curve (pi-A isotherm), and their results were consistent and supported the difference. PMID:18048050

  6. Relationship between the electrochemical activity of Raney nickel and the rate of hydrogenation of maleic acid

    SciTech Connect

    Pervii, E.N.; Sofronkov, A.N.; Fedyshina, N.M.

    1986-02-10

    The purpose of this investigation was to determine the conditions in which a direct correlation exists between the rate of hydrogenation of maleic acid and the electrochemical activity of catalysts of hydrogen ionization. The rate of maleic acid hydrogenation in presence of Raney nickel catalyst was studied by a combination of volumetric and potentiometric methods.

  7. The effect of linoleic acid on the whole body synthesis rates of polyunsaturated fatty acids from α-linolenic acid and linoleic acid in free-living rats.

    PubMed

    Domenichiello, Anthony F; Kitson, Alex P; Chen, Chuck T; Trépanier, Marc-Olivier; Stavro, P Mark; Bazinet, Richard P

    2016-04-01

    Docosahexaenoic acid (DHA) is thought to be important for brain function. The main dietary source of DHA is fish, however, DHA can also be synthesized from precursor omega-3 polyunsaturated fatty acids (n-3 PUFA), the most abundantly consumed being α-linolenic acid (ALA). The enzymes required to synthesize DHA from ALA are also used to synthesize longer chain omega-6 (n-6) PUFA from linoleic acid (LNA). The large increase in LNA consumption that has occurred over the last century has led to concern that LNA and other n-6 PUFA outcompete n-3 PUFA for enzymes involved in DHA synthesis, and therefore, decrease overall DHA synthesis. To assess this, rats were fed diets containing LNA at 53 (high LNA diet), 11 (medium LNA diet) or 1.5% (low LNA diet) of the fatty acids with ALA being constant across all diets (approximately 4% of the fatty acids). Rats were maintained on these diets from weaning for 8 weeks, at which point they were subjected to a steady-state infusion of labeled ALA and LNA to measure DHA and arachidonic acid (ARA) synthesis rates. DHA and ARA synthesis rates were generally highest in rats fed the medium and high LNA diets, while the plasma half-life of DHA was longer in rats fed the low LNA diet. Therefore, increasing dietary LNA, in rats, did not impair DHA synthesis; however, low dietary LNA led to a decrease in DHA synthesis with tissue concentrations of DHA possibly being maintained by a longer DHA half-life. PMID:27012633

  8. Measurement of the rates of oxindole-3-acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings

    NASA Technical Reports Server (NTRS)

    Nonhebel, H. M.; Bandurski, R. S. (Principal Investigator)

    1986-01-01

    Oxindole-3-acetic acid is the principal catabolite of indole-3-acetic acid in Zea mays seedlings. In this paper measurements of the turnover of oxindole-3-acetic acid are presented and used to calculate the rate of indole-3-acetic acid oxidation. [3H]Oxindole-3-acetic acid was applied to the endosperm of Zea mays seedlings and allowed to equilibrate for 24 h before the start of the experiment. The subsequent decrease in its specific activity was used to calculate the turnover rate. The average half-life of oxindole-3-acetic acid in the shoots was found to be 30 h while that in the kernels had an average half-life of 35h. Using previously published values of the pool sizes of oxindole-3-acetic acid in shoots and kernels from seedlings of the same age and variety, and grown under the same conditions, the rate of indole-3-acetic acid oxidation was calculated to be 1.1 pmol plant-1 h-1 in the shoots and 7.1 pmol plant-1 h-1 in the kernels.

  9. Amino acid residue Y196E substitution and C-terminal peptide synergistically alleviate the toxicity of Clostridium perfringens epsilon toxin.

    PubMed

    Yao, Wenwu; Kang, Lin; Gao, Shan; Zhuang, Xiangjin; Zhang, Tao; Yang, Hao; Ji, Bin; Xin, Wenwen; Wang, Jinglin

    2015-06-15

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains, and is the causative agent of a lethal enterotoxemia in livestock animals and possibly in humans. However, many details of ETX structure and activity are not known. Therefore, it is important to clarify the relationship between ETX structure and activity. To explore the effect and mechanism of ETX amino acid residue Y196E substitution and C-terminal peptide on toxicity, four recombinant proteins, rETX (without 13 N-terminal peptides and 23 C-terminal peptides), rETX-C (rETX with 23 C-terminal peptides), rETX(Y196E) (rETX with an amino acid residue substitution at Y196) and rETX(Y196E)-C (rETX-C with a Y196E mutation), were constructed in this study. Both the amino acid residue Y196E substitution and the C-terminal peptide reduce ETX toxicity to a similar extent, and the two factors synergistically alleviate ETX toxicity. In addition, we demonstrated that the C-terminal peptides and Y196E amino acid mutation reduce the toxin toxicity in two different pathways: the C-terminal peptides inhibit the binding activity of toxins to target cells, and the Y196E amino acid mutation slightly inhibits the pore-forming or heptamer-forming process. Interaction between the two factors was not observed in pore-forming or binding assays but toxicity assays, which demonstrated that the relationship between domains of the toxin is more complicated than previously appreciated. However, the exact mechanism of synergistic action is not yet clarified. PMID:25912943

  10. The effect of organic acids on plagioclase dissolution rates and stoichiometry

    NASA Astrophysics Data System (ADS)

    Welch, Susan A.; Ullman, William J.

    1993-06-01

    The rates of plagioclase dissolution in solutions containing organic acids are up to ten times greater than the rates determined in solutions containing inorganic acids at the same acidity. Initial rates of dissolution are poorly reproduced in replicate experiments. After a day, however, the rates of plagioclase dissolution calculated from the rates of silicon release are reproducible and constant for up to nineteen days. Steady-state rates of dissolution are highest (up to 1.3 × 10 -8 mol/m 2/sec) in acidic solutions (pH ≈ 3) and decrease (to 1 × 10 -11 mol/m 2/sec) as acidity decreases toward neutral pH. The polyfunctional acids, oxalate, citrate, succinate, pyruvate, and 2-ketoglutarate, are the most effective at promoting dissolution. Acetate and propionate are not as effective as the other organic acids but are nonetheless more effective than solutions containing only inorganic acids. The degree of ligand-promoted enhancement of dissolution rate (rate in organic-containing solution/rate in inorganic solution at the same pH) decreases as acidity increases, indicating that the ligand-promoted dissolution mechanism becomes relatively more important as the rate of proton-promoted dissolution decreases. The stoichiometry of release to solution indicates that dissolution is selective even after the rates of dissolution become constant. As in previously published studies, Na and Ca are rapidly released from the plagioclase feldspar, leaving a surface enriched in Si and/or Al. The ratio of Al/Si released to solution indicates that the stoichiometry of the residual plagioclase surface is a function of pH and the nature of the organic ligand. The ligands which remove Al in preference to Si from the dissolving mineral surface are also those which enhance overall plagioclase dissolution rates.

  11. Determination of small halogenated carboxylic acid residues in drug substances by high performance liquid chromatography-diode array detection following derivatization with nitro-substituted phenylhydrazines.

    PubMed

    Hou, Desheng; Fan, Jingjing; Han, Lingfei; Ruan, Xiaoling; Feng, Feng; Liu, Wenyuan; Zheng, Feng

    2016-03-18

    A method for the determination of small halogenated carboxylic acid (HCA) residues in drug substances is urgently needed because of the potential of HCAs for genotoxicity and carcinogenicity in humans. We have now developed a simple method, involving derivatization followed by high performance liquid chromatography-diode array detection (HPLC-DAD), for the determination of six likely residual HCAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, 2-chloropropionic acid, 2-bromopropionic acid and 3-chloropropionic acid) in drug substances. Different nitro-substituted phenylhydrazines (NPHs) derivatization reagents were systematically compared and evaluated. 2-Nitrophenylhydrazine hydrochloride (2-NPH·HCl) was selected as the most suitable choice since its derivatives absorb strongly at 392 nm, a region of the spectrum where most drug substances and impurities absorb very weakly. During the derivatization process, the commonly used catalyst, pyridine, caused rapid dechlorination or chlorine substitution of α-halogenated derivatives. To avoid these unwanted side reactions, a reliable derivatization method that did not use pyridine was developed. Reaction with 2-NPH·HCl using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as coupling agent in acetonitrile-water (70:30) at room temperature for 2h gave complete reaction and avoided degradation products. The derivatives were analyzed, without any pretreatment, using gradient HPLC with detection in the near visible region. Organic acids commonly found in drug substances and other impurities did not interfere with the analysis. Good linearity (r>0.999) and low limits of quantitation (0.05-0.12 μg mL(-1)) were obtained. The mean recoveries were in the range of 80-115% with RSD <5.81% except for 3-CPA in ibuprofen which was 78.5%. The intra- and inter-day precisions were expressed as RSD <1.98% and <4.39%, respectively. Finally, the proposed method was successfully used for the residue

  12. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    SciTech Connect

    Zhang, Xinsheng; Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan; Anzala, Omu; Sanders, Eduard J.; Kamali, Anatoli; Allen, Susan; Fast, Pat; Gilmour, Jill; Price, Matt A.; Parks, Christopher L.

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  13. Sensory evaluation ratings and melting characteristics show that okra gum is an acceptable milk-fat ingredient substitute in chocolate frozen dairy dessert.

    PubMed

    Romanchik-Cerpovicz, Joelle E; Costantino, Amanda C; Gunn, Laura H

    2006-04-01

    Reducing dietary fat intake may lower the risk of developing coronary heart disease. This study examined the feasibility of substituting okra gum for 25%, 50%, 75%, or 100% milk fat in frozen chocolate dairy dessert. Fifty-six consumers evaluated the frozen dairy desserts using a hedonic scale. Consumers rated color, smell, texture, flavor, aftertaste, and overall acceptability characteristics of all products as acceptable. All ratings were similar among the products except for the aftertaste rating, which was significantly lower for chocolate frozen dairy dessert containing 100% milk-fat replacement with okra gum compared with the control (0% milk-fat replacement) (P<0.05). Whereas melting points of all products were similar, melting rates slowed significantly as milk-fat replacement with okra gum increased, suggesting that okra gum may increase the stability of frozen dairy desserts (P<0.05). Overall, this study shows that okra gum is an acceptable milk-fat ingredient substitute in chocolate frozen dairy dessert. PMID:16567157

  14. Dissolution rates of carbonated hydroxyapatite in hydrochloric acid.

    PubMed

    Hankermeyer, Christine R; Ohashi, Kevin L; Delaney, David C; Ross, John; Constantz, Brent R

    2002-02-01

    Osteoclasts have been shown to dissolve efficiently and effectively the mineral phase of bone by locally controlling the environment surrounding the cell. Although this mineral phase has been identified and well characterized as carbonated hydroxyapatite, there is little understanding of the factors that affect the dissolution properties of this mineral phase. Mimicking the mechanism by which osteoclasts dissolve the mineral phase of bone may provide insight into methods for the decalcification of atherosclerotic mineral deposits in the vascular system. Accordingly, a detailed characterization of the effects of various chemical and mechanical parameters on the dissolution of carbonated hydroxyapatite mineral was investigated in this study. Increases in the mineral dissolution rate (2-10 times) were associated with increases in dissolving solution [H+], osmolality, temperature, and flow rate. Mineral dissolution rate increases (5-8 times) were associated with greater surface area of the mineral and mechanical agitation of the dissolving solution. PMID:11771694

  15. Modulation of internal conversion rate and nonlinear absorption in meso-tetraphenylporphyrins by donor/acceptor substitutes

    NASA Astrophysics Data System (ADS)

    Ao, Guanghong; Qian, Xuemin; Xiao, Zhengguo; Li, Zhongguo; Nie, Zhongquan; Wang, Yuxiao; Zhang, Xueru; Song, Yinglin

    2015-08-01

    Meso-tetraphenylporphyrin (TPP), (meso-tetrakis(4-cyanophenyl)porphyrin [TPP(CN)4)] and (meso-tetrakis(4-methoxyphenyl)porphyrin [TPP(OMe)4]) are synthesized. The donor methoxy substitute (OMe) and acceptor cyano substitute (CN) are introduced into TPP in order to examine their influence on the photophysical properties of TPP. The nonlinear absorption properties of these three porphyrins are studied using open-aperture Z-scan and time-resolved pump-probe techniques in picosecond and nanosecond regimes, and the results are elucidated successfully by a five-level model. As compared to TPP, TPP(OMe)4 exhibits an accelerated internal conversion from S1 to S0 state (0.02 ns), and S1 to T1 state (0.03 ns). Moreover, a changeover from saturable absorption to reverse saturable absorption is observed in TPP(OMe)4 in the ps regime. Whereas the nonlinear absorption behavior of TPP(CN)4 is similar to that of TPP. The results imply that these two substituents, especially the OMe group, could modulate the photophysical properties of TPP, which may provide a useful option for porphyrin-related applications.

  16. Potential of a cryopreserved cultured dermal substitute composed of hyaluronic acid and collagen to release angiogenic cytokine.

    PubMed

    Sawa, Manami; Kuroyanagi, Yoshimitsu

    2013-01-01

    An allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a spongy matrix of hyaluronic acid (HA) and collagen (Col), which was then cryopreserved. This cryopreserved allogeneic CDS (CDS-1; cryopreserved for 1 month, CDS-6; cryopreserved for 6 months) was thawed and re-cultured for a period of 7 days to investigate the potential of the CDS for wound treatment. The cell metabolic activity in the CDS and their cytokine production were measured using an MTT assay and ELISA. Fibroblast metabolic activity in each CDS-1 and CDS-6 immediately after thawing and following 3 and 7 days of re- cultivation was 56, 67 and 93%, and 49, 64 and 86%, respectively, of that before cryopreservation. The amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released from the CDS-1 on days 1, 3 and 7 of re-cultivation after thawing was 8, 44 and 92% (VEGF) and 3, 7 and 28% (HGF), respectively, of that before cryopreservation. The amount of VEGF and HGF released from the CDS-6 on days 1, 3 and 7 of re-cultivation after thawing was 9, 32 and 45% (VEGF) and 6, 10 and 27% (HGF), respectively, of that before cryopreservation. These findings showed that the potential of the CDS was restored to some extent over the first 3 days of re-cultivation after thawing. The potential of the CDS for wound treatment was then evaluated using a wound surface model, in which the each CDS-1 and CDS-6 that was re-cultured for 3 days after thawing was elevated at the air/culture medium interface, and a wound dressing was placed on top, and then cultured for 5 days. Two different types of wound dressing were tested. Fibroblasts in the CDS in Group II (placing a wound dressing with EGF) released increased amount of VEGF and HGF compared with that in Group I (placing a wound dressing without EGF). These findings suggest that re-culture of the CDS for 3 days following thawing results in a CDS with improved wound healing potential and that an EGF

  17. Differences in the relative myocardial/organ ratios of iodine-123-BMIPP and the dimethyl-substituted iodine 123-DMIPP fatty acid analogue in humans

    SciTech Connect

    Sloof, G.W.; Comans, E.F.I.; Visser, F.C.

    1997-05-01

    Radioiodinated fatty acid analogues, modified by methyl-substitution are used for SPECT imaging of the heart. The effect of mono- and dimethyl-substitution on biodistribution was investigated in humans to evaluate their relative merits for SPECT image quality. Planar total body scans were performed in fasting patients with coronary artery disease, but without heart failure, one hour after administration of 111 MBq 15-(p-[I-123]-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP, n=7) or III MBq 15-(p-[I-123]-iodophenyl)-3,3-dimethylpentaderanoic acid (DMIPP, n=4). Because these branched fatty acids are used for cardiac imaging, we focussed on heart/organ ratios, by comparing small roi-counts in heart, liver, lung, muscle (thigh) and bladder. Statistical analysis: t-test for unpaired data. Both tracers showed good visualization of the heart. While DMIPP showed a relatively high liver uptake, increased background, ie lung, activity was found for BMIPP. In contrast to DMIPP, BMIPP also showed elevated activity in the bladder.

  18. Influence of chlorine substitution on intramolecular hydrogen bond energy and ESIPT barrier: Experimental and theoretical measurements on the photophysics of 3,5-dichlorosalicylic acid

    NASA Astrophysics Data System (ADS)

    Paul, Bijan Kumar; Samanta, Anuva; Guchhait, Nikhil

    2010-08-01

    The effect of chlorine atom on the intramolecular hydrogen bond strength and excited state proton transfer barrier in pharmaceutically important chloro-substituted derivative of salicylic acid viz., 3,5-dichlorosalicylic acid (3,5DCSA) has been explored through steady-state absorption, emission and time-resolved fluorescence spectroscopy. Stokes shifted emission band with negligible solvent polarity dependency corresponds to the spectroscopic signature of excited state intramolecular proton transfer (ESIPT) reaction. The spectral signature was compared with its parent molecule salicylic acid (SA) and 5-chlorosalicylic acid (5ClSA). Quantum chemical calculations by ab initio Hartree-Fock (HF) and Density Functional Theory (DFT) methods have been fruitfully employed to correlate experimental findings. Calculated S0 and S1 states potential energy surfaces across the proton transfer co-ordinate substantiates the experimental evidence for the occurrence of ESIPT process and negates the ground state intramolecular proton transfer (GSIPT) reaction. Weakening of intramolecular hydrogen bond (IMHB) energy and subsequent enhancement of barrier to ESIPT reaction in 3,5DCSA as compared to SA and 5ClSA appears to be a reflection of conjugate impact of electron withdrawing inductive and electron donating resonance effects of chlorine substitutions depending on its location on the aromatic benzene nucleus.

  19. Structural and clinical implications of amino acid substitutions in α-L-iduronidase: insight into the basis of mucopolysaccharidosis type I.

    PubMed

    Saito, Seiji; Ohno, Kazuki; Maita, Nobuo; Sakuraba, Hitoshi

    2014-02-01

    Allelic mutations, predominantly missense ones, of the α-l-iduronidase (IDUA) gene cause mucopolysaccharidosis type I (MPS I), which exhibits heterogeneous phenotypes. These phenotypes are basically classified into severe, intermediate, and attenuated types. We previously examined the structural changes in IDUA due to MPS I by homology modeling, but the reliability was limited because of the low sequence identity. In this study, we built new structural models of mutant IDUAs due to 57 amino acid substitutions that had been identified in 27 severe, 1 severe-intermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients based on the crystal structure of human IDUA, which was recently determined by us. The structural changes were examined by calculating the root-mean-square distances (RMSD) and the number of atoms influenced by the amino acid replacements. The results revealed that the structural changes of the enzyme protein tended to be correlated with the severity of the disease. Then we focused on the structural changes resulting from amino acid replacements in the immunoglobulin-like domain and adjacent region, of which the structure had been missing in the IDUA model previously built. Coloring of atoms influenced by an amino acid substitution was performed in each case and the results revealed that the structural changes occurred in a region far from the active site of IDUA, suggesting that they affected protein folding. Structural analysis is thus useful for elucidation of the basis of MPS I. PMID:24480078

  20. Substitution of DNA-Contacting Amino Acids with Functional Variants in the Gata-1 Zinc Finger: A Structurally and Phylogenetically Guided Mutagenesis

    PubMed Central

    Vonderfecht, Tyson R.; Schroyer, Daniel L.; Schenck, Brandy L.; McDonough, Virginia M.; Pikaart, Michael J.

    2008-01-01

    DNA binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)4 metal binding sites which recognize the protein’s eponymous binding site, GATA. Unlike other conserved DNA binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence specific DNA binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3’ and 5’ flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers. PMID:18328814

  1. Acid-catalyzed synthesis of 10-substituted triazolyl artemisinins and their growth inhibitory activity against various cancer cells.

    PubMed

    Oh, Sangtae; Shin, Woon-Seob; Ham, Jungyeob; Lee, Seokjoon

    2010-07-15

    A diastereomeric and regioisomeric library of 10-substituted triazolyl artemisinin compounds (6a-6h, 7a-7h, and 8a-8h) with a potent growth inhibitory activities against various cancer cell lines was established. These compounds were synthesized by a reaction with dihydroartemisinin (2) and various substituted triazoles (5a-5h) in methylene chloride using a BF(3)Et(2)O catalyst. Most of the compounds exhibited a strong potency in the submicromolar range, and, in particular, 6f, 7f, and 8f, which have a pentylphenyltriazole moiety, proved to be promising candidates for preclinical trials. PMID:20538462

  2. Enhancement of the Luminance Efficiency in Organic Light-Emitting Devices with p-Substituted Phenylphosphonic-Acid Self-Assembled Monolayers.

    PubMed

    Kim, Min Sung; Jeon, Young Pyo; Kim, Youngwoo; Noh, Jaegeun; Kim, Tae Whan

    2015-07-01

    Organic light-emitting devices (OLEDs) containing self-assembled monolayers (SAMs) prepared by using p-substituted phenylphosponic acids on indium-tin-oxide electrodes were fabricated and examined to understand the substituent effect of the SAMs on the device performance. OLEDs modified by using (4-methoxyphenyl)phosphonic acid (MOPPA) SAMs or (4-chlorophenyl)phosphonic acid (CPPA) SAMs, both with electron withdrawing groups, had enhanced hole injection, reduced operating voltage, and remarkably increased current density and luminance efficiency compared with those without SAMs. The luminance efficiency which was the ratio of luminous flux to power for OLEDs containing CPPA SAMs and that for the OLEDs containing MOPPA SAMs were enhanced 2.2 and 1.9 times, respectively, in comparison with that of OLEDs without SAMs. CPPA SAMs significantly reduced the operating voltage of OLED by 24.8% compared with OLEDs without SAMs. PMID:26373078

  3. Palladium-catalyzed C–N and C–O bond formation of N-substituted 4-bromo-7-azaindoles with amides, amines, amino acid esters and phenols

    PubMed Central

    Surasani, Rajendra; Rao, A V Dhanunjaya; Chandrasekhar, K B

    2012-01-01

    Summary Simple and efficient procedures for palladium-catalyzed cross-coupling reactions of N-substituted 4-bromo-7-azaindole (1H-pyrrole[2,3-b]pyridine), with amides, amines, amino acid esters and phenols through C–N and C–O bond formation have been developed. The C–N cross-coupling reaction of amides, amines and amino acid esters takes place rapidly by using the combination of Xantphos, Cs2CO3, dioxane and palladium catalyst precursors Pd(OAc)2/Pd2(dba)3. The combination of Pd(OAc)2, Xantphos, K2CO3 and dioxane was found to be crucial for the C–O cross-coupling reaction. This is the first report on coupling of amides, amino acid esters and phenols with N-protected 4-bromo-7-azaindole derivatives. PMID:23209536

  4. A single amino-acid substitution toggles chloride dependence of the alpha-amylase paralog amyrel in Drosophila melanogaster and Drosophila virilis species.

    PubMed

    Claisse, Gaëlle; Feller, Georges; Bonneau, Magalie; Da Lage, Jean-Luc

    2016-08-01

    In animals, most α-amylases are chloride-dependent enzymes. A chloride ion is required for allosteric activation and is coordinated by one asparagine and two arginine side chains. Whereas the asparagine and one arginine are strictly conserved, the main chloride binding arginine is replaced by a glutamine in some rare instances, resulting in the loss of chloride binding and activation. Amyrel is a distant paralogue of α-amylase in Diptera, which was not characterized biochemically to date. Amyrel shows both substitutions depending on the species. In Drosophila melanogaster, an arginine is present in the sequence but in Drosophila virilis, a glutamine occurs at this position. We have investigated basic enzymological parameters and the dependence to chloride of Amyrel of both species, produced in yeast, and in mutants substituting arginine to glutamine or glutamine to arginine. We found that the amylolytic activity of Amyrel is about thirty times weaker than the classical Drosophila α-amylase, and that the substitution of the arginine by a glutamine in D. melanogaster suppressed the chloride-dependence but was detrimental to activity. In contrast, changing the glutamine into an arginine rendered D. virilis Amyrel chloride-dependent, and interestingly, significantly increased its catalytic efficiency. These results show that the chloride ion is not mandatory for Amyrel but stimulates the reaction rate. The possible phylogenetic origin of the arginine/glutamine substitution is also discussed. PMID:27312592

  5. Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge

    PubMed Central

    Ashok raj, Kattur Venkatachalam; He, Fang; Kwang, Jimmy

    2015-01-01

    Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against

  6. Synthesis of densely substituted 1,3-butadienes through acid-catalyzed alkenylations of α-oxoketene dithioacetals with aldehydes.

    PubMed

    Liu, Changhui; Gu, Yanlong

    2014-10-17

    Aldehydes were proved to be viable reagents for implementing alkenylation of α-oxoketene dithioacetals. AlCl3 was found to be the best catalyst. The established reaction opened an avenue to access densely substituted 1,3-butadiene derivatives. The obtained product bears multiple reactive sites that can be converted into various valuable molecules. PMID:25247719

  7. Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.

    PubMed

    Fukuda, Yasuhisa; Abe, Asuka; Tamura, Takashi; Kishimoto, Takahide; Sogabe, Atsushi; Akanuma, Satoshi; Yokobori, Shin-Ichi; Yamagishi, Akihiko; Imada, Katsumi; Inagaki, Kenji

    2016-05-01

    Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. PMID:26493633

  8. A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice▿

    PubMed Central

    Jiao, Peirong; Tian, Guobin; Li, Yanbing; Deng, Guohua; Jiang, Yongping; Liu, Chang; Liu, Weilong; Bu, Zhigao; Kawaoka, Yoshihiro; Chen, Hualan

    2008-01-01

    In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell. PMID:18032512

  9. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    SciTech Connect

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  10. Liquid-Phase Heat-Release Rates of the Systems Hydrazine-Nitric Acid and Unsymmetrical Dimethylhydrazine-Nitric Acid

    NASA Technical Reports Server (NTRS)

    Somogyi, Dezso; Feiler, Charles E.

    1960-01-01

    The initial rates of heat release produced by the reactions of hydrazine and unsymmetrical dimethylhydrazine with nitric acid were determined in a bomb calorimeter under conditions of forced mixing. Fuel-oxidant weight ratio and injection velocity were varied. The rate of heat release apparently depended on the interfacial area between the propellants. Above a narrow range of injection velocities representing a critical amount of interfacial area, the rates reached a maximum and were almost constant with injection velocity. The maximum rate for hydrazine was about 70 percent greater than that for unsymmetrical dimethylhydrazine. The total heat released did not vary with mixture ratio over the range studied.

  11. Amino acid substitutions in the hepatitis C virus core region of genotype 1b are the important predictor of severe insulin resistance in patients without cirrhosis and diabetes mellitus.

    PubMed

    Akuta, Norio; Suzuki, Fumitaka; Hirakawa, Miharu; Kawamura, Yusuke; Yatsuji, Hiromi; Sezaki, Hitomi; Suzuki, Yoshiyuki; Hosaka, Tetsuya; Kobayashi, Masahiro; Kobayashi, Mariko; Saitoh, Satoshi; Arase, Yasuji; Ikeda, Kenji; Kumada, Hiromitsu

    2009-06-01

    Previous studies provided a direct experimental evidence for the contribution of HCV core protein in the development of insulin resistance (IR), but the clinical impact of HCV core region on IR is still not clear. The present study evaluated the impact of Amino acid (aa) substitutions of HCV-1b core region on IR in 123 Japanese patients infected with HCV-1b without cirrhosis and diabetes mellitus, and investigated the treatment efficacy of 48-week pegylated interferon (PEG-IFN) plus ribavirin (RBV) according to HOMA-IR values. Patients with IR (HOMA-IR > or = 2.5) and severe IR (HOMA-IR > or = 3.5) were present in 51.2% and 27.6%, respectively. Multivariate analysis identified body mass index (> or = 25 kg/m(2)) and hepatocyte steatosis (> or = 5%) as significant determinants of IR. Furthermore, multivariate analysis identified hepatocyte steatosis (> or = 5%), aa substitutions of the core region (Gln70 (His70) and/or Met91), and age (> or = 55 years) as significant determinants of severe IR. Especially, significantly lower proportions of patients with Gln70 (His70) and/or Met91 were noted among those without severe IR (59.6%) than those with severe IR (82.4%). The rates of sustained virological response in patients with IR (50.0%) were not significantly different from those without IR (52.9%). Furthermore, the rates of non-virological response in patients with IR (28.9%) were not significantly also different from those without IR (20.6%). In conclusion, the present study indicated that substitutions of HCV-1b core region were the important predictor of severe IR in patients without cirrhosis and diabetes mellitus, but HOMA-IR values might be not useful as predictors of 48-week PEG-IFN plus RBV therapy. PMID:19382270

  12. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/. PMID:26982818

  13. Structural and Functional Characteristics of Oxysterol 7α-Hydroxylase with Amino-Acid Substitution R486C and Their Relation to the Appearance of Neurodegenerative Diseases

    NASA Astrophysics Data System (ADS)

    Dichenko, Ya. V.; Yantsevich, A. V.; Usanov, S. A.

    2015-03-01

    The influence of the amino-acid substitution Arg486Cys on the conformational stability of recombinant cytochrome P450 7B1 (CYP7B1, oxysterol 7α-hydroxylase) was studied. The single base change was shown to decrease the free energy of the transition of the heme-protein from its native state to a denatured one, which pointed to a lower thermodynamic stability for the mutant form of the enzyme. This could be the cause of the metabolic disruption of neurosteroids and, as a consequence, the appearance of neurodegenerative diseases.

  14. Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates.

    PubMed

    Garner, Matthew L; Georgiadis, Taxiarchis M; Li, Jessica Bo; Wang, Tianxiu; Long, Eric C

    2014-05-01

    Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-amidine conjugates was developed to facilitate installation of phenylbenzimidazole-amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N'-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N'-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl2·2H2O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-amidine ring system. Employing this protocol, a series of amino acid-phenylbenzimidazole-amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-amidine and Xaa is any amino acid). PMID:24562478

  15. Protodeboronation of Heteroaromatic, Vinyl, and Cyclopropyl Boronic Acids: pH-Rate Profiles, Autocatalysis, and Disproportionation.

    PubMed

    Cox, Paul A; Leach, Andrew G; Campbell, Andrew D; Lloyd-Jones, Guy C

    2016-07-27

    pH-rate profiles for aqueous-organic protodeboronation of 18 boronic acids, many widely viewed as unstable, have been studied by NMR and DFT. Rates were pH-dependent, and varied substantially between the boronic acids, with rate maxima that varied over 6 orders of magnitude. A mechanistic model containing five general pathways (k1-k5) has been developed, and together with input of [B]tot, KW, Ka, and KaH, the protodeboronation kinetics can be correlated as a function of pH (1-13) for all 18 species. Cyclopropyl and vinyl boronic acids undergo very slow protodeboronation, as do 3- and 4-pyridyl boronic acids (t0.5 > 1 week, pH 12, 70 °C). In contrast, 2-pyridyl and 5-thiazolyl boronic acids undergo rapid protodeboronation (t0.5 ≈ 25-50 s, pH 7, 70 °C), via fragmentation of zwitterionic intermediates. Lewis acid additives (e.g., Cu, Zn salts) can attenuate (2-pyridyl) or accelerate (5-thiazolyl and 5-pyrazolyl) fragmentation. Two additional processes compete when the boronic acid and the boronate are present in sufficient proportions (pH = pKa ± 1.6): (i) self-/autocatalysis and (ii) sequential disproportionations of boronic acid to borinic acid and borane. PMID:27355973

  16. Measurement of Fatty Acid Oxidation Rates in Animal Tissues and Cell Lines

    PubMed Central

    Huynh, Frank K.; Green, Michelle F.; Koves, Timothy R.; Hirschey, Matthew D.

    2014-01-01

    While much oncological research has focused on metabolic shifts in glucose and amino acid oxidation, recent evidence suggests that fatty acid oxidation (FAO) may also play an important role in the metabolic reprogramming of cancer cells. Here, we present a simple method for measuring FAO rates using radiolabeled palmitate, common laboratory reagents, and standard supplies. This protocol is broadly applicable for measuring FAO rates in cultured cancer cells as well as in both malignant and nontransformed animal tissues. PMID:24862277

  17. Arsenic Scavenging by Al-Substituted Ferrihydrites in a Circumneutral pH River Impacted by the Acid Mine Drainage of Carnoulès, Gard, France

    NASA Astrophysics Data System (ADS)

    ADRA, A.; Morin, G.; ona-Nguema, G.; Maillot, F.; Casiot, C.; Bruneel, O.

    2013-12-01

    Ferrihydrite (Fh) is a nanocrystalline ferric oxyhydroxide involved in the retention of pollutants in natural systems and in water-treatment processes. The status and properties of major chemical impurities in natural Fh is however still scarcely documented. Here we investigated the structure and reactivity of aluminum-rich Fh from river-bed sediments collected in a circumneutral river (pH 6-7) impacted by an arsenic-rich acid mine drainage (AMD). Extended X-ray absorption fine structure (EXAFS) spectroscopy at the Fe K-edge shows that Fh is the predominant mineral phase forming after neutralization of the AMD, in association with minor amount of schwertmannite transported from the AMD. EXAFS analysis indicates that Al(III) substitutes for Fe(III) ions into the Fh structure in the natural sediment samples, with local aluminum concentration within the 20-37×7 mol%Al range, in agreement with bulk chemical compositions. Synthetic aluminous Fh analogues prepared in the present study are found to be less Al-substituted (14-18×4 mol%Al). Finally, EXAFS analysis at the arsenic K-edge indicates that As(V) form similar inner-sphere surface complexes on the natural and synthetic Al-substituted Fh studied. Our results provide direct evidences for the scavenging of arsenic by natural Al- Fh, with possible implications for other pollutants in natural or engineered systems.

  18. New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket.

    PubMed

    Perera, Erick; Pons, Tirso; Hernandez, Damir; Moyano, Francisco J; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2010-09-01

    Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed. PMID:20649906

  19. 40 CFR 721.10202 - Benzoic acid, 4-chloro-2- [(substituted)azo]-, strontium salt (1:1) (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Benzoic acid, 4-chloro-2- -, strontium... New Uses for Specific Chemical Substances § 721.10202 Benzoic acid, 4-chloro-2- -, strontium salt (1:1... identified generically as benzoic acid, 4-chloro-2- -, strontium salt (1:1) (PMN P-08-701) is subject...

  20. 40 CFR 721.10202 - Benzoic acid, 4-chloro-2- [(substituted)azo]-, strontium salt (1:1) (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Benzoic acid, 4-chloro-2- -, strontium... New Uses for Specific Chemical Substances § 721.10202 Benzoic acid, 4-chloro-2- -, strontium salt (1:1... identified generically as benzoic acid, 4-chloro-2- -, strontium salt (1:1) (PMN P-08-701) is subject...

  1. 40 CFR 721.10202 - Benzoic acid, 4-chloro-2- [(substituted)azo]-, strontium salt (1:1) (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzoic acid, 4-chloro-2- -, strontium... New Uses for Specific Chemical Substances § 721.10202 Benzoic acid, 4-chloro-2- -, strontium salt (1:1... identified generically as benzoic acid, 4-chloro-2- -, strontium salt (1:1) (PMN P-08-701) is subject...

  2. 40 CFR 721.10202 - Benzoic acid, 4-chloro-2- [(substituted)azo]-, strontium salt (1:1) (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzoic acid, 4-chloro-2- -, strontium... New Uses for Specific Chemical Substances § 721.10202 Benzoic acid, 4-chloro-2- -, strontium salt (1:1... identified generically as benzoic acid, 4-chloro-2- -, strontium salt (1:1) (PMN P-08-701) is subject...

  3. The Prevalence of Hepatitis C Virus Core Amino Acid 70 Substitution and Genotypes of Polymorphisms Near the IFNL3 Gene in Iranian Patients With Chronic Hepatitis C

    PubMed Central

    Kadjbaf, Danesh; Keshvari, Maryam; Alavian, Seyed Moayed; Pouryasin, Ali; Behnava, Bita; Salimi, Shima; Mehrnoush, Leila; Karimi Elizee, Pegah; Sharafi, Heidar

    2016-01-01

    Background Molecular studies have demonstrated that the hepatitis C virus (HCV) genotype and host genetics play predictive roles in the management of patients infected with HCV. Objectives This study aimed to investigate the HCV genotype, core amino acid (aa) 70 substitution, and polymorphisms near the IFNL3 gene (including rs12979860 and rs8099917) among Iranian patients with chronic hepatitis C (CHC). Patients and Methods In this cross-sectional study, the molecular profiles of the HCV genotype, core aa 70 substitution, and rs12979860 and rs8099917 polymorphisms and plasma HCV RNA levels were determined in 429 CHC patients including 141 hemophilic, 84 thalassemic, and 204 non-hemophilic, non-thalassemic patients. Results The hepatitis C virus subtype 1a was the most common subtype in the study population. Core aa substitution Arg70Gln was strongly associated with cirrhosis (OR = 2.49; 95% CI = 1.13 - 5.50; P = 0.020). Core aa 70 substitutions were more frequently observed in patients with the HCV subtype 1b than in patients with any other HCV subtypes (P < 0.001). Core aa 70 substitutions were also more common in patients with the rs12979860 TT genotype than in patients with non-TT genotypes (17.3% vs. 8.5%, P = 0.022) and also in rs8099917 non-TT genotypes than in the TT genotype (14.0% vs. 7.0%, P = 0.026). The HCV genotypes and rs8099917 polymorphisms were correlated in which HCV subtype 1b was in favor of rs8099917 GG and HCV subtype 3a favored rs8099917 TT (P = 0.021). Furthermore, the rs12979860 TT and rs8099917 GG genotypes showed significantly lower HCV RNA levels than the other genotypes (P < 0.001). Conclusions There is an as yet unexplained association between HCV and host parameters with unknown mechanisms in patients with chronic HCV infection. The assessments of core aa 70 substitution and polymorphisms near the IFNL3 gene could offer promising steps to improve the management of patients with HCV.

  4. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

    PubMed Central

    Suarez, Javier; Schramm, Vern L.

    2015-01-01

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon–deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects. PMID:26305965

  5. Effects of varying media, temperature, and growth rates on the intracellular concentrations of yeast amino acids.

    PubMed

    Martínez-Force, E; Benítez, T

    1995-01-01

    Variations of the yeast free amino acid pool under different culture conditions were studied in two Saccharomyces strains, the laboratory haploid strain S288C and the industrial fermentative yeast IFI256. The internal amino acid pool of both strains was measured when grown in laboratory (minimal and complete) versus semiindustrial (molasses with or without added biotin and/or diammonium phosphate) media, in fermentable (glucose, fructose, sucrose) versus respirable (glycerol) carbon sources, in different temperatures (22, 30, and 37 degrees C), pHs (2.0-4.75), and growth rates (0.018-0.24 h-1) in continuous culture, and at different phases of the growth curve in batch culture (lag, exponential, early and late stationary). Results indicated that environmental conditions, particularly the presence of amino acids in the media, enormously influenced the intracellular amino acid concentration. Higher values were detected in molasses than in laboratory media and in fermentable carbon sources (glucose, fructose, sucrose) than in glycerol. Variations in the amino acid pool along the growth curve were greater at 37 degrees C than at other temperatures; in all cases, the highest values were measured at the beginning of the exponential phase. In continuous culture and at different growth rates, intracellular free amino acid concentrations increased by 3-10-fold when the growth rate was lower than 0.05 h-1, representing 20-35% of the total (free plus protein) amino acid content and indicating that amino acid yield was a partly growth-linked parameter. PMID:7654310

  6. Support Effects on Bronsted acid site densities and alcohol dehydration turnover rates on tungsten oxide domains

    SciTech Connect

    Macht, Josef; Baertsch, Chelsey D.; May-Lozano, Marcos; Soled, Stuart L.; Wang, Yong; Iglesia, Enrique

    2005-03-01

    Initial activity and acid site density of several WAl, WSi (MCM41) and one WSn sample were determined. Trans/cis 2-butene selectivity is dependent on the support. Presumably, these differences are due to subtle differences in base strengths. 2-Butanol dehydration rates (per W-atom) reached maximum values at intermediate WOx surface densities on WAl, as reported for 2-butanol dehydration reactions on WZr. Titration results indicate that Bronsted acid sites are required for 2-butanol dehydration on WAl, WSi and WSn. UV-visible studies suggest that WAl is much more difficult to reduce than WZr. The detection of reduced centers on WAl, the number of which correlates to Bronsted acid site density and catalyst activity, as well as the temperature dependence of Bronsted acid site density indicate the in-situ formation of these active sites. We infer that this mechanism is common among all supported WOx samples described in this study. Turnover rates are a function of Bronsted acid site density only. High acid site densities lead to high turnover rates. Higher active site densities may cause stronger conjugate bases, as a higher electron density has to be stabilized, and thus weaker acidity, enabling a faster rate of product desorption. The maximum achievable active site density is dependent on the support. WZr reaches a higher active site density than WAl.

  7. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    PubMed Central

    2012-01-01

    Background Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. Results Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. Conclusion Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the

  8. The DNA binding specificity of the basic region of the yeast transcriptional activator GCN4 can be changed by substitution of a single amino acid.

    PubMed Central

    Suckow, M; von Wilcken-Bergmann, B; Müller-Hill, B

    1993-01-01

    The X-ray structure of a GCN4 DNA complex (1) shows, that specific DNA binding of the GCN4 basic region is mediated by a complicated network of base pair and DNA backbone contacts. According to the X-ray structure, alanine -14 of the basic region of GCN4 (we define the first leucine of the leucine zipper as +1) makes a hydrophobic contact to the methyl group of the thymine next to the center of the GCN4 binding site 5' ATGACTCAT 3'. We tested the DNA binding properties of the nineteen derivatives of GCN4, which carry all possible amino acids in position -14 of the basic region. Substitution of alanine -14 of GCN4 by either asparagine or cysteine changes the DNA binding specificity. Serine in this position broadens the specificity for position 1 of the target, whereas other amino acids either retain or decrease GCN4 specificity. Images PMID:8502548

  9. An Amino Acid Substitution Inhibits Specialist Herbivore Production of an Antagonist Effector and Recovers Insect-Induced Plant Defenses1[W][OA

    PubMed Central

    Schmelz, Eric A.; Huffaker, Alisa; Carroll, Mark J.; Alborn, Hans T.; Ali, Jared G.; Teal, Peter E.A.

    2012-01-01

    Plants respond to insect herbivory through the production of biochemicals that function as either direct defenses or indirect defenses via the attraction of natural enemies. While attack by closely related insect pests can result in distinctive levels of induced plant defenses, precise biochemical mechanisms responsible for differing responses remain largely unknown. Cowpea (Vigna unguiculata) responds to Fall armyworm (Spodoptera frugiperda) herbivory through the detection of fragments of chloroplastic ATP synthase γ-subunit proteins, termed inceptin-related peptides, present in larval oral secretions (OS). In contrast to generalists like Fall armyworm, OS of the legume-specializing velvetbean caterpillar (VBC; Anticarsia gemmatalis) do not elicit ethylene production and demonstrate significantly lower induced volatile emission in direct herbivory comparisons. Unlike all other Lepidoptera OS examined, which preferentially contain inceptin (Vu-In; +ICDINGVCVDA−), VBC OS contain predominantly a C-terminal truncated peptide, Vu-In−A (+ICDINGVCVD−). Vu-In−A is both inactive and functions as a potent naturally occurring antagonist of Vu-In-induced responses. To block antagonist production, amino acid substitutions at the C terminus were screened for differences in VBC gut proteolysis. A valine-substituted peptide (Vu-InΔV; +ICDINGVCVDV−) retaining full elicitor activity was found to accumulate in VBC OS. Compared with the native polypeptide, VBC that previously ingested 500 pmol of the valine-modified chloroplastic ATP synthase γ-subunit precursor elicited significantly stronger plant responses in herbivory assays. We demonstrate that a specialist herbivore minimizes the activation of defenses by converting an elicitor into an antagonist effector and identify an amino acid substitution that recovers these induced plant defenses to a level observed with generalist herbivores. PMID:23008466

  10. Lipoteichoic acid of Streptococcus oralis Uo5: a novel biochemical structure comprising an unusual phosphorylcholine substitution pattern compared to Streptococcus pneumoniae

    PubMed Central

    Gisch, Nicolas; Schwudke, Dominik; Thomsen, Simone; Heß, Nathalie; Hakenbeck, Regine; Denapaite, Dalia

    2015-01-01

    Members of the Mitis group of streptococci possess teichoic acids (TAs) as integral components of their cell wall that are unique among Gram-positive bacteria. Both, lipoteichoic (LTA) and wall teichoic acid, are formed by the same biosynthetic pathway, are of high complexity and contain phosphorylcholine (P-Cho) residues. These residues serve as anchors for choline-binding proteins (CBPs), some of which have been identified as virulence factors of the human pathogen Streptococcus pneumoniae. We investigated the LTA structure of its close relative Streptococcus oralis. Our analysis revealed that S. oralis Uo5 LTA has an overall architecture similar to pneumococcal LTA (pnLTA) and can be considered as a subtype of type IV LTA. Its structural complexity is even higher than that of pnLTA and its composition differs in number and type of carbohydrate moieties, inter-residue connectivities and especially the P-Cho substitution pattern. Here, we report the occurrence of a saccharide moiety substituted with two P-Cho residues, which is unique as yet in bacterial derived surface carbohydrates. Finally, we could link the observed important structural variations between S. oralis and S. pneumoniae LTA to the divergent enzymatic repertoire for their TA biosynthesis. PMID:26577602

  11. The folded conformation of phage P22 coat protein is affected by amino acid substitutions that lead to a cold-sensitive phenotype.

    PubMed

    Fong, D G; Doyle, S M; Teschke, C M

    1997-04-01

    Three cold-sensitive mutants in phage P22 coat protein have been characterized to determine the effects of the amino acid substitutions that cause cold sensitivity on the folding pathway and the conformation of refolded coat protein. Here we find that the three cold-sensitive mutants which have the threonine residue at position 10 changed to isoleucine (T10I), the arginine residue at position 101 changed to cysteine (R101C), or the asparagine residue at position 414 changed to serine (N414S) were capable of folding from a denatured state into a soluble monomeric species, but in each case, the folded conformation was altered. Changes in the kinetics of folding were observed by both tryptophan and bisANS fluorescence. In contrast to the temperature-sensitive for folding coat protein mutants which can be rescued at nonpermissive temperatures in vivo by the overproduction of molecular chaperones GroEL and GroES [Gordon, C. L., Sather, S. K., Casjens, S., & King, J. (1994) J. Biol. Chem. 269, 27941-27951], the folding defects associated with the cold-sensitive amino acid substitutions were not recognized by GroEL and GroES. PMID:9092827

  12. Properties of Rhodobacter sphaeroides photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L.

    PubMed

    Fufina, T Yu; Vasilieva, L G; Khatypov, R A; Shuvalov, V A

    2011-04-01

    Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacteriochlorophyll (BChl) B(B) Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacteriopheophytin appears in the B(B) binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386-7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B(B) Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B(B) Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl B(B) Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position. PMID:21585320

  13. Synthesis, structure-activity, and structure-stability relationships of 2-substituted-N-(4-oxo-3-oxetanyl) N-acylethanolamine acid amidase (NAAA) inhibitors.

    PubMed

    Vitale, Romina; Ottonello, Giuliana; Petracca, Rita; Bertozzi, Sine Mandrup; Ponzano, Stefano; Armirotti, Andrea; Berteotti, Anna; Dionisi, Mauro; Cavalli, Andrea; Piomelli, Daniele; Bandiera, Tiziano; Bertozzi, Fabio

    2014-02-01

    N-Acylethanolamine acid amidase (NAAA) is a cysteine amidase that preferentially hydrolyzes saturated or monounsaturated fatty acid ethanolamides (FAEs), such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), which are endogenous agonists of nuclear peroxisome proliferator-activated receptor-α (PPAR-α). Compounds that feature an α-amino-β-lactone ring have been identified as potent and selective NAAA inhibitors and have been shown to exert marked anti-inflammatory effects that are mediated through FAE-dependent activation of PPAR-α. We synthesized and tested a series of racemic, diastereomerically pure β-substituted α-amino-β-lactones, as either carbamate or amide derivatives, investigating the structure-activity and structure-stability relationships (SAR and SSR) following changes in β-substituent size, relative stereochemistry at the α- and β-positions, and α-amino functionality. Substituted carbamate derivatives emerged as more active and stable than amide analogues, with the cis configuration being generally preferred for stability. Increased steric bulk at the β-position negatively affected NAAA inhibitory potency, while improving both chemical and plasma stability. PMID:24403170

  14. Formation rates, stability and reactivity of sulfuric acid - amine clusters predicted by computational chemistry

    NASA Astrophysics Data System (ADS)

    Kurtén, Theo; Ortega, Ismael; Kupiainen, Oona; Olenius, Tinja; Loukonen, Ville; Reiman, Heidi; McGrath, Matthew; Vehkamäki, Hanna

    2013-04-01

    Despite the importance of atmospheric particle formation for both climate and air quality, both experiments and non-empirical models using e.g. sulfuric acid, ammonia and water as condensing vapors have so far been unable to reproduce atmospheric observations using realistic trace gas concentrations. Recent experimental and theoretical evidence has shown that this mystery is likely resolved by amines. Combining first-principles evaporation rates for sulfuric acid - dimethylamine clusters with cluster kinetic modeling, we show that even sub-ppt concentrations of amines, together with atmospherically realistic concentrations of sulfuric acid, result in formation rates close to those observed in the atmosphere. Our simulated cluster formation rates are also close to, though somewhat larger than, those measured at the CLOUD experiment in CERN for both sulfuric acid - ammonia and sulfuric acid - dimethylamine systems. A sensitivity analysis indicates that the remaining discrepancy for the sulfuric acid - amine particle formation rates is likely caused by steric hindrances to cluster formation (due to alkyl groups of the amine molecules) rather than by significant errors in the evaporation rates. First-principles molecular dynamic and reaction kinetic modeling shed further light on the microscopic physics and chemistry of sulfuric acid - amine clusters. For example, while the number and type of hydrogen bonds in the clusters typically reach their equilibrium values on a picosecond timescale, and the overall bonding patterns predicted by traditional "static" quantum chemical calculations seem to be stable, the individual atoms participating in the hydrogen bonds continuously change at atmospherically realistic temperatures. From a chemical reactivity perspective, we have also discovered a surprising phenomenon: clustering with sulfuric acid molecules slightly increases the activation energy required for the abstraction of alkyl hydrogens from amine molecules. This implies

  15. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. II. ACID AND GENERAL BASE CATALYZED HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to calculate acid and neutral hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition states of a ...

  16. Calibration of a molecular clock in tits (Paridae)--do nucleotide substitution rates of mitochondrial genes deviate from the 2% rule?

    PubMed

    Päckert, Martin; Martens, Jochen; Tietze, Dieter Thomas; Dietzen, Christian; Wink, Michael; Kvist, Laura

    2007-07-01

    The ongoing debate on the reliability of avian molecular clocks is actually based on only a small number of calibrations carried out under different assumptions with respect to the choice and constraints of calibration points or to the use of substitution models. In this study, we provide substitution rate estimates for two mitochondrial genes, cytochrome b and the control region, and age estimates for lineage splits within four subgenera of tits (Paridae: Parus, Cyanistes, Poecile and Periparus). Overall sequence divergence between cytochrome b lineages covers a range of 0.4-1.8% per million years and is thus consistent with the frequently adopted approximation for a sequence divergence between avian lineages of 1.6-2% per my. Overall rate variation is high and encompasses the 2% value in a 95% CI for model corrected data. Mean rate estimates for cytochrome b range between 1.9 and 8.9 x 10(-3) substitutions per site per lineage. Local rates differ significantly between taxonomic levels with lowest estimates for haplotype lineages. At the population/subspecies level mean sequence divergence between lineages matches the 2% rule best for most cytochrome b datasets (1.5-1.9% per my) with maximum estimates for small isolated populations like those of the Canarian P. teneriffae complex (up to 3.9% per my). Overall rate estimates for the control region range at similar values like those for cytochrome b (2.7-8.8 x 10(-3), 0.5-1.8% per my), however, within some subgenera mean rates are higher than those for cytochrome b for uncorrected sequence data. The lowest rates for both genes were calculated for coal tits of subgenus Periparus (0.04-0.6% per my). Model-corrected sequence data tend to result in higher rate estimates than uncorrected data. Increase of the gamma shape parameter goes along with a significant decrease of rate and partly age estimates, too. Divergence times for earliest deep splits within tit subgenera Periparus and Parus were dated to the mid Miocene at

  17. The effect of limestone treatments on the rate of acid generation from pyritic mine gangue.

    PubMed

    Burt, R A; Caruccio, F T

    1986-09-01

    Surface water enters the Haile Gold Mine, Lancaster County, South Carolina by means of a small stream and is ponded behind a dam and in an abandoned pit. This water is affected by acidic drainage. In spite of the large exposures of potentially acid producing pyritic rock, the flux of acid to the water is relatively low. Nevertheless, the resulting pH values of the mine water are low (around 3.5) due to negligible buffering capacity. In view of the observed low release of acidity, the potential for acid drainage abatement by limestone ameliorants appears feasible.This study investigated the effects of limestone treatment on acid generation rates of the Haile mine pyritic rocks through a series of leaching experiments. Below a critical alkalinity threshold value, solutions of dissolved limestone were found consistently to accelerate the rate of pyrite oxidation by varying degrees. The oxidation rates were further accelerated by admixing solid limestone with the pyritic rock. However, after a period of about a month, the pyrite oxidation rate of the admixed samples declined to a level lower than that of untreated pyrite. Leachates produced by the pyrite and limestone mixtures contained little if any iron. Further, in the mixtures, an alteration of the pyrite surface was apparent.The observed behaviour of the treated pyrite appears to be related to the immersion of the pyrite grains within a high alkalinity/high pH environment. The high pH increases the rate of oxidation of ferrous iron which results in a higher concentration of ferric iron at the pyrite surface. This, in turn, increases the rate of pyrite oxidation. Above a threshold alkalinity value, the precipitation of hydrous iron oxides at the pyrite surface eventually outpaces acid generation and coats the pyrite surface, retarding the rate of pyrite oxidation. PMID:24214013

  18. Implications of amino acid substitutions in GyrA at position 83 in terms of oxolinic acid resistance in field isolates of Burkholderia glumae, a causal agent of bacterial seedling rot and grain rot of rice.

    PubMed

    Maeda, Yukiko; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2004-09-01

    Oxolinic acid (OA), a quinolone, inhibits the activity of DNA gyrase composed of GyrA and GyrB and shows antibacterial activity against Burkholderia glumae. Since B. glumae causes bacterial seedling rot and grain rot of rice, both of which are devastating diseases, the emergence of OA-resistant bacteria has important implications on rice cultivation in Japan. Based on the MIC of OA, 35 B. glumae field isolates isolated from rice seedlings grown from OA-treated seeds in Japan were divided into sensitive isolates (OSs; 0.5 microg/ml), moderately resistant isolates (MRs; 50 microg/ml), and highly resistant isolates (HRs; > or =100 microg/ml). Recombination with gyrA of an OS, Pg-10, led MRs and HRs to become OA susceptible, suggesting that gyrA mutations are involved in the OA resistance of field isolates. The amino acid at position 83 in the GyrA of all OSs was Ser, but in all MRs and HRs it was Arg and Ile, respectively. Ser83Arg and Ser83Ile substitutions in the GyrA of an OS, Pg-10, resulted in moderate and high OA resistance, respectively. Moreover, Arg83Ser and Ile83Ser substitutions in the GyrA of MRs and HRs, respectively, resulted in susceptibility to OA. These results suggest that Ser83Arg and Ser83Ile substitutions in GyrA are commonly responsible for resistance to OA in B. glumae field isolates. PMID:15345450

  19. Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

    PubMed Central

    Jelokhani-Niaraki, Saber; Hashemnejad, Khadije; Kamalzadeh, Morteza; Lotfi, Mohsen

    2011-01-01

    The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3Dpol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3Dpol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp26→Glu substitution in a beta sheet located within a small groove of the 3Dpol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment. PMID:22122902

  20. Flip-flop of oleic acid in a phospholipid membrane: rate and mechanism.

    PubMed

    Wei, Chenyu; Pohorille, Andrew

    2014-11-13

    Flip-flop of protonated oleic acid molecules dissolved at two different concentrations in membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine is studied with the aid of molecular dynamics simulations at a time scale of several microseconds. Direct, single-molecule flip-flop events are observed at this time scale, and the flip-flop rate is estimated at 0.2-0.3 μs(-1). As oleic acid molecules move toward the center of the bilayer during flip-flop, they undergo gradual, correlated translational, and rotational motion. Rare, double-flipping events of two hydrogen-bonded oleic acid molecules are also observed. A two-dimensional free energy surface is obtained for the translational and rotational degree of freedom of the oleic acid molecule, and the minimum energy path on this surface is determined. A barrier to flip-flop of ~4.2 kcal/mol is found at the center of the bilayer. A two-dimensional diffusion model is found to provide a good description of the flip-flop process. The fast flip-flop rate lends support to the proposal that fatty acids permeate membranes without assistance of transport proteins. It also suggests that desorption rather than flip-flop is the rate-limiting step in fatty acid transport through membranes. The relation of flip-flop rates to the evolution of ancestral cellular systems is discussed. PMID:25319959

  1. Peeking through the trapdoor: Historical biogeography of the Aegean endemic spider Cyrtocarenum Ausserer, 1871 with an estimation of mtDNA substitution rates for Mygalomorphae.

    PubMed

    Kornilios, P; Thanou, E; Kapli, P; Parmakelis, A; Chatzaki, M

    2016-05-01

    The Aegean region, located in the Eastern Mediterranean, is an area of rich biodiversity and endemism. Its position, geographical configuration and complex geological history have shaped the diversification history of many animal taxa. Mygalomorph spiders have drawn the attention of researchers, as excellent model systems for phylogeographical investigations. However, phylogeographic studies of spiders in the Aegean region are scarce. In this study, we focused on the phylogeography of the endemic ctenizid trap-door spider Cyrtocarenum Ausserer, 1871. The genus includes two morphologically described species: C. grajum (C.L. Koch, 1836) and C. cunicularium (Olivier, 1811). We sampled 60 specimens from the distributions of both species and analyzed four mitochondrial and two nuclear markers. Cyrtocarenum served as an example to demonstrate the importance of natural history traits in the inference of phylogeographic scenarios. The mtDNA substitution rates inferred for the genus are profoundly higher compared to araneomorph spiders and other arthropods, which seems tightly associated with their biology. We evaluate published mtDNA substitution rates followed in the literature for mygalomorph spiders and discuss potential pitfalls. Following gene tree (maximum likelihood, Bayesian inference) and species tree approaches ((*)BEAST), we reconstructed a time-calibrated phylogeny of the genus. These results, combined with a biogeographical ancestral-area analysis, helped build a biogeographic scenario that describes how the major palaeogeographic and palaeoclimatic events of the Aegean may have affected the distribution of Cyrtocarenum lineages. The diversification of the genus seems to have begun in the Middle Miocene in the present west Aegean area, while major phylogenetic events occurred at the Miocene-Pliocene boundary for C. cunicularium, probably related to the Messinian Salinity Crisis. Our results also demonstrate the clear molecular distinction of the two

  2. Influence of fatty acid oxidation rate on glycerol release from cardiac myocytes

    SciTech Connect

    Larsen, T.S.; Severson, D.L.

    1986-03-05

    Quiescent cardiac myocytes are characterized by low rates of fatty acid oxidation due to the reduced energy demand compared with beating hearts. The accumulation of intracellular fatty acid metabolites may, therefore, result in feed-back inhibition of the cardiac lipase responsible for the mobilization of triacylglycerols (lipolysis). The objective of this study was to examine if interventions that increase fatty acid oxidation rates in myocytes have an effect on lipolysis. Addition of 100 ..mu..M dinitrophenol (DNP) to calcium-tolerant rat ventricular myocytes caused an increase in the rate of /sup 14/C-oleic acid oxidation from 1.11 +/- 0.06 to 2.38 +/- 0.17 nmol /sup 14/CO/sub 2//10/sup 6/ cells/min (115% stimulation; mean +/- S.D., n = 3). In parallel incubations, DNP increased the rate of lipolysis from 4.4 +/- 1.7 to 13.6 +/- 3.2 nmol glycerol/10/sup 6/ cells/30 min (215% stimulation). The addition of 1 mM barium to a modified Ringer's incubation medium produced an increase in the contractile activity of the myocytes, and increased the rates of oleic acid oxidation from 0.62 +/- 0.16 to 0.88 +/- 0.23 nmol/10/sup 6/ cells/min (42% stimulation; n = 6) and lipolysis from 13.1 +/- 6.5 to 22.2 +/- 6.4 nmol/10/sup 6/ cells/30 min (70% stimulation). These data show that stimulation of fatty acid oxidation in myocardial myocytes is accompanied by increased lipolytic rates, the latter probably due to release of feed-back inhibition of cardiac lipases by accumulated fatty acid metabolites.

  3. Effects of dose, flow rate, and bile acid on diclofenac disposition in the perfused rat liver.

    PubMed

    Uraki, Misato; Kawase, Atsushi; Matsushima, Yuka; Iwaki, Masahiro

    2016-06-01

    An in situ perfused rat liver system is useful for studying the hepatic disposition of drugs and their metabolites. However, the effects of the perfusion conditions on drug disposition are unclear. We examined the effects of conditions such as flow rate (13 or 26 mL/min) and bile acid on disposition of diclofenac (DF) as a model drug and DF metabolites [diclofenac-1-O-acyl glucuronide (DF-Glu) or 4'-hydroxydiclofenac (DF-4'OH)] in the absence of albumin. DF, DF-Glu, and DF-4'OH concentrations in the perfusate and cumulative amounts of DF-Glu excreted in bile were measured using high-performance liquid chromatography methods. DF in the perfusate was rapidly eliminated as the perfusate flow rate increased. The area under the plasma concentration-time curve from 0 to 60 min (AUC0-60) for DF-Glu and DF-4'OH in a perfusate containing bile acid was lower at a flow rate of 26 and 13 mL/min, respectively. The bile flow rate at 26 mL/min with 24 μM of bile acid in the perfusate was significantly higher (ca. 3.5 times) compared with that at 13 mL/min without bile acid. Cumulative biliary DF-Glu excretion was also dramatically affected by the flow rate and addition of bile acid. This study indicated that the flow rate and bile acid in the perfused rat liver were key factors for bile flow rate and DF, DF-Glu, and DF-4'OH disposition in the absence of albumin. PMID:25656736

  4. Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

    PubMed Central

    Gross, Christian H.; Parsons, Jonathan D.; Grossman, Trudy H.; Charifson, Paul S.; Bellon, Steven; Jernee, James; Dwyer, Maureen; Chambers, Stephen P.; Markland, William; Botfield, Martyn; Raybuck, Scott A.

    2003-01-01

    DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli. PMID:12604539

  5. Skin Substitutes

    PubMed Central

    Howe, Nicole; Cohen, George

    2014-01-01

    In a relatively short timespan, a wealth of new skin substitutes made of synthetic and biologically derived materials have arisen for the purpose of wound healing of various etiologies. This review article focuses on providing an overview of skin substitutes including their indications, contraindications, benefits, and limitations. The result of this overview was an appreciation of the vast array of options available for clinicians, many of which did not exist a short time ago. Yet, despite the rapid expansion this field has undergone, no ideal skin substitute is currently available. More research in the field of skin substitutes and wound healing is required not only for the development of new products made of increasingly complex biomolecular material, but also to compare the existing skin substitutes. PMID:25371771

  6. Chiral Phosphoric Acid Catalyzed [3 + 2] Cycloaddition and Tandem Oxidative [3 + 2] Cycloaddition: Asymmetric Synthesis of Substituted 3-Aminodihydrobenzofurans.

    PubMed

    Gelis, Coralie; Bekkaye, Mathieu; Lebée, Clément; Blanchard, Florent; Masson, Géraldine

    2016-07-15

    Asymmetric [3 + 2] cycloaddition of quinones with ene- and thioene-carbamates was achieved by chiral phosphoric acid catalysis, providing the corresponding 3-amino-2,3-dihydrobenzofurans in excellent yields with moderate to good diastereoselectivities and excellent enantioselectivities. An asymmetric tandem oxidative cycloaddition protocol starting from hydroquinones was also accomplished with phenyliodine(III) diacetate and a chiral phosphoric acid in the same reaction vessel. PMID:27352020

  7. Helix propensities of conformationally restricted amino acids. Non-natural substitutes for helix breaking proline and helix forming alanine.

    PubMed

    Alías, Miriam; Ayuso-Tejedor, Sara; Fernández-Recio, Juan; Cativiela, Carlos; Sancho, Javier

    2010-02-21

    Alpha helices are useful scaffolds to build biologically active peptides. The intrinsic stability of an alpha-helix is a key feature that can be successfully designed, and it is governed by the constituting amino acid residues. Their individual contributions to helix stability are given, according to Lifson-Roig theory, by their w parameters, which are known for all proteinogenic amino acids, but not for non-natural ones. On the other hand, non-natural, conformationally-restricted amino acids can be used to impart biochemical stability to peptides intended for in vivo administration. Efficient design of peptides based on these amino acids requires the previous determination of their w parameters. We begin here this task by determining the w parameters of two restricted analogs of alanine: (alpha-methyl)alanine and 1-aminocyclopropanecarboxylic acid. According to their w values (alpha-methyl)alanine is almost as good a helix forming residue as alanine, while 1-aminocyclopropanecarboxylic acid is, similarly to proline, a helix breaker. PMID:20135035

  8. Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: an attempt to improve the activity against Mycobacterium tuberculosis.

    PubMed

    Andaloussi, Mounir; Lindh, Martin; Björkelid, Christofer; Suresh, Surisetti; Wieckowska, Anna; Iyer, Harini; Karlén, Anders; Larhed, Mats

    2011-09-15

    Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC(50) values in the micromolar range, with a best value of 41 μM. PMID:21824775

  9. Antimicrobial and antioxidant screening of N¢-substituted sulphonyl and benzoyl derivatives of 4-Pyridine carboxylic acid hydrazide.

    PubMed

    Naeem, Sabahat; Akhtar, Shamim; Asghar, Nadia; Sherwani, Sikander Khan; Mushtaq, Nousheen; Kamil, Arfa; Zafar, Shaista; Arif, Mohammad; Saify, Zafar Saeed

    2015-11-01

    In this research program, the antibacterial, antifungal and antioxidant activities of six N'-substituted sulfonyl and benzoyl derivatives of lead molecule PCH were reported. Out of these compounds, sulphonyl derivatives 2,3 and benzoyl derivative 5 showed moderate to good activity against different strains of gram-positive and gram-negative bacteria including B. cereus, B. subtilis, B. thruingiensis and S. pyogenes, S. fecalis and E. coli ATCC 8739. Moreover, upon antifungal screening, the compound, N¢-[(2,4,6-trimethylbenzene) sulfonyl]pyridine-4-carbohydrazide possessed good antifungal activity against Candida species, a causative agent of systemic fungal infections. Antioxidant study demonstrated more than 50% inhibition in DPPH assay for sulphonyl derivative 2 indicating its potential as antioxidant while the other derivatives expressed low level of radical scavenging property. PMID:26639506

  10. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    SciTech Connect

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-09-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  11. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

    PubMed Central

    Nakagawa, Yoshiko; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    ABSTRACT Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  12. Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice.

    PubMed

    Nakagawa, Yoshiko; Sakuma, Tetsushi; Nishimichi, Norihisa; Yokosaki, Yasuyuki; Yanaka, Noriyuki; Takeo, Toru; Nakagata, Naomi; Yamamoto, Takashi

    2016-01-01

    Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency. PMID:27387532

  13. Quantitative Structure-Property Relationship (QSPR) Models for a Local Quantum Descriptor: Investigation of the 4- and 3-Substituted-Cinnamic Acid Esterification.

    PubMed

    Rodrigues-Santos, Cláudio E; Echevarria, Aurea; Sant'Anna, Carlos M R; Bitencourt, Thiago B; Nascimento, Maria G; Bauerfeldt, Glauco F

    2015-01-01

    In this work, the theoretical description of the 4- and 3-substituted-cinnamic acid esterification with different electron donating and electron withdrawing groups was performed at the B3LYP and M06-2X levels, as a two-step process: the O-protonation and the nucleophile attack by ethanol. In parallel, an experimental work devoted to the synthesis and characterization of the substituted-cinnamate esters has also been performed. In order to quantify the substituents effects, quantitative structure-property relationship (QSPR) models based on the atomic charges, Fukui functions and the Frontier Effective-for-Reaction Molecular Orbitals (FERMO) energies were investigated. In fact, the Fukui functions, ƒ⁺C and ƒ(-)O, indicated poor correlations for each individual step, and in contrast with the general literature, the O-protonation step is affected both by the FERMO energies and the O-charges of the carbonyl group. Since the process was shown to not be totally described by either charge- or frontier-orbitals, it is proposed to be frontier-charge-miscere controlled. Moreover, the observed trend for the experimental reaction yields suggests that the electron withdrawing groups favor the reaction and the same was observed for Step 2, which can thus be pointed out as the determining step. PMID:26402661

  14. Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality

    PubMed Central

    Wang, Tao; Haagenson, Michael; Spellman, Stephen R.; Askar, Medhat; Battiwalla, Minoo; Baxter-Lowe, Lee Ann; Bitan, Menachem; Fernandez-Viña, Marcelo; Gandhi, Manish; Jakubowski, Ann A.; Maiers, Martin; Marino, Susana R.; Marsh, Steven G. E.; Oudshoorn, Machteld; Palmer, Jeanne; Prasad, Vinod K.; Reddy, Vijay; Ringden, Olle; Saber, Wael; Santarone, Stella; Schultz, Kirk R.; Setterholm, Michelle; Trachtenberg, Elizabeth; Turner, E. Victoria; Woolfrey, Ann E.; Lee, Stephanie J.; Anasetti, Claudio

    2013-01-01

    HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death. PMID:23982174

  15. Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

    SciTech Connect

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

    2008-07-28

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg{sup 2+} as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 {angstrom} (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

  16. Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analog benzoylphosphonic acid methyl ester

    PubMed Central

    Brandt, Gabriel S.; Nemeria, Natalia; Chakraborty, Sumit; McLeish, Michael J.; Yep, Alejandra; Kenyon, George L.; Petsko, Gregory A.; Jordan, Frank; Ringe, Dagmar

    2009-01-01

    Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these type of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analog of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (PDB ID: 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase. PMID:18570438

  17. Evolution of the capsid protein genes of foot-and-mouth disease virus: antigenic variation without accumulation of amino acid substitutions over six decades.

    PubMed Central

    Martínez, M A; Dopazo, J; Hernández, J; Mateu, M G; Sobrino, F; Domingo, E; Knowles, N J

    1992-01-01

    The genetic diversification of foot-and-mouth disease virus (FMDV) of serotype C over a 6-decade period was studied by comparing nucleotide sequences of the capsid protein-coding regions of viruses isolated in Europe, South America, and The Philippines. Phylogenetic trees were derived for VP1 and P1 (VP1, VP2, VP3, and VP4) RNAs by using the least-squares method. Confidence intervals of the derived phylogeny (significance levels of nodes and standard deviations of branch lengths) were placed by application of the bootstrap resampling method. These procedures defined six highly significant major evolutionary lineages and a complex network of sublines for the isolates from South America. In contrast, European isolates are considerably more homogeneous, probably because of the vaccine origin of several of them. The phylogenetic analysis suggests that FMDV CGC Ger/26 (one of the earliest FMDV isolates available) belonged to an evolutionary line which is now apparently extinct. Attempts to date the origin (ancestor) of the FMDVs analyzed met with considerable uncertainty, mainly owing to the stasis noted in European viruses. Remarkably, the evolution of the capsid genes of FMDV was essentially associated with linear accumulation of silent mutations but continuous accumulation of amino acid substitutions was not observed. Thus, the antigenic variation attained by FMDV type C over 6 decades was due to fluctuations among limited combinations of amino acid residues without net accumulation of amino acid replacements over time. PMID:1316467

  18. Single-channel studies on linear gramicidins with altered amino acid sequences. A comparison of phenylalanine, tryptophane, and tyrosine substitutions at positions 1 and 11.

    PubMed Central

    Mazet, J L; Andersen, O S; Koeppe, R E

    1984-01-01

    The relation between chemical structure and permeability characteristics of transmembrane channels has been investigated with the linear gramicidins (A, B, and C), where the amino acid at position 1 was chemically replaced by phenylalanine, tryptophane or tyrosine. The purity of most of the compounds was estimated to be greater than 99.99%. The modifications resulted in a wide range of conductance changes in NaCl solutions: sixfold from tryptophane gramicidin A to tyrosine gramicidin B. The conductance changes induced by a given amino acid substitution at position 1 are not the same as at position 11. The only important change in the Na+ affinity was observed when the first amino acid was tyrosine. No major conformational changes of the polypeptide backbone structure could be detected on the basis of experiments with mixtures of different analogues and valine gramicidin A (except possibly with tyrosine at position 1), as all the compounds investigated could form hybrid channels with valine gramicidin A. The side chains are not in direct contact with the permeating ions. The results were therefore interpreted in terms of modifications of the energy profile for ion movement through the channel, possibly due to an electrostatic interaction between the dipoles of the side chains and ions in the channel. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:6201199

  19. Synthesis and antimicrobial evaluation of L-phenylalanine-derived C5-substituted rhodanine and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone.

    PubMed

    Jin, Xin; Zheng, Chang-Ji; Song, Ming-Xia; Wu, Yan; Sun, Liang-Peng; Li, Yin-Jing; Yu, Li-Jun; Piao, Hu-Ri

    2012-10-01

    Four novel series of compounds, including the l-phenylalanine-derived C5-substituted rhodanine (6a-q, 7a-j) and chalcone derivatives containing thiobarbituric acid or 2-thioxo-4-thiazolidinone (9a-e, 11a-e) have been designed, synthesized, characterized, and evaluated for their antibacterial activity. Some of these compounds showed significant antibacterial activity against Gram-positive bacterias, especially against the strains of multidrug-resistant clinical isolates, among which compounds 6c-e, 6g, 6i, 6j and 6q exhibiting high levels of antimicrobial activity against Staphylococcus aureus RN4220 with minimum inhibitory concentration (MIC) values of 2 μg/mL. Compound 6q showed the most potent activity of all of the compounds against all of the test multidrug-resistant clinical isolates tested. Unfortunately, however, none of the compounds were active against Gram-negative bacteria at 64 μg/mL. PMID:22982124

  20. Technical note: Synergistic effect of iodide ions on inhibitive performance of substituted dithiobiurets during corrosion of mild steel in hot hydrochloric acid

    SciTech Connect

    Quraishi, M.A.; Rawat, J.; Ajmal, M.

    1999-10-01

    Four substituted dithiobiurets (i.e., 1,5-diphenyl-2,4-dithiobiuret [DPDTB]; 1-anisidyl-5-phenyl 2,4-dithiodiuret [APDTB]; 1-tolyl-5-phenyl 2,4-dithiobiuret [TPDTB]; and 1-chlorophenyl-5-phenyl 2,4-dithiobiuret [CPDTB]) were synthesized to study their inhibiting effect on mild steel (MS) corrosion in 5 N hot hydrochloric acid (HCl). The synergistic effect of these compounds with potassium iodide (KI) was studied at different concentrations, temperatures, and immersion periods by weight loss and potentiodynamic polarization methods. All compounds showed good inhibition efficiency (IE) at all temperatures and showed the enhancement in IE with the addition of small amounts of KI. Potentiodynamic polarization studies showed that APDTB and DPDTB are predominantly cathodic inhibitors, whereas TPDTB and CPDTB are mixed inhibitors. The adsorption of all these compounds followed Temkin's adsorption isotherm.

  1. Synthesis and biological evaluation of novel N-substituted 1H-dibenzo[a,c]carbazole derivatives of dehydroabietic acid as potential antimicrobial agents.

    PubMed

    Gu, Wen; Qiao, Chao; Wang, Shi-Fa; Hao, Yun; Miao, Ting-Ting

    2014-01-01

    A series of new N-substituted 1H-dibenzo[a,c]carbazole derivatives were synthesized from dehydroabietic acid, and their structures were characterized by IR, (1)H NMR and HRMS spectral data. All compounds were evaluated for their antibacterial and antifungal activities against four bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas fluorescens) and three fungi (Candida albicans, Candida tropicalis and Aspergillus niger) by serial dilution technique. Some of the synthesized compounds displayed pronounced antimicrobial activity against tested strains with low MIC values ranging from 0.9 to 15.6μg/ml. Among them, compounds 6j and 6r exhibited potent inhibitory activity comparable to reference drugs amikacin and ketoconazole. PMID:24300736

  2. Rational Design, Synthesis, and Preliminary Structure-Activity Relationships of α-Substituted-2-Phenylcyclopropane Carboxylic Acids as Inhibitors of Salmonella typhimurium O-Acetylserine Sulfhydrylase.

    PubMed

    Pieroni, Marco; Annunziato, Giannamaria; Beato, Claudia; Wouters, Randy; Benoni, Roberto; Campanini, Barbara; Pertinhez, Thelma A; Bettati, Stefano; Mozzarelli, Andrea; Costantino, Gabriele

    2016-03-24

    Cysteine is a building block for several biomolecules that are crucial for living organisms. The last step of cysteine biosynthesis is catalyzed by O-acetylserine sulfydrylase (OASS), a highly conserved pyridoxal 5'-phosphate (PLP)-dependent enzyme, present in different isoforms in bacteria, plants, and nematodes, but absent in mammals. Beside the biosynthesis of cysteine, OASS exerts a series of "moonlighting" activities in bacteria, such as transcriptional regulation, contact-dependent growth inhibition, swarming motility, and induction of antibiotic resistance. Therefore, the discovery of molecules capable of inhibiting OASS would be a valuable tool to unravel how this protein affects the physiology of unicellular organisms. As a continuation of our efforts toward the synthesis of OASS inhibitors, in this work we have used a combination of computational and spectroscopic approaches to rationally design, synthesize, and test a series of substituted 2-phenylcyclopropane carboxylic acids that bind to the two S. typhymurium OASS isoforms at nanomolar concentrations. PMID:26894308

  3. Analytical Enantioseparation of β-Substituted-2-Phenylpropionic Acids by High-Performance Liquid Chromatography with Hydroxypropyl-β-Cyclodextrin as Chiral Mobile Phase Additive.

    PubMed

    Tong, Shengqiang; Zhang, Hu; Yan, Jizhong

    2016-04-01

    Analytical enantioseparation of five β-substituted-2-phenylpropionic acids by high-performance liquid chromatography with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral mobile phase additive was established in this paper, and chromatographic retention mechanism was studied. The effects of various factors such as the organic modifier, different ODS C18 columns and concentration of HP-β-CD were investigated. The chiral mobile phase was composed of methanol or acetonitrile and 0.5% triethylamine acetate buffer at pH 3.0 added with 25 mmol L(-1) of HP-β-CD, and baseline separations could be reached for all racemates. As for chromatographic retention mechanism, it was found that there was a negative correlation between the concentration of HP-β-CD in mobile phase and the retention factor under constant pH value and column temperature. PMID:26755500

  4. LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

    NASA Astrophysics Data System (ADS)

    Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

    2009-03-01

    Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

  5. A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity.

    PubMed

    Zhang, Lan-Yue; Cai, Jie; Li, Ru-Jie; Liu, Wei; Wagner, Christian; Wong, Kam-Bo; Xie, Zhi-Ping; Staehelin, Christian

    2016-07-01

    The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization. PMID:27383628

  6. A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity

    PubMed Central

    Zhang, Lan-Yue; Cai, Jie; Li, Ru-Jie; Liu, Wei; Wagner, Christian; Wong, Kam-Bo; Xie, Zhi-Ping; Staehelin, Christian

    2016-01-01

    The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization. PMID:27383628

  7. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian . E-mail: yhe@nybloodcenter.org; Li Jingjing; Jiang Shibo

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  8. Occurrence and metabolism of 4-substituted glutamic acids in the seedlings of various species of legumes. [Sophora japonica

    SciTech Connect

    Winter, H.C.; Dekker, E.E.

    1987-04-01

    The authors measured the levels of 4-methyleneglutamic acid (Meglu), 4-methyleneglutamine (Megln), erythro-4-methylglutamic acid (e-Mglu), and threo-4-methylglutamic acid (t-Mglu) in seedlings of various species of legumes by HPLC and ion exchange chromatography. High levels of e-Mglu and Megln but no t-Mglu or Meglu are present in Sophora japonica. Peanut seedling contain both e-Mglu and t-Mglu at 20-50% and 5%, resp., of the level of Meglu whereas only traces of Meglu and Mglu occur in soybean seedlings. Excised peanut embryos germinated on Linsmaier and Skoog medium + (U-/sup 14/C)-leucine incorporated isotope into e-Mglu, Meglu, and Megln; (U-/sup 14/C)-proline or glycine was not so incorporated. Soybean embryos rapidly converted added (2-/sup 14/C)-Meglu to a variety of non-amino acid products; peanut embryos, in contrast, retain 25% of added Meglu unchanged and 50% as Megln. These results suggest that in a variety of legumes leucine may serve as a precursor of Mglu and Meglu during germination; also, whereas Meglu remains as such or as Megln in some species, it is rapidly metabolized in others.

  9. A two-amino acid substitution in the 1918 influenza virus hemagglutinin abolishes transmission of the pandemic virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Herein we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch f...

  10. Factors affecting the rate of hydrolysis of phenylboronic acid in lab-scale precipitate reactor studies

    SciTech Connect

    Bannochie, C.J.; Marek, J.C.; Eibling, R.E.; Baich, M.A.

    1992-10-01

    Removing aromatic carbon from an aqueous slurry of cesium-137 and other alkali tetraphenylborates by acid hydrolysis will be an important step in preparing high-level radioactive waste for vitrification at the Savannah River Site`s Defense Waste Processing Facility (DWPF). Kinetic data obtained in bench-scale precipitate hydrolysis reactors suggest changes in operating parameters to improve product quality in the future plant-scale radioactive operation. The rate-determining step is the removal of the fourth phenyl group, i.e. hydrolysis of phenylboronic acid. Efforts to maximize this rate have established the importance of several factors in the system, including the ratio of copper(II) catalyst to formic acid, the presence of nitrite ion, reactions of diphenylmercury, and the purge gas employed in the system.

  11. Factors affecting the rate of hydrolysis of phenylboronic acid in lab-scale precipitate reactor studies

    SciTech Connect

    Bannochie, C.J.; Marek, J.C.; Eibling, R.E.; Baich, M.A.

    1992-01-01

    Removing aromatic carbon from an aqueous slurry of cesium-137 and other alkali tetraphenylborates by acid hydrolysis will be an important step in preparing high-level radioactive waste for vitrification at the Savannah River Site's Defense Waste Processing Facility (DWPF). Kinetic data obtained in bench-scale precipitate hydrolysis reactors suggest changes in operating parameters to improve product quality in the future plant-scale radioactive operation. The rate-determining step is the removal of the fourth phenyl group, i.e. hydrolysis of phenylboronic acid. Efforts to maximize this rate have established the importance of several factors in the system, including the ratio of copper(II) catalyst to formic acid, the presence of nitrite ion, reactions of diphenylmercury, and the purge gas employed in the system.

  12. [*C]octanoic acid breath test to measure gastric emptying rate of solids.

    PubMed

    Maes, B D; Ghoos, Y F; Rutgeerts, P J; Hiele, M I; Geypens, B; Vantrappen, G

    1994-12-01

    We have developed a breath test to measure solid gastric emptying using a standardized scrambled egg test meal (250 kcal) labeled with [14C]octanoic acid or [13C]octanoic acid. In vitro incubation studies showed that octanoic acid is a reliable marker of the solid phase. The breath test was validated in 36 subjects by simultaneous radioscintigraphic and breath test measurements. Nine healthy volunteers were studied after intravenous administration of 200 mg erythromycin and peroral administration of 30 mg propantheline, respectively. Erythromycin significantly enhanced gastric emptying, while propantheline significantly reduced gastric emptying rates. We conclude that the [*C]octanoic breath test is a promising and reliable test for measuring the gastric emptying rate of solids. PMID:7995200

  13. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

    NASA Technical Reports Server (NTRS)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.

    1981-01-01

    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  14. Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

    PubMed Central

    Marín-Navarro, Julia; Roupain, Nicole; Talens-Perales, David; Polaina, Julio

    2015-01-01

    Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described. PMID:26642312

  15. Substituting CF2 for O4' in Components of Nucleic Acids: Towards Systems with Reduced Propensity to Form Abasic Lesions.

    PubMed

    Yurenko, Yevgen P; Novotný, Jan; Sklenář, Vladimir; Marek, Radek

    2015-12-01

    Intrinsic structural features and energetics of nucleotides containing variously fluorinated sugars as potential building blocks of DNA duplexes and quadruplexes are explored systematically using the modern methods of density functional theory (DFT) and quantum chemical topology (QCT). Our results suggest that fluorination at the 2'-β or 2'-α,β positions somewhat stabilizes in vacuo the AI relative to the BI conformations. In contrast, substitution of the CF2 group for the O4' atom (O4'-CF2 modification) leads to a preference of the BI relative to AI DNA-like conformers. All the studied modifications result in a noticeable increase in the stability of the glycosidic bond [estimated by the relaxed force constants (RFC) approach], with particularly encouraging results for the O4'-CF2 derivative. Consequently, the O4'-CF2 modified systems are suggested and explored as promising scaffolds for the development of duplex and quadruplex structures with reduced propensity to form abasic lesions and to undergo DNA damage. PMID:26493955

  16. Fundamental Study on Temperature Dependence of Deposition Rate of Silicic Acid - 13270

    SciTech Connect

    Shinmura, Hayata; Niibori, Yuichi; Mimura, Hitoshi

    2013-07-01

    The dynamic behavior of the silicic acid is one of the key factors to estimate the condition of the repository system after the backfill. This study experimentally examined the temperature dependence of dynamic behavior of supersaturated silicic acid in the co-presence of solid phase, considering Na ions around the repository, and evaluated the deposition rate constant, k, of silicic acid by using the first-order reaction equation considering the specific surface area. The values of k were in the range of 1.0x10{sup -11} to 1.0x10{sup -9} m/s in the temperature range of 288 K to 323 K. The deposition rate became larger with increments of temperature under the Na ion free condition. Besides, in the case of Na ions 0.6 M, colloidal silicic acid decreased dramatically at a certain time. This means that the diameter of the colloidal silicic acid became larger than the pore size of filter (0.45 μm) due to bridging of colloidal silicic acid. Furthermore, this study estimated the range of altering area and the aperture of flow-path in various value of k corresponding to temperature by using advection-dispersion model. The concentration in the flow-path became lower with increments of temperature, and when the value of k is larger than 1.0x10{sup -11} m/s, the deposition range of supersaturated silicic acid was estimated to be less than 20 m around the repository. In addition, the deposition of supersaturated silicic acid led the decrement of flow-path aperture, which was remarkable under the condition of relatively high temperature. Such a clogging in flow paths is expected as a retardation effect of radionuclides. (authors)

  17. The effect of random precipitation times on the scavenging rate for tropospheric nitric acid

    NASA Technical Reports Server (NTRS)

    Stewart, Richard W.

    1988-01-01

    A model for the effective scavenging rate of a soluble species has been developed. The model takes into account the possibility of positive as well as negative correlations between departures from the mean of the scavenging rate and species concentration. The model is demonstrated for the case of late afternoon rainout of nitric acid occurring just prior to the nighttime cessation of its chemical production. The calculations give effective scavenging rates which are about a factor of 2 to 3 greater than those calculated using the models of Rodhe and Grandell (1972) and Giorgi and Chameides (1985).

  18. Single substitutions to closely related amino acids contribute to the functional diversification of an insect-inducible, positively selected plant cystatin.

    PubMed

    Rasoolizadeh, Asieh; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

    2016-04-01

    A causal link has been reported between positively selected amino acids in plant cystatins and the inhibitory range of these proteins against insect digestive cysteine (Cys) proteases. Here we assessed the impact of single substitutions to closely related amino acids on the contribution of positive selection to cystatin diversification. Cystatin sequence alignments, while confirming hypervariability, indicated a preference for related amino acids at positively selected sites. For example, the non-polar residues leucine (Leu), isoleucine (Ile) and valine (Val) were shown to predominate at positively selected site 2 in the N-terminal region, unlike selected sites 6 and 10, where polar residues are preferred. The model cystatin SlCYS8 and single variants with Leu, Ile or Val at position 2 were compared with regard to their ability to bind digestive proteases of the coleopteran pest Leptinotarsa decemlineata and to induce compensatory responses in this insect. A functional proteomics procedure to capture target Cys proteases in midgut extracts allowed confirmation of distinct binding profiles for the cystatin variants. A shotgun proteomics procedure to monitor whole Cys protease complements revealed protease family specific compensatory responses in the insect, dependent on the variant ingested. Our data confirm the contribution of closely related amino acids to the functional diversity of positively selected plant cystatins in a broader structure/function context imposing physicochemical constraints to primary structure alterations. They also underline the complexity of protease/inhibitor interactions in plant-insect systems, and the challenges still to be met in order to harness the full potential of ectopically expressed protease inhibitors in crop protection. PMID:26833679

  19. New molecular markers for prostate tumor imaging: a study on 2-methylene substituted fatty acids as new AMACR inhibitors.

    PubMed

    Morgenroth, Agnieszka; Urusova, Elizaveta A; Dinger, Cornelia; Al-Momani, Ehab; Kull, Thomas; Glatting, Gerhard; Frauendorf, Holm; Jahn, Olaf; Mottaghy, Felix M; Reske, Sven N; Zlatopolskiy, Boris D

    2011-08-29

    The development of prostate carcinoma is associated with alterations in fatty acid metabolism. α-Methylacyl-CoA racemase (AMACR) is a peroxisomal and mitochondrial enzyme that catalyses interconversion between the (S)/(R)-isomers of a range of α-methylacyl-CoA thioesters. AMACR is involved in the β-oxidation of the dietary branched-chain fatty acids and bile acid intermediates. It is highly expressed in prostate (more than 95 %), colon (92 %), and breast cancers (44 %) but not in the respective normal or hyperplastic tissues. Thus, targeting of AMACR could be a new strategy for molecular imaging and therapy of prostate and some other cancers. Unlabeled 2-methylenacyl-CoA thioesters (12 a-c) were designed as AMACR binding ligands. The thioesters were tested for their ability to inhibit the AMACR-mediated epimerization of (25R)-THC-CoA and were found to be strong AMACR inhibitors. Radioiodinated (E)-(131) I-13-iodo-2-methylentridec-12-enoic acid ((131) I-7 c) demonstrated preferential retention in AMACR-positive prostate tumor cells (LNCaP, LNCaP C4-2wt and DU145) compared with both AMACR-knockout LNCaP C4-2 AMACR-siRNA and benign BPH1 prostate cell lines. A significant protein-bound radioactive fraction with main bands at 47 (sum of molecular weights of AMACR plus 12 c), 70, and 75 kDa was detected in LNCaP C4-2 wt cells. In contrast, only negligible amounts of protein-bound radioactivity were found in LNCaP C4-2 AMACR-siRNA cells. PMID:21812041

  20. Synthesis and Evaluation of 2-Alkylthio-4-(N-substituted sulfonamide)pyrimidine Hydroxamic Acids as Anti-myeloma Agents.

    PubMed

    Xiang, Jinbao; Leung, Crystal; Zhang, Zhuoqi; Hu, Cassie; Geng, Chao; Liu, Lili; Yi, Lang; Li, Zhiwei; Berenson, James; Bai, Xu

    2016-03-01

    A series of pyrimidine hydroxamic acids with a sulfide substituent at the second position and a sulfonamide substituent at the fourth position have been synthesized and evaluated for their activity against human myeloma cell line RPMI 8226. Several compounds exhibited significant anti-cancer potency. It was found that representative compound 6a selectively killed cancerous but not normal cells. Moreover, compound 6a was effective in causing apoptosis in RPMI 8226 cells and exhibited promising HDAC-inhibitory activities. PMID:26518472

  1. Comparison of parameterized nitric acid rainout rates using a coupled stochastic-photochemical tropospheric model

    NASA Technical Reports Server (NTRS)

    Stewart, Richard W.; Thompson, Anne M.; Owens, Melody A.; Herwehe, Jerold A.

    1989-01-01

    A major tropospheric loss of soluble species such as nitric acid results from scavenging by water droplets. Several theoretical formulations have been advanced which relate an effective time-independent loss rate for soluble species to statistical properties of precipitation such as the wet fraction and length of a precipitation cycle. In this paper, various 'effective' loss rates that have been proposed are compared with the results of detailed time-dependent model calculations carried out over a seasonal time scale. The model is a stochastic precipitation model coupled to a tropospheric photochemical model. The results of numerous time-dependent seasonal model runs are used to derive numerical values for the nitric acid residence time for several assumed sets of preciptation statistics. These values are then compared with the results obtained by utilizing theoretical 'effective' loss rates in time-independent models.

  2. Towards Sustainable Aquafeeds: Complete Substitution of Fish Oil with Marine Microalga Schizochytrium sp. Improves Growth and Fatty Acid Deposition in Juvenile Nile Tilapia (Oreochromis niloticus).

    PubMed

    Sarker, Pallab K; Kapuscinski, Anne R; Lanois, Alison J; Livesey, Erin D; Bernhard, Katie P; Coley, Mariah L

    2016-01-01

    We conducted a 84-day nutritional feeding experiment with dried whole cells of DHA-rich marine microalga Schizochytrium sp. (Sc) to determine the optimum level of fish-oil substitution (partial or complete) for maximum growth of Nile tilapia. When we fully replaced fish oil with Schizochytrium (Sc100 diet), we found significantly higher weight gain and protein efficiency ratio (PER), and lower (improved) feed conversion ratio (FCR) and feed intake compared to a control diet containing fish oil (Sc0); and no significant change in SGR and survival rate among all diets. The Sc100 diet had the highest contents of 22:6n3 DHA, led to the highest DHA content in fillets, and consequently led to the highest DHA:EPA ratios in tilapia fillets. Schizochytrium sp. is a high quality candidate for complete substitution of fish oil in juvenile Nile tilapia feeds, providing an innovative means to formulate and optimize the composition of tilapia juvenile feed while simultaneously raising feed efficiency of tilapia aquaculture and to further develop environmentally and socially sustainable aquafeeds. Results show that replacing fish oil with DHA-rich marine Sc improves the deposition of n3 LC PUFA levels in tilapia fillet. These results support further studies to lower Schizochytrium production costs and to combine different marine microalgae to replace fish oil and fishmeal into aquafeeds. PMID:27258552

  3. Towards Sustainable Aquafeeds: Complete Substitution of Fish Oil with Marine Microalga Schizochytrium sp. Improves Growth and Fatty Acid Deposition in Juvenile Nile Tilapia (Oreochromis niloticus)

    PubMed Central

    Sarker, Pallab K.; Kapuscinski, Anne R.; Lanois, Alison J.; Livesey, Erin D.; Bernhard, Katie P.; Coley, Mariah L.

    2016-01-01

    We conducted a 84-day nutritional feeding experiment with dried whole cells of DHA-rich marine microalga Schizochytrium sp. (Sc) to determine the optimum level of fish-oil substitution (partial or complete) for maximum growth of Nile tilapia. When we fully replaced fish oil with Schizochytrium (Sc100 diet), we found significantly higher weight gain and protein efficiency ratio (PER), and lower (improved) feed conversion ratio (FCR) and feed intake compared to a control diet containing fish oil (Sc0); and no significant change in SGR and survival rate among all diets. The Sc100 diet had the highest contents of 22:6n3 DHA, led to the highest DHA content in fillets, and consequently led to the highest DHA:EPA ratios in tilapia fillets. Schizochytrium sp. is a high quality candidate for complete substitution of fish oil in juvenile Nile tilapia feeds, providing an innovative means to formulate and optimize the composition of tilapia juvenile feed while simultaneously raising feed efficiency of tilapia aquaculture and to further develop environmentally and socially sustainable aquafeeds. Results show that replacing fish oil with DHA-rich marine Sc improves the deposition of n3 LC PUFA levels in tilapia fillet. These results support further studies to lower Schizochytrium production costs and to combine different marine microalgae to replace fish oil and fishmeal into aquafeeds. PMID:27258552

  4. Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate.

    PubMed

    Mookerjee, Shona A; Brand, Martin D

    2015-01-01

    Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis(1). Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate(2), the same is not true for extracellular acid production and its relationship to glycolytic rate (3-6). Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate(-) + H(+) and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3(-) + H(+) is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification (6). Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model. PMID:26709455

  5. Straightforward and effective synthesis of γ-aminobutyric acid transporter subtype 2-selective acyl-substituted azaspiro[4.5]decanes.

    PubMed

    Ma, Xiaofeng; Lubin, Hodney; Ioja, Enikő; Kékesi, Orsolya; Simon, Ágnes; Apáti, Ágota; Orbán, Tamás I; Héja, László; Kardos, Julianna; Markó, István E

    2016-01-15

    Supply of major metabolites such as γ-aminobutyric acid (GABA), β-alanine and taurine is an essential instrument that shapes signalling, proper cell functioning and survival in the brain and peripheral organs. This background motivates the synthesis of novel classes of compounds regulating their selective transport through various fluid-organ barriers via the low-affinity γ-aminobutyric acid (GABA) transporter subtype 2 (GAT2). Natural and synthetic spirocyclic compounds or therapeutics with a range of structures and biological activity are increasingly recognised in this regard. Based on pre-validated GABA transport activity, straightforward and efficient synthesis method was developed to provide an azaspiro[4.5]decane scaffold, holding a variety of charge, substituent and 3D constrain of spirocyclic amine. Investigation of the azaspiro[4.5]decane scaffold in cell lines expressing the four GABA transporter subtypes led to the discovery of a subclass of a GAT2-selective compounds with acyl-substituted azaspiro[4.5]decane core. PMID:26706177

  6. Adaptive amino acid substitutions enhance the virulence of an H7N7 avian influenza virus isolated from wild waterfowl in mice.

    PubMed

    Chen, Qiang; Yu, Zhijun; Sun, Weiyang; Li, Xue; Chai, Hongliang; Gao, Xiaolong; Guo, Jiao; Zhang, Kun; Feng, Na; Zheng, Xuexing; Wang, Hualei; Zhao, Yongkun; Qin, Chuan; Huang, Geng; Yang, Songtao; Qian, Jun; Gao, Yuwei; Xia, Xianzhu; Wang, Tiecheng; Hua, Yuping

    2015-05-15

    Although H7N7 AIVs primarily circulate in wild waterfowl, documented cases of human infection with H7N7 viruses suggest they may pose a pandemic threat. Here, we generated mouse-adapted variants of a wild waterfowl-origin H7N7 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD50) of the adapted variants were reduced >5000-fold compared to the parental virus. Mouse-adapted variants viruses displayed enhanced replication in vitro and in vivo, and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H7N7 AIVs in mice. Genomic analysis of the adapted variant viruses revealed amino acid changes in the PB2 (E627K), PB1 (R118I), PA (L550M), HA (G214R), and NA (S372N) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H7N7 virus to replicate and cause severe disease in mammals. PMID:25769645

  7. Identifiability of the unrooted species tree topology under the coalescent model with time-reversible substitution processes, site-specific rate variation, and invariable sites.

    PubMed

    Chifman, Julia; Kubatko, Laura

    2015-06-01

    The inference of the evolutionary history of a collection of organisms is a problem of fundamental importance in evolutionary biology. The abundance of DNA sequence data arising from genome sequencing projects has led to significant challenges in the inference of these phylogenetic relationships. Among these challenges is the inference of the evolutionary history of a collection of species based on sequence information from several distinct genes sampled throughout the genome. It is widely accepted that each individual gene has its own phylogeny, which may not agree with the species tree. Many possible causes of this gene tree incongruence are known. The best studied is the incomplete lineage sorting, which is commonly modeled by the coalescent process. Numerous methods based on the coalescent process have been proposed for the estimation of the phylogenetic species tree given DNA sequence data. However, use of these methods assumes that the phylogenetic species tree can be identified from DNA sequence data at the leaves of the tree, although this has not been formally established. We prove that the unrooted topology of the n-leaf phylogenetic species tree is generically identifiable given observed data at the leaves of the tree that are assumed to have arisen from the coalescent process under a time-reversible substitution process with the possibility of site-specific rate variation modeled by the discrete gamma distribution and a proportion of invariable sites. PMID:25791286

  8. The Effect of Ring Substitution Position on the Structural Conformation of Mercaptobenzoic Acid Self-Assembled Monolayers on Au(111)

    SciTech Connect

    Lee, J; Willey, T; Nilsson, J; Terminello, L; De Yoreo, J; van Buuren, T

    2006-04-12

    Near edge X-ray absorption fine structure (NEX-AFS) spectroscopy, photoemission spectroscopy (PES) and contact angle measurements have been used to examine the structure and bonding of self-assembled monolayers (SAMs) prepared on Au(111) from the positional isomers of mercaptobenzoic acid (MBA). The isomer of MBA and solvent chosen in SAM preparation has considerable bearing upon film morphology. Carbon K-edge NEXAFS measurements indicate that the monomers of 2-, 3- and 4-MBA have well-defined orientations within their respective SAMs. Monomers of 3- and 4-MBA assume an upright orientation on the Au substrates in monolayers prepared using an acetic acid in ethanol solvent. The aryl ring and carboxyl group of these molecules are tilted from the surface normal by a colatitudal angle of {approx} 30{sup o}. Preparation of 4-MBA SAMs using pure ethanol solvent, a more traditional means of synthesis, had no appreciable effect upon the monomer orientation. Nonetheless, S(2p) PES measurements illustrate that it results in extensive bilayer formation via carboxyl group hydrogen-bonding between 4-MBA monomers. In 2-MBA monolayers prepared using acetic acid/ethanol solvent, the monomers adopt a more prostrate orientation on the Au substrates, in which the aryl ring and carboxyl group of the molecules are tilted {approx} 50{sup o} from the surface normal. This configuration is consistent with an interaction between both the mercaptan sulfur and carboxyl group of 2-MBA with the underlying substrate. S(2p) and C(1s) PES experiments provide supporting evidence for a bidentate interaction between 2-MBA and Au(111).

  9. Synthesis of 5-Substituted Derivatives of Isophthalic Acid as Non-Polymeric Amphiphilic Coating for Metal Oxide Nanoparticles

    PubMed Central

    Nilov, Denis; Kucheryavy, Pavel; Walker, Verina; Kidd, Clayton; Kolesnichenko, Vladimir L.; Goloverda, Galina Z.

    2014-01-01

    In the course of development of novel capping ligands with variable steric factor, which will be used as an organic coating for metal oxide nanoparticles, a base-catalyzed nucleophilic oxirane ring-opening addition reaction between dimethyl 5-hydroxyisophthalate and allyl glycidyl ether was studied. The allyl-terminated 1-1, 1-2 and 1-3 adducts and dihydroxylated derivative of the 1-1 adduct, 5-diglyceroxy isophthalic acid, were synthesized. The latter binds to the surface of 5 nm γ-Fe2O3 nanoparticles in reaction with their surfactant-free diethylene glycol colloids. PMID:25152545

  10. Synthesis, antiproliferative and antifungal activities of 1,2,3-triazole-substituted carnosic Acid and carnosol derivatives.

    PubMed

    Pertino, Mariano Walter; Theoduloz, Cristina; Butassi, Estefania; Zacchino, Susana; Schmeda-Hirschmann, Guillermo

    2015-01-01

    Abietane diterpenes exhibit an array of interesting biological activities, which have generated significant interest among the pharmacological community. Starting from the abietane diterpenes carnosic acid and carnosol, twenty four new triazole derivatives were synthesized using click chemistry. The compounds differ in the length of the linker and the substituent on the triazole moiety. The compounds were assessed as antiproliferative and antifungal agents. The antiproliferative activity was determined on normal lung fibroblasts (MRC-5), gastric epithelial adenocarcinoma (AGS), lung cancer (SK-MES-1) and bladder carcinoma (J82) cells while the antifungal activity was assessed against Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32264. The carnosic acid γ-lactone derivatives 1-3 were the most active antiproliferative compounds of the series, with IC50 values in the range of 43.4-46.9 μM and 39.2-48.9 μM for MRC-5 and AGS cells, respectively. Regarding antifungal activity, C. neoformans was the most sensitive fungus, with nine compounds inhibiting more than 50% of its fungal growth at concentrations ≤250 µg∙mL-1. Compound 22, possessing a p-Br-benzyl substituent on the triazole ring, showed the best activity (91% growth inhibition) at 250 µg∙mL-1 In turn, six compounds inhibited 50% C. albicans growth at concentrations lower than 250 µg∙mL-1. PMID:26007173

  11. Amino acid substitutions in the coat protein result in loss of insect transmissibility of a plant virus.

    PubMed Central

    Atreya, P L; Atreya, C D; Pirone, T P

    1991-01-01

    Amino acids near the N terminus of the coat protein of tobacco vein mottling virus were deleted or altered by site-directed mutagenesis to determine the effect on aphid transmissibility of the virus. Deletion of a three amino acid sequence Asp-Ala-Gly, which is conserved in aphid-transmissible potyvirus isolates, abolished transmission. The mutation Ala----Thr in this triplet drastically reduced transmission, whereas the mutation Asp----Asn had no effect, and the mutation Asp----Lys consistently reverted to the wild-type residue. The mutation Lys----Glu, in the residue adjacent to the glycine of the triplet, drastically reduced transmission, whereas the mutation Gln----Pro, seven residues downstream from the glycine had no effect. Comparison of the sequences of other potyviruses suggests that the presence of a glycine residue at the third position of the Asp-Ala-Gly triplet is critical for aphid transmissibility and that certain changes in the residues adjacent to this position abolish or greatly reduce aphid transmissibility. PMID:1881922

  12. Modulation of Enzymatic Activity and Biological Function of Listeria monocytogenes Broad-Range Phospholipase C by Amino Acid Substitutions and by Replacement with the Bacillus cereus Ortholog

    PubMed Central

    Zückert, Wolfram R.; Marquis, Hélène; Goldfine, Howard

    1998-01-01

    The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium’s ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens α-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells. PMID:9746585

  13. Design, Synthesis and Crystal Structures of 6-Alkylidene-2’-Substituted Penicillanic Acid Sulfones as Potent Inhibitors of Acinetobacter baumannii OXA-24 Carbapenemase

    PubMed Central

    Bou, German; Santillana, Elena; Sheri, Anjaneyulu; Beceiro, Alejandro; Sampson, Jared; Kalp, Matthew; Bethel, Christopher R.; Distler, Anne M.; Drawz, Sarah M.; Pagadala, Sundar Ram Reddy; van den Akker, Focco; Bonomo, Robert A.; Romero, Antonio; Buynak, John D.

    2010-01-01

    Class D β-lactamases represent a growing and diverse class of penicillin inactivating enzymes that are usually resistant to commercial β-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel β-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2’-substituted penicillanic acid sulfones (1, 2, 3, 4, and 5) were synthesized and tested against OXA-24, a clinically important β-lactamase that inactivates carbapenems and found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 β-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC50 values, against OXA-24, and two OXA-24 β-lactamase variants ranged from 10 ± 1 (4 vs. WT) to 338 ± 20 nM (5 vs. Tyr112Ala, Met223Ala). Compound 4 possessed the lowest Ki (500 ± 80 nM vs. WT) and 1 possessed the highest inactivation efficiency (kinact/Ki = 0.21 ± 0.02 μM-1s-1). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal there is formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2’-substituted penicillin sulfones are effective mechanism-based inactivators of class D β-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D β-lactamases is proposed. PMID:20822105

  14. Experimental and theoretical study of [N-substituted] p-aminoazobenzene derivatives as corrosion inhibitors for mild steel in sulfuric acid solution

    NASA Astrophysics Data System (ADS)

    Shihab, Mehdi Salih; Al-Doori, Hanan Hussien

    2014-11-01

    [N-substituted] p-aminoazobenzene derivatives (1), (2), (3), (4) and (5) were prepared and investigated as corrosion inhibitors for mild steel in 1 M H2SO4 solution by weight loss measurements. It has been observed that the corrosion rate decreases, inhibition efficiencies increase and surface coverage degree increases with increasing inhibitor concentration. Inhibition efficiencies for prepared compounds were ordered: (1) > (2) > (5) > (4) > (3) with the highest inhibiting efficiency of 63% for 10-3 M. The values of ΔGadso are showing physisorption effect for all prepared compounds. Semiempirical molecular orbital calculations for (1), (2), (3), (4) and (5) could be used as a useful tool to obtain information for explaining the nature of interaction between the metal surface and the organic molecule as a corrosion inhibitor.

  15. Fluorogenic Thorium Sensors Based on 2,6-Pyridinedicarboxylic Acid-Substituted Tetraphenylethenes with Aggregation-Induced Emission Characteristics.

    PubMed

    Wen, Jun; Dong, Liang; Hu, Sheng; Li, Weiyi; Li, Shuo; Wang, Xiaolin

    2016-01-01

    A novel fluorescent sensor based on tetraphenylethene (TPE) modified with 2,6-pyridinedicarboxylic acid (PDA) that shows aggregation-induced emission (AIE) characteristics for thorium recognition with remarkable fluoresence enhancement response has been synthesized. This sensor is capable of visually distinguishing Th(4+) among lanthanides, transition metals, and alkali metals under UV light. Th(4+) can be detected by the naked eye at ppb levels owing to the AIE phenomenon. The sensor showed high selectivity for Th(4+) compared to all other metals tested, and this recognition displayed good anti-interference qualities. This study represents the first application of a AIE fluorescence sensor in actinide metal recognition and it has potential applications in environmental systems for thorium ion detection. PMID:26419754

  16. ModelOMatic: fast and automated model selection between RY, nucleotide, amino acid, and codon substitution models.

    PubMed

    Whelan, Simon; Allen, James E; Blackburne, Benjamin P; Talavera, David

    2015-01-01

    Molecular phylogenetics is a powerful tool for inferring both the process and pattern of evolution from genomic sequence data. Statistical approaches, such as maximum likelihood and Bayesian inference, are now established as the preferred methods of inference. The choice of models that a researcher uses for inference is of critical importance, and there are established methods for model selection conditioned on a particular type of data, such as nucleotides, amino acids, or codons. A major limitation of existing model selection approaches is that they can only compare models acting upon a single type of data. Here, we extend model selection to allow comparisons between models describing different types of data by introducing the idea of adapter functions, which project aggregated models onto the originally observed sequence data. These projections are implemented in the program ModelOMatic and used to perform model selection on 3722 families from the PANDIT database, 68 genes from an arthropod phylogenomic data set, and 248 genes from a vertebrate phylogenomic data set. For the PANDIT and arthropod data, we find that amino acid models are selected for the overwhelming majority of alignments; with progressively smaller numbers of alignments selecting codon and nucleotide models, and no families selecting RY-based models. In contrast, nearly all alignments from the vertebrate data set select codon-based models. The sequence divergence, the number of sequences, and the degree of selection acting upon the protein sequences may contribute to explaining this variation in model selection. Our ModelOMatic program is fast, with most families from PANDIT taking fewer than 150 s to complete, and should therefore be easily incorporated into existing phylogenetic pipelines. ModelOMatic is available at https://code.google.com/p/modelomatic/. PMID:25209223

  17. Decomposition of phenylarsonic acid by AOP processes: degradation rate constants and by-products.

    PubMed

    Jaworek, K; Czaplicka, M; Bratek, Ł

    2014-10-01

    The paper presents results of the studies photodegradation, photooxidation, and oxidation of phenylarsonic acid (PAA) in aquatic solution. The water solutions, which consist of 2.7 g dm(-3) phenylarsonic acid, were subjected to advance oxidation process (AOP) in UV, UV/H2O2, UV/O3, H2O2, and O3 systems under two pH conditions. Kinetic rate constants and half-life of phenylarsonic acid decomposition reaction are presented. The results from the study indicate that at pH 2 and 7, PAA degradation processes takes place in accordance with the pseudo first order kinetic reaction. The highest rate constants (10.45 × 10(-3) and 20.12 × 10(-3)) and degradation efficiencies at pH 2 and 7 were obtained at UV/O3 processes. In solution, after processes, benzene, phenol, acetophenone, o-hydroxybiphenyl, p-hydroxybiphenyl, benzoic acid, benzaldehyde, and biphenyl were identified. PMID:24824504

  18. Swfoldrate: predicting protein folding rates from amino acid sequence with sliding window method.

    PubMed

    Cheng, Xiang; Xiao, Xuan; Wu, Zhi-cheng; Wang, Pu; Lin, Wei-zhong

    2013-01-01

    Protein folding is the process by which a protein processes from its denatured state to its specific biologically active conformation. Understanding the relationship between sequences and the folding rates of proteins remains an important challenge. Most previous methods of predicting protein folding rate require the tertiary structure of a protein as an input. In this study, the long-range and short-range contact in protein were used to derive extended version of the pseudo amino acid composition based on sliding window method. This method is capable of predicting the protein folding rates just from the amino acid sequence without the aid of any structural class information. We systematically studied the contributions of individual features to folding rate prediction. The optimal feature selection procedures are adopted by means of combining the forward feature selection and sequential backward selection method. Using the jackknife cross validation test, the method was demonstrated on the large dataset. The predictor was achieved on the basis of multitudinous physicochemical features and statistical features from protein using nonlinear support vector machine (SVM) regression model, the method obtained an excellent agreement between predicted and experimentally observed folding rates of proteins. The correlation coefficient is 0.9313 and the standard error is 2.2692. The prediction server is freely available at http://www.jci-bioinfo.cn/swfrate/input.jsp. PMID:22933332

  19. Effect of dietary fatty acids on metabolic rate and nonshivering thermogenesis in golden hamsters.

    PubMed

    Jefimow, Małgorzata; Wojciechowski, Michał S

    2014-02-01

    Hibernating rodents prior to winter tend to select food rich in polyunsaturated fatty acids (PUFA). Several studies found that such diet may positively affect their winter energy budget by enhancing torpor episodes. However, the effect of composition of dietary fatty acids (FA) on metabolism of normothermic heterotherms is poorly understood. Thus we tested whether diets different in FA composition affect metabolic rate (MR) and the capacity for nonshivering thermogenesis (NST) in normothermic golden hamsters (Mesocricetus auratus). Animals were housed in outdoor enclosures from May 2010 to April 2011 and fed a diet enriched with PUFA (i.e., standard food supplemented weekly with sunflower and flax seeds) or with saturated and monounsaturated fatty acids (SFA/MUFA, standard food supplemented with mealworms). Since diet rich in PUFA results in lower MR in hibernating animals, we predicted that PUFA-rich diet would have similar effect on MR of normothermic hamsters, that is, normothermic hamsters on the PUFA diet would have lower metabolic rate in cold and higher NST capacity than hamsters supplemented with SFA/MUFA. Indeed, in winter resting metabolic rate (RMR) below the lower critical temperature was higher and NST capacity was lower in SFA/MUFA-supplemented animals than in PUFA-supplemented ones. These results suggest that the increased capacity for NST in PUFA-supplemented hamsters enables them lower RMR below the lower critical temperature of the thermoneural zone. PMID:24151228

  20. Solvent substitution

    SciTech Connect

    Not Available

    1990-01-01

    The DOE Environmental Restoration and Waste Management Office of Technology Development and the Air Force Engineering and Services Center convened the First Annual International Workshop on Solvent Substitution on December 4--7, 1990. The primary objectives of this joint effort were to share information and ideas among attendees in order to enhance the development and implementation of required new technologies for the elimination of pollutants associated with industrial use of hazardous and toxic solvents; and to aid in accelerating collaborative efforts and technology transfer between government and industry for solvent substitution. There were workshop sessions focusing on Alternative Technologies, Alternative Solvents, Recovery/Recycling, Low VOC Materials and Treatment for Environmentally Safe Disposal. The 35 invited papers presented covered a wide range of solvent substitution activities including: hardware and weapons production and maintenance, paint stripping, coating applications, printed circuit boards, metal cleaning, metal finishing, manufacturing, compliance monitoring and process control monitoring. This publication includes the majority of these presentations. In addition, in order to further facilitate information exchange and technology transfer, the US Air Force and DOE solicited additional papers under a general Call for Papers.'' These papers, which underwent review and final selection by a peer review committee, are also included in this combined Proceedings/Compendium. For those involved in handling, using or managing hazardous and toxic solvents, this document should prove to be a valuable resource, providing the most up-to-date information on current technologies and practices in solvent substitution. Individual papers are abstracted separated.

  1. Separation of 1,3-substituted imidazoles for quality control of a Lewis acidic ionic liquid for aluminum electroplating.

    PubMed

    Kosmus, Patrick; Steiner, Oliver; Goessler, Walter; Gollas, Bernhard

    2014-05-01

    Ionic liquids (ILs) are already used or have great potential in many industrial applications. Knowledge about their unique physicochemical characteristics makes ILs suitable for the electrodeposition of metals with very low negative potentials. Aluminum with its good corrosion protection behavior has great capability to be electroplated from IL electrolytes on steel substrates. The stability of the chosen electrolyte is very important to ensure industrial applicability. In this study, temperature and electrochemical long-term stability from electrolytes based on a Lewis acidic mixture of AlCl3 and 1-ethyl-3-methylimidazolium chloride are investigated. A published method was modified to identify possible degradation products using mass spectrometric detection. The optimized method used an Agilent Zorbax SB-Phenyl column (2.0 × 150 mm, 5 μm particles) with a 20 mmol TFA and 5% ACN mobile phase. This method allowed the quantification of several imidazoles from 0.1 to 100 mg/L. When analyzing the long-term stressed electrolytes, no significant changes in electrolyte composition could be observed. PMID:24254472

  2. Cytochrome c Trp65Ser substitution results in inhibition of acetic acid-induced programmed cell death in Saccharomyces cerevisiae.

    PubMed

    Guaragnella, Nicoletta; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2011-11-01

    To gain further insight into the role of cytochrome c (cyt c) in yeast programmed cell death induced by acetic acid (AA-PCD), comparison was made between wild type and two mutant cells, one lacking cyt c and the other (W65Scyc1) expressing a mutant iso-1-cyt c in a form unable to reduce cyt c oxidase, with respect to occurrence of AA-PCD, cyt c release, ROS production and caspase-like activity. We show that in W65Scyc1 cells: i. no release of mutant cyt c occurs with inhibition of W65Scyc1 cell AA-PCD shown to be independent on impairment of electron flow, ii. there is a decrease in ROS production and an increase in caspase-like activity. We conclude that cyt c release does not depend on cyt c function as an electron carrier and that when still associated to the mitochondrial membrane, cyt c in its reduced form has a role in AA-PCD, by regulating ROS production and caspase-like activity. PMID:21907312

  3. SAVANNAH RIVER SITE TANK CLEANING: CORROSION RATE FOR ONE VERSUS EIGHT PERCENT OXALIC ACID SOLUTION

    SciTech Connect

    Ketusky, E.; Subramanian, K.

    2011-01-20

    Until recently, the use of oxalic acid for chemically cleaning the Savannah River Site (SRS) radioactive waste tanks focused on using concentrated 4 and 8-wt% solutions. Recent testing and research on applicable dissolution mechanisms have concluded that under appropriate conditions, dilute solutions of oxalic acid (i.e., 1-wt%) may be more effective. Based on the need to maximize cleaning effectiveness, coupled with the need to minimize downstream impacts, SRS is now developing plans for using a 1-wt% oxalic acid solution. A technology gap associated with using a 1-wt% oxalic acid solution was a dearth of suitable corrosion data. Assuming oxalic acid's passivation of carbon steel was proportional to the free oxalate concentration, the general corrosion rate (CR) from a 1-wt% solution may not be bound by those from 8-wt%. Therefore, after developing the test strategy and plan, the corrosion testing was performed. Starting with the envisioned process specific baseline solvent, a 1-wt% oxalic acid solution, with sludge (limited to Purex type sludge-simulant for this initial effort) at 75 C and agitated, the corrosion rate (CR) was determined from the measured weight loss of the exposed coupon. Environmental variations tested were: (a) Inclusion of sludge in the test vessel or assuming a pure oxalic acid solution; (b) acid solution temperature maintained at 75 or 45 C; and (c) agitation of the acid solution or stagnant. Application of select electrochemical testing (EC) explored the impact of each variation on the passivation mechanisms and confirmed the CR. The 1-wt% results were then compared to those from the 8-wt%. The immersion coupons showed that the maximum time averaged CR for a 1-wt% solution with sludge was less than 25-mils/yr for all conditions. For an agitated 8-wt% solution with sludge, the maximum time averaged CR was about 30-mils/yr at 50 C, and 86-mils/yr at 75 C. Both the 1-wt% and the 8-wt% testing demonstrated that if the sludge was removed from

  4. Dating the origin of hepatitis B virus reveals higher substitution rate and adaptation on the branch leading to F/H genotypes.

    PubMed

    Paraskevis, Dimitrios; Angelis, Konstantinos; Magiorkinis, Gkikas; Kostaki, Evangelia; Ho, Simon Y W; Hatzakis, Angelos

    2015-12-01

    The evolution of hepatitis B virus (HBV), particularly its origins and evolutionary timescale, has been the subject of debate. Three major scenarios have been proposed, variously placing the origin of HBV in humans and great apes from some million years to only a few thousand years ago (ka). To compare these scenarios, we analyzed 105 full-length HBV genome sequences from all major genotypes sampled globally. We found a high correlation between the demographic histories of HBV and humans, as well as coincidence in the times of origin of specific subgenotypes with human migrations giving rise to their host indigenous populations. Together with phylogenetic evidence, this suggests that HBV has co-expanded with modern humans. Based on the co-expansion, we conducted a Bayesian dating analysis to estimate a precise evolutionary timescale for HBV. Five calibrations were used at the origins of F/H genotypes, D4, C3 and B6 from respective indigenous populations in the Pacific and Arctic and A5 from Haiti. The estimated time for the origin of HBV was 34.1ka (95% highest posterior density interval 27.6-41.3ka), coinciding with the dispersal of modern non-African humans. Our study, the first to use full-length HBV sequences, places a precise timescale on the HBV epidemic and also shows that the "branching paradox" of the more divergent genotypes F/H from Amerindians is due to an accelerated substitution rate, probably driven by positive selection. This may explain previously observed differences in the natural history of HBV between genotypes F1 and A2, B1, and D. PMID:26220838

  5. A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance.

    PubMed

    Ali, Akhtar; Raddatz, Natalia; Aman, Rashid; Kim, Songmi; Park, Hyeong Cheol; Jan, Masood; Baek, Dongwon; Khan, Irfan Ullah; Oh, Dong-Ha; Lee, Sang Yeol; Bressan, Ray A; Lee, Keun Woo; Maggio, Albino; Pardo, Jose M; Bohnert, Hans J; Yun, Dae-Jin

    2016-07-01

    A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K(+) TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na(+) from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K(+) transporter in the presence of Na(+) in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1(N-D)) complemented K(+)-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N) (-) (D) and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1 Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na(+) and K(+) based on the n/d variance in the pore region. This change also dictated inward-rectification for Na(+) transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats. PMID:27208305

  6. A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance1[OPEN

    PubMed Central

    Ali, Akhtar; Aman, Rashid; Park, Hyeong Cheol; Jan, Masood; Baek, Dongwon; Khan, Irfan Ullah; Oh, Dong-Ha; Lee, Sang Yeol; Bressan, Ray A.; Lee, Keun Woo; Maggio, Albino; Yun, Dae-Jin

    2016-01-01

    A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K+ TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na+ from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K+ transporter in the presence of Na+ in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T. salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1N-D) complemented K+-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1N-D and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1. Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na+ and K+ based on the n/d variance in the pore region. This change also dictated inward-rectification for Na+ transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats. PMID:27208305

  7. Comparison and preparation of multilayered polylactic acid fabric strengthen calcium phosphate-based bone substitutes for orthopedic applications.

    PubMed

    Chen, Wen-Cheng; Ko, Chia-Ling; Yang, Jia-Kai; Wu, Hui-Yu; Lin, Jia-Horng

    2016-03-01

    An attempt to maintain the three-dimensional space into restorative sites through the conveniently pack porous fillers are general used strategy. Advancement in the manufacturing protective shells in the scaffolds, which would be filled with brittle ceramic grafts for the development of highly connective pores provides the approach to solve crack problem for generating the tissues. Therefore, multilayered braided and alkalized poly(lactic acid) (PLA) composites with calcium phosphate bone cement (CPC) were synthesized and compared. The PLA/CPC composites were divided into various groups according to a series of heat-treatment temperatures (100-190 °C) and periods (1-3 h) and then characterized. The effects of 24-h immersion on the strength decay resistance of the samples were compared. Results showed that the residual oil capped on the surfaces of alkalized PLA braid was removed, and the structure was unaltered. However, the reduced tensile stress of alkalized PLA braids was due to ester-group formation by hydrolysis. Mechanical test results of PLA/CPC composites showed that the strength significantly increased after heat treatment, except when the heating temperature was higher than the PLA melting point at approximately 160-170 °C. The degree of PLA after recrystallization became higher than that of unheated composites, thereby leading to reduced strength and toughness of the specimen. Braiding fibers of biodegradable PLA reinforced and toughened the structure particularly of the extra-brittle material of thin-sheet CPC after implantation. PMID:26280316

  8. Amino acid substitutions occurring during adaptation of an emergent H5N6 avian influenza virus to mammals.

    PubMed

    Peng, Xiuming; Wu, Haibo; Peng, Xiaorong; Wu, Xiaoxin; Cheng, Linfang; Liu, Fumin; Ji, Shujing; Wu, Nanping

    2016-06-01

    Avian influenza viruses (AIVs) are known to cross species barriers, and emergent highly pathogenic H5N6 AIVs pose a serious threat to human health and the poultry industry. Here, we serially passaged an H5N6 virus 10 times in BALB/c mice. The pathogenicity of the wild-type 6D2 (WT-6D2) and mammal-adapted 6D2 strain (MA-6D2) were compared. The viral titer in multiple organs and the death rate for MA-6D2 were significantly higher than for WT-6D2. We provide evidence that the mutations HA A150V, NA R143K and G147E, PB2 E627K, and PA A343T may be important for adaptation of H5N6 AIVs to mammals. PMID:26997612

  9. Amino acid substitutions of conserved residues in the carboxyl-terminal domain of the [alpha]I(X) chain of type X collagen occur in two unrelated families with metaphyseal chondrodysplasia type Schmid

    SciTech Connect

    Wallis, G.A.; Rash, B.; Sweetman, W.A.; Thomas, J.T.; Grant, M.E.; Boot-Handford, R.P. ); Super, M. ); Evans, G. )

    1994-02-01

    Type X collagen is a homotrimeric, short-chain, nonfibrillar extracellular-matrix component that is specifically and transiently synthesized by hypertrophic chondrocytes at the site of endochondral ossification. The precise function of type X collagen is not known, but its specific pattern of expression suggests that mutations within the encoding gene (COL10A1) that alter the structure or synthesis of the protein may cause heritable forms of chondrodysplasia. The authors used the PCR and the SSCP techniques to analyze the coding and upstream promoter regions of the COL10A1 gene in a number of individuals with forms of chondrodysplasia. Using this approach, they identified two individuals with metaphyseal chondrodysplasia type Schmid (MCDS) with SSCP changes in the region of the gene encoding the carboxyl-terminal domain. Sequence analysis demonstrated that the individuals were heterozygous for two unique single-base-pair transitions that led to the substitution of the highly conserved amino acid residue tyrosine at position 598 by aspartic acid in one person and of leucine at position 614 by proline in the other. The substitution at residue 598 segregated with the phenotype in a family of eight (five affected and three unaffected) related persons. The substitutions at residue 614 occurred in a sporadically affected individual but not in her unaffected mother and brother. Additional members of this family were not available for further study. These results suggest that certain amino acid substitutions within the carboxyl-terminal domain of the chains of the type X collagen molecule cause MCDS. These amino acid substitutions are likely to alter either chain recognition or assembly of the type X collagen molecule, thereby depleting the amount of normal type X collagen deposited in the extracellular matrix, with consequent aberrations in bone growth and development. 36 refs., 5 figs.

  10. Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: identification of diagnostic marker products and biological implications.

    PubMed

    Sikora, Adam; Zielonka, Jacek; Adamus, Jan; Debski, Dawid; Dybala-Defratyka, Agnieszka; Michalowski, Bartosz; Joseph, Joy; Hartley, Richard C; Murphy, Michael P; Kalyanaraman, Balaraman

    2013-06-17

    Aromatic boronic acids react rapidly with peroxynitrite (ONOO(-)) to yield phenols as major products. This reaction was used to monitor ONOO(-) formation in cellular systems. Previously, we proposed that the reaction between ONOO(-) and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O(•-)···(•)NO2 (minor pathway). [Sikora, A. et al. (2011) Chem. Res. Toxicol. 24, 687-697]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO(-) and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO(-) was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O(•-) radical anion with subsequent reaction of the resulting phenyl radical with (•)NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of the o-MitoPhB(OH)2O(•-) radical anion is significantly lower than that of m-MitoPhB(OH)2O(•-) and p-MitoPhB(OH)2O(•-) radical anions. The nitrated product, o-MitoPhNO2, is not formed by the nitrogen dioxide radical generated by myeloperoxidase in the presence of the nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO(-). Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO(-) yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO(-) in biological systems. PMID:23611338

  11. Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers–Identification of diagnostic marker products and biological implications

    PubMed Central

    Sikora, Adam; Zielonka, Jacek; Adamus, Jan; Debski, Dawid; Dybala-Defratyka, Agnieszka; Michalowski, Bartosz; Joseph, Joy; Hartley, Richard C.; Murphy, Michael P.; Kalyanaraman, Balaraman

    2013-01-01

    Aromatic boronic acids react rapidly with peroxynitrite (ONOO−) to yield phenols as major products. This reaction was used to monitor ONOO− formation in cellular systems. Previously, we proposed that the reaction between ONOO− and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O•−…•NO2 (minor pathway). [Sikora A. et al., Chem Res Toxicol 24, 687-97, 2011]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO− and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO− was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O•− radical anion with subsequent reaction of the resulting phenyl radical with •NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of o-MitoPhB(OH)2O•− radical anion is significantly lower than that of m-MitoPhB(OH)2O•− and p-MitoPhB(OH)2O•− radical anions. The nitrated product, o-MitoPhNO2, is not formed by nitrogen dioxide radical generated by myeloperoxidase in the presence of nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO−. Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO− yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO− in biological systems. PMID:23611338

  12. The evolutionary significance of certain amino acid substitutions and their consequences for HIV-1 immunogenicity toward HLA's A*0201 and B*27.

    PubMed

    Hecht, Luke; Dormer, Anton

    2013-01-01

    In silico tools are employed to examine the evolutionary relationship to possible vaccine peptide candidates' development. This perspective sheds light on the proteomic changes affecting the creation of HLA specific T-cell stimulating peptide vaccines for HIV. Full-length sequences of the envelope protein of the HIV subtypes A, B, C and D were obtained through the NCBI Protein database were aligned using CLUSTALW. They were then analyzed using RANKPEP specific to Human Leukocyte Antigen A*02 and B*27. Geneious was used to catalogue the collected gp160 sequences and to construct a phylogenic tree. Mesquite was employed for ancestral state reconstruction to infer the order of amino acid substitutions in the epitopes examined. The results showed that consensus peptide identified SLAEKNITI had changes that indicated predicted escape mutation in strains of HIV responding to pressure exerted by CD8+ cells expressing HLA A*02. The predominating 9-mers IRIGPGQAF of gp120 are significantly less immunogenic toward HLA B*27 than to HLA A*02. The data confirms previous findings on the importance for efficacious binding, of an arginine residue at the 2(nd) position of the gag SL9 epitope, and extends this principle to other epitopes which interacts with HLA B*27. This study shows that the understanding of viral evolution relating T-cell peptide vaccine design is a development that has much relevance for the creation of personalized therapeutics for HIV treatment. PMID:23745018

  13. Development of 2-(Substituted Benzylamino)-4-Methyl-1, 3-Thiazole-5-Carboxylic Acid Derivatives as Xanthine Oxidase Inhibitors and Free Radical Scavengers.

    PubMed

    Ali, Md Rahmat; Kumar, Suresh; Afzal, Obaid; Shalmali, Nishtha; Sharma, Manju; Bawa, Sandhya

    2016-04-01

    A series of 2-(substituted benzylamino)-4-methylthiazole-5-carboxylic acid was designed and synthesized as structural analogue of febuxostat. A methylene amine spacer was incorporated between the phenyl ring and thiazole ring in contrast to febuxostat in which the phenyl ring was directly linked with the thiazole moiety. The purpose of incorporating methylene amine was to provide a heteroatom which is expected to favour hydrogen bonding within the active site residues of the enzyme xanthine oxidase. The structure of all the compounds was established by the combined use of FT-IR, NMR and MS spectral data. All the compounds were screened in vitro for their ability to inhibit the enzyme xanthine oxidase as per the reported procedure along with DPPH free radical scavenging assay. Compounds 5j, 5k and 5l demonstrated satisfactory potent xanthine oxidase inhibitory activities with IC50 values, 3.6, 8.1 and 9.9 μm, respectively, whereas compounds 5k, 5n and 5p demonstrated moderate antioxidant activities having IC50 15.3, 17.6 and 19.6 μm, respectively, along with xanthine oxidase inhibitory activity. Compound 5k showed moderate xanthine oxidase inhibitory activity as compared with febuxostat along with antioxidant activity. All the compounds were also studied for their binding affinity in active site of enzyme (PDB ID-1N5X). PMID:26575582

  14. The Amino Acid Substitution Q65H in the 2C Protein of Swine Vesicular Disease Virus Confers Resistance to Golgi Disrupting Drugs

    PubMed Central

    Vázquez-Calvo, Ángela; Caridi, Flavia; González-Magaldi, Mónica; Saiz, Juan-Carlos; Sobrino, Francisco; Martín-Acebes, Miguel A.

    2016-01-01

    Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the species Enterovirus B within the Picornaviridae family. Brefeldin A (BFA) is an inhibitor of guanine nucleotide exchange factors of Arf proteins that induces Golgi complex disassembly and alters the cellular secretory pathway. Since BFA has been shown to inhibit the RNA replication of different enteroviruses, including SVDV, we have analyzed the effect of BFA and of golgicide A (GCA), another Golgi disrupting drug, on SVDV multiplication. BFA and GCA similarly inhibited SVDV production. To investigate the molecular basis of the antiviral effect of BFA, SVDV mutants with increased resistance to BFA were isolated. A single amino acid substitution, Q65H, in the non-structural protein 2C was found to be responsible for increased resistance to BFA. These results provide new insight into the relationship of enteroviruses with the components of the secretory pathway and on the role of SVDV 2C protein in this process. PMID:27199941

  15. Synthesis and biological activities of novel 5-substituted-1,3,4-oxadiazole Mannich bases and bis-Mannich bases as ketol-acid reductoisomerase inhibitors.

    PubMed

    Zhang, Yan; Liu, Xing-Hai; Zhan, Yi-Zhou; Zhang, Li-Yuan; Li, Zheng-Ming; Li, Yong-Hong; Zhang, Xiao; Wang, Bao-Lei

    2016-10-01

    A series of novel 5-substituted-1,3,4-oxadiazole Mannich bases and bis-Mannich bases have been conveniently synthesized in good yields. Their structures were characterized by IR, (1)H NMR, (13)C NMR and elemental analysis. The preliminary bioassay results indicated that some of the compounds showed promising in vitro fungicidal activities towards several test plant fungi; some of them exhibited significant herbicidal activities against Brassica campestris and excellent in vitro inhibitory activities against rice ketol-acid reductoisomerase (KARI). Among 14 novel compounds, 8c, 8d and 8m showed potent KARI inhibitory activities with Ki value of (0.96±0.42), (3.86±0.49) and (3.10±0.71) μmol/L, respectively, and were comparable with IpOHA. These compounds could be novel KARI inhibitors for further investigation. The density functional theory (DFT) calculations and molecular docking were carried out to study the structure-activity relationship (SAR) of the active inhibitors in this Letter. PMID:27575481

  16. Master Amino acid Pattern as sole and total substitute for dietary proteins during a weight-loss diet to achieve the body's nitrogen balance equilibrium.

    PubMed

    Lucà-Moretti, M; Grandi, A; Lucà, E; Muratori, G; Nofroni, M G; Mucci, M P; Gambetta, P; Stimolo, R; Drago, P; Giudice, G; Tamburlin, N; Karbalai, M; Valente, C; Moras, G

    2003-01-01

    Results of this multicentric study have shown that by giving Master Amino acid Pattern (MAP) as a sole and total substitute of dietary proteins to 500 overweight participants undergoing the American Nutrition Clinics/Overweight Management Program (ANC/OMP), the participants' body nitrogen balance could be maintained in equilibrium with essentially no calories (MAP 1 g=0.04 kcal), thereby preserving the body's structural and functional proteins, eliminating excessive water retention from the interstitial compartment, and preventing the sudden weight increase after study conclusion commonly known as the yo-yo effect. Study results have shown that the use of MAP, in conjunction with the ANC/OMP regimen, has proven to be safe and effective by preventing those adverse effects associated with a negative nitrogen balance, such as oversized or flabby tissue, stretch marks, the sagging of breast tissue, increased hair loss, faded hair color, and fragile or brittle nails. Also prevented were those anomalies commonly associated with weight-loss diets, such as hunger, weakness, headache caused by ketosis, constipation, and decreased libido. The use of MAP in conjunction with the ANC/OMP also allowed for mean weight loss of 2.5 kg (5.5 lb) per week, achieved through reduction of excessive fat tissue and elimination of excessive water retention from the interstitial compartment. PMID:14964347

  17. Master Amino acid Pattern as substitute for dietary proteins during a weight-loss diet to achieve the body's nitrogen balance equilibrium with essentially no calories.

    PubMed

    Lucà-Moretti, M; Grandi, A; Lucà, E; Muratori, G; Nofroni, M G; Mucci, M P; Gambetta, P; Stimolo, R; Drago, P; Giudice, G; Tamburlin, N

    2003-01-01

    Results of this multicentric study have shown that by giving 10 g (10 tablets) of Master Amino acid Pattern (MAP) as a substitute for dietary proteins, once a day, to 114 overweight participants undergoing the American Nutrition Clinics/Overweight Management Program (ANC/OMP), the participants' nitrogen balance could be maintained in equilibrium with essentially no calories (MAP 1 g=0.04 kcal), thereby preserving the body's structural and functional proteins, eliminating excessive water retention from the interstitial compartment, and preventing the sudden weight increase after study conclusion commonly known as the yo-yo effect. Study results have shown that the use of MAP, in conjunction with the ANC/OMP, has proven to be safe and effective by preventing those adverse effects associated with a negative nitrogen balance, such as oversized or flabby tissue, stretch marks, sagging of breast tissue, increased hair loss, faded hair color, and fragile or brittle nails. Also preventing those anomalies commonly associated with weight-loss diets, such as hunger, weakness, headache caused by ketosis, constipation, or decreased libido, the use of MAP, in conjunction with the ANC/OMP, allowed for mean weight loss of 1.4 kg (3 lb) per week. PMID:14964348

  18. Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds.

    PubMed Central

    Tomassini, J; Selnick, H; Davies, M E; Armstrong, M E; Baldwin, J; Bourgeois, M; Hastings, J; Hazuda, D; Lewis, J; McClements, W

    1994-01-01

    Synthesis of influenza virus mRNA is primed by capped and methylated (cap 1, m7GpppXm) RNAs which the virus derives by endonucleolytic cleavage from RNA polymerase II transcripts in host cells. The conserved nature of the endonucleolytic processing provides a unique target for the development of antiviral agents for influenza viruses. A series of 4-substituted 2,4-dioxobutanoic acid compounds has been identified as selective inhibitors of this activity in both influenza A and B viruses. These inhibitors exhibited 50% inhibitory concentrations in the range of 0.2 to 29.0 microM for cap-dependent influenza virus transcription and had no effect on the activity of other viral and cellular polymerases when tested at 100- to 500-fold higher concentrations. The compounds did not inhibit the initiation or elongation of influenza virus mRNA synthesis but specifically inhibited the cleavage of capped RNAs by the influenza virus endonuclease and were not inhibitory to the activities of other nucleases. Additionally, the compounds specifically inhibited replication of influenza A and B viruses in cell culture with potencies comparable to the 50% inhibitory concentrations obtained for transcription. Images PMID:7695269

  19. N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase.

    PubMed

    Pescatori, Luca; Métifiot, Mathieu; Chung, Suhman; Masoaka, Takashi; Cuzzucoli Crucitti, Giuliana; Messore, Antonella; Pupo, Giovanni; Madia, Valentina Noemi; Saccoliti, Francesco; Scipione, Luigi; Tortorella, Silvano; Di Leva, Francesco Saverio; Cosconati, Sandro; Marinelli, Luciana; Novellino, Ettore; Le Grice, Stuart F J; Pommier, Yves; Marchand, Christophe; Costi, Roberta; Di Santo, Roberto

    2015-06-11

    Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3'-processing (3'-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC50 = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was negligible, with only a few compounds active at concentrations higher than 10 μM. The binding mode of the most potent IN inhibitor 11j within the IN catalytic core domain (CCD) is described as well as its binding mode within the RNase H catalytic site to rationalize its selectivity. PMID:25961960

  20. Attenuation of Chikungunya Virus Vaccine Strain 181/Clone 25 Is Determined by Two Amino Acid Substitutions in the E2 Envelope Glycoprotein

    PubMed Central

    Gorchakov, Rodion; Wang, Eryu; Leal, Grace; Forrester, Naomi L.; Plante, Kenneth; Rossi, Shannan L.; Partidos, Charalambos D.; Adams, A. Paige; Seymour, Robert L.; Weger, James; Borland, Erin M.; Sherman, Michael B.; Powers, Ann M.; Osorio, Jorge E.

    2012-01-01

    Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies. PMID:22457519