Sample records for lys2 hmg2 his3

  1. Quantitative changes of high mobility group non-histone chromosomal proteins HMG1 and HMG2 during rooster spermatogenesis. (United States)

    Chiva, M; Mezquita, C


    The quantitative changes of a group of non-histone chromosomal proteins identified by its solubility, electrophoretic mobility and amino acid analysis as the high mobility group proteins HMG1 and HMG2, were studied throughout rooster spermatogenesis. The ratio HMG1/HMG2 remained constant (0.66 +/- 0.04) during the transition from dividing meiotic and premeiotic cells to nondividing spermatids and from transcriptionally active cells (spermatogonia, spermatocytes and early spermatids) to transcriptionally inactive late spermatids. The ratios HMG1/nucleosomal histone and HMG2/nucleosomal histone increased markedly at the end of spermiogenesis during the transition from nucleohistone to nucleoprotamine when nucleosomes are being disassembled. The high mobility group chromosomal proteins HMG1 and HMG2 were not detectable in the nuclei of rooster spermatozoa.

  2. High affinity binding of proteins HMG1 and HMG2 to semicatenated DNA loops

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    Strauss François


    Full Text Available Abstract Background Proteins HMG1 and HMG2 are two of the most abundant non histone proteins in the nucleus of mammalian cells, and contain a domain of homology with many proteins implicated in the control of development, such as the sex-determination factor Sry and the Sox family of proteins. In vitro studies of interactions of HMG1/2 with DNA have shown that these proteins can bind to many unusual DNA structures, in particular to four-way junctions, with binding affinities of 107 to 109 M-1. Results Here we show that HMG1 and HMG2 bind with a much higher affinity, at least 4 orders of magnitude higher, to a new structure, Form X, which consists of a DNA loop closed at its base by a semicatenated DNA junction, forming a DNA hemicatenane. The binding constant of HMG1 to Form X is higher than 5 × 1012 M-1, and the half-life of the complex is longer than one hour in vitro. Conclusions Of all DNA structures described so far with which HMG1 and HMG2 interact, we have found that Form X, a DNA loop with a semicatenated DNA junction at its base, is the structure with the highest affinity by more than 4 orders of magnitude. This suggests that, if similar structures exist in the cell nucleus, one of the functions of these proteins might be linked to the remarkable property of DNA hemicatenanes to associate two distant regions of the genome in a stable but reversible manner.

  3. Characterization of the lys2 gene of Acremonium chrysogenum encoding a functional alpha-aminoadipate activating and reducing enzyme. (United States)

    Hijarrubia, M J; Aparicio, J F; Casqueiro, J; Martín, J F


    A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient mutant of P. chrysogenum (lysine auxotroph L-G-). Enzyme assays showed that the alpha-aminoadipate reductase activity was restored in all the transformants tested. The lys2-encoded enzyme catalyzed both the activation and reduction of alpha-aminoadipic acid to its semialdehyde, as shown by reaction of the product with p-dimethylaminobenzaldehyde. The reaction required NADPH, and was not observed in the presence of NADH. Sequence analysis revealed that the gene encodes a protein with relatively high similarity to members of the superfamily of acyladenylate-forming enzymes. The Lys2 protein contained all nine motifs that are conserved in the adenylating domain of this enzyme family, a peptidyl carrier domain, and a reduction domain. In addition, a new NADP-binding motif located at the N-terminus of the reduction domain that may form a Rossmann-like betaalphabeta-fold has been identified and found to be shared by all known Lys2 proteins. The lys2 gene was mapped to chromosome I (2.2 Mb, the smallest chromosome) of A. chrysogenum C10 (the chromosome that contains the "late" cephalosporin cluster) and is transcribed as a monocistronic 4.5-kb mRNA although at relatively low levels compared with the beta-actin gene.

  4. The chimeric peptide [Lys(-2)-Arg(-1)]-sarafotoxin-S6b, composed of the endothelin pro-sequence and sarafotoxin, retains the salt-bridge staple between Arg(-1) and Asp8 previously observed in [Lys(-2)-Arg(-1)]-endothelin. Implications of this salt-bridge in the contractile activity and the oxidative folding reaction. (United States)

    Aumelas, A; Chiche, L; Kubo, S; Chino, N; Watanabe, T X; Kobayashi, Y


    The chimeric peptide [Lys(-2)-Arg(-1)]-sarafotoxin-S6b (KR-SRTb) designed from the Lys-2-Arg-1 dipeptide of the endothelin pro-sequence and the sarafotoxin-S6b sequence was synthesized. Its contractile activity was found to be decreased markedly when compared with that of the parent SRTb. In contrast, the extension by the Lys-Arg dipeptide was found to increase the formation of the native disulfide isomer (82/18 versus 96/4) when the reaction was carried out in the presence of redox reagents. The solution structure of KR-SRTb was determined by NMR as a function of pH. In the carboxylic acid state, the structure consists of the cystine-stabilized alpha-helical motif, with the alpha-helical part spanning residues 9-15, and of an unstructured C-terminal tail. In the carboxylate state, the structure is characterized by a salt-bridge between Arg(-1) and Asp8, which we identified previously in the [Lys(-2)-Arg(-1)]-endothelin-1 peptide (KR-ET-1). The fact that this salt-bridge is commonly observed in KR-SRTb and KR-ET-1, despite the 33% sequence difference between the corresponding parental peptides, highlights the remarkable adaptability of the Lys-Arg extension for the formation of a special salt-bridge. As a consequence, this salt-bridge, which does not depend on either the 4-7 sequence of the loop or the C-terminal sequence, appears to be particularly well suited to improve the stability of the cystine-stabilized alpha-helical motif. Therefore, because of its high yield in the native disulfide arrangement and its high permissiveness for sequence mutation in the 4-7 loop, such a stabilized cystine-stabilized alpha-helical motif could be a valuable scaffold for the presentation of a library of constrained short peptides.

  5. High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide. (United States)

    Hoh, François; Cerdan, Rachel; Kaas, Quentin; Nishi, Yoshinori; Chiche, Laurent; Kubo, Shigeru; Chino, Naoyoshi; Kobayashi, Yuji; Dumas, Christian; Aumelas, André


    Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that

  6. Identification and characterization of cDNA sequences encoding the HIS3 and LEU2 genes of the fungus Alternaria tenuissima

    Institute of Scientific and Technical Information of China (English)

    Ying Wan; Xuli Wang; Yun Huang; Dewen Qiu; Linghuo Jiang


    Alternaria tenuissima is a fungus widely present in the environment and could cause diseases in plants and humans.In this study,through a yeast genetic approach,cDNA sequences were isolated and characterized for the AtHIS3 and AtLEU2 genes.AtHIS3 cDNA encodes a protein of 238 amino acids,while AtLEU2 cDNA encodes a protein of 363 amino acids.Based on the phylogenetic analysis of amino acid sequences of AtHis3p and AtLeu2p,A.tenuissima is closely related to the plant pathogenic fungus Phaeosphaeria nodorum.This study provides two genetic markers for studies of functions of genes regulating development,morphology,and virulence of A.tenuissima.

  7. The [Lys(-2)-Arg(-1)-des(17-21)]-endothelin-1 peptide retains the specific Arg(-1)-Asp8 salt bridge but reveals discrepancies between NMR data and molecular dynamics simulations. (United States)

    Kaas, Quentin; Aumelas, André; Kubo, Shigeru; Chino, Naoyoshi; Kobayashi, Yuji; Chiche, Laurent


    The [des(17-21)]-endothelin-1 (CSH-ET) and [Lys(-)(2)-Arg(-)(1)-des(17-21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys(-)(2)-Arg(-)(1)]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg(-)(1) and Asp(8) side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg(-)(1)-Asp(8) salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation-pi interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation-pi interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 series.

  8. [Lys(-2)-Arg(-1)]endothelin-1 solution structure by two-dimensional 1H-NMR: possible involvement of electrostatic interactions in native disulfide bridge formation and in biological activity decrease. (United States)

    Aumelas, A; Chiche, L; Kubo, S; Chino, N; Tamaoki, H; Kobayashi, Y


    Addition of the Lys(-2)-Arg(-1) dipeptide, present in the precursor protein, to the N-terminus of endothelin-1 (ET-1), to form a 23-residue peptide (KR-ET-1) has been shown to greatly improve formation of native disulfide bridges and to dramatically decrease biological activity. Conformational analysis was carried out on this peptide. During protonation of the carboxyl groups, CD spectra showed a decrease in the helical contribution, and NMR spectra displayed strong chemical shift modifications, suggesting the importance of electrostatic interactions in the KR-ET-1 conformation. CD spectra and two-dimensional NMR experiments were performed to investigate the KR-ET-1 three-dimensional structure in water in the carboxylic acid and carboxylate states. Distance and angle constraints were used as input for distance geometry calculations. The KR-ET-1 carboxylic acid conformation was found to be very similar to ET-1, with a helix spanning residues 9-15 and an unconstrained C-terminal part. In contrast, in the carboxylate state, large changes in Arg(-1) and Phe14 chemical shifts and long-range NOEs were consistent with a conformation characterized by a helix extension to Leu17 and a stabilized C-terminal section folded back toward the N-terminus. In addition, thanks to NOEs with Cys11 and Phe14, the Arg(-1) side chain appeared well-defined. Simulated annealing and molecular dynamics calculations, supported an Arg(-1)-Glu10 salt bridge and an electrostatic network involving the charged groups of Trp21, Asp18, and Lys(-2). Moreover, stabilization of the KR-ET-1 C-terminal part is probably reinforced by hydrophobic interactions involving the Val12, Tyr13, Phe14, Leu17, Ile19, Ile20, and Trp21 side chains. In vitro, native disulfide bond formation improvement observed for KR-ET-1 could be ascribed to electrostatic interactions and more specifically to the Arg(-1)-Glu10 salt bridge. In vivo, similar interactions could play an important role in the native folding of the ET-1

  9. Experiment list: SRX455442 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available azole || genotype=MATa can1-100 his3-11 leu2-3,112 lys2delta trp1-1 ura3-1 hta2-htb2delta::TRP1 HA-6His-HTB1::HIS3 || treatment=3-aminotri

  10. Experiment list: SRX455444 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available azole || genotype=MATa can1-100 his3-11 leu2-3,112 lys2delta trp1-1 ura3-1 hta2-htb2delta::TRP1 HA-6His-HTB1::HIS3 || treatment=3-aminotri

  11. Saccharomyces cerevisiae single-copy plasmids for auxotrophy compensation, multiple marker selection, and for designing metabolically cooperating communities [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Michael Mülleder


    Full Text Available Auxotrophic markers are useful tools in cloning and genome editing, enable a large spectrum of genetic techniques, as well as facilitate the study of metabolite exchange interactions in microbial communities. If unused background auxotrophies are left uncomplemented however, yeast cells need to be grown in nutrient supplemented or rich growth media compositions, which precludes the analysis of biosynthetic metabolism, and which leads to a profound impact on physiology and gene expression. Here we present a series of 23 centromeric plasmids designed to restore prototrophy in typical Saccharomyces cerevisiae laboratory strains. The 23 single-copy plasmids complement for deficiencies in HIS3, LEU2, URA3, MET17 or LYS2 genes and in their combinations, to match the auxotrophic background of the popular functional-genomic yeast libraries that are based on the S288c strain. The plasmids are further suitable for designing self-establishing metabolically cooperating (SeMeCo communities, and possess a uniform multiple cloning site to exploit multiple parallel selection markers in protein expression experiments.

  12. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae. (United States)

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji


    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

  13. Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress. (United States)

    Swinnen, Steve; Goovaerts, Annelies; Schaerlaekens, Kristien; Dumortier, Françoise; Verdyck, Pieter; Souvereyns, Kris; Van Zeebroeck, Griet; Foulquié-Moreno, María R; Thevelein, Johan M


    Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Arabidopsis CDS blastp result: AK065847 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK065847 J013042H05 At1g04880.1 high mobility group (HMG1/2) family protein / ARID/BRIGHT...ein 4 (HMG-4) (High mobility group protein 2a) (HMG-2a) {Homo sapiens}; contains Pfam profiles PF00505: HMG (high mobility group) box, PF01388: ARID/BRIGHT DNA binding domain 7e-74 ...

  15. Arabidopsis CDS blastp result: AK069900 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK069900 J023035E20 At5g23420.1 high mobility group (HMG1/2) family protein similar to high... mobility group protein 2 HMG2 [Ipomoea nil] GI:1052956; contains Pfam profile PF00505: HMG (high mobility group) box 8e-32 ...

  16. Sequence Classification: 889429 [

    Lifescience Database Archive (English)

    Full Text Available which is the fifth step in biosynthesis of lysine; activation requires posttranslational phosphopantetheinylation by Lys5p; Lys2p || ...

  17. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

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    Ping Cui


    Full Text Available The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt- phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS within the ∂ element as the position of Spt- transcription in tested Spt- mutants.

  18. Arachidonic acid alters tomato HMG expression and fruit growth and induces 3-hydroxy-3-methylglutaryl coenzyme A reductase-independent lycopene accumulation

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    Rodriguez-Concepcion, M.; Gruissem, W. [Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology


    Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, the authors manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Their results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.

  19. Experimental Study of the Burners for Liquefied Petroleum Gas%液化石油气烧嘴的试验研究

    Institute of Scientific and Technical Information of China (English)

    王璋保; 陶保国


    介绍了LYS2液化石油气烧嘴的结构特点和热工特点。它具有空-煤气混合好、调节比大、助燃空气压力低、可用于冷风或热风、适应性强等特点。%In this paper,the design and thermal characteristics of LYS2 type burner for liquefied petroleum gas are introduced. This kind of burner has good air/gas mixture ,large regulating ratio and low combustion air pressure ,and is applicable to cold or hot air.

  20. Features of Mg2Si Layer Growth in Si/Mg2Si Multilayers

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    L.E. Konotopskyi


    Full Text Available Features of magnesium siliced layer growth in Si/Mg2Si multilayers in initial state and after thermal annealing were studied by methods of transmission electron microscopy and X-Ray scattering. As-deposited magnesium silicide layers are amorphous with nanocrystal inclusions of metastable h-Mg2Si. Formation of Mg2Si in hexagonal modification occurs under the influence of stress produced by silicon layers. At T = 723 К Mg2Si layers finished crystallizes in hexagonal modification, with some coarsening of grains. That is accompanied with 7.3 % reduction in period of the Si/Mg2Si multilayer.

  1. Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination


    Ciubotaru, Mihai; Schatz, David G.


    V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed R...

  2. Molecular phylogeny of the higher and lower taxonomy of the Fusarium genus and differences in the evolutionary histories of multiple genes (United States)


    Background Species of the Fusarium genus are important fungi which is associated with health hazards in human and animals. The taxonomy of this genus has been a subject of controversy for many years. Although many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, their phylogenetic relationships remain unclear only few comprehensive phylogenetic analyses of the Fusarium genus and a lack of suitable nucleotides and amino acid substitution rates. A previous stugy with whole genome comparison among Fusairum species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history, and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need not only to check substitution rates of genes but also to perform the exact evaluation of each gene-evolution. Results We performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region (rDNA cluster), and the β-tubulin gene (β-tub), the elongation factor 1α gene (EF-1α), and the aminoadipate reductase gene (lys2). Although incongruence of the tree topologies between lys2 and the other genes was detected, all genes supported the classification of Fusarium species into 7 major clades, I to VII. To obtain a reliable phylogeny for Fusarium species, we excluded the lys2 sequences from our dataset, and re-constructed a maximum likelihood (ML) tree based on the combined data of the rDNA cluster, β-tub, and EF-1α. Our ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera. Moreover, we observed a novel evolutionary history of lys2. We suggest that the unique tree topologies of lys2 are not due to an analytical artefact, but due to differences in the evolutionary history of genomes caused by positive selection of particular lineages. Conclusion This study showed the reliable species tree of the higher and lower taxonomy

  3. Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation. (United States)

    Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette


    To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

  4. 78 FR 70839 - Day of Remembrance for President John F. Kennedy (United States)


    ... history. In his 3 years as President of the United States, John F. Kennedy weathered some of the most perilous tests of the Cold War and led America to the cusp of a bright new age. His leadership through the...

  5. Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon of Neurospora crassa

    Indian Academy of Sciences (India)

    Kandasamy Pitchaimani; Ramesh Maheshwari


    Heterokaryons of Neurospora crassa were generated by transformation of multinucleate conidia of a histidine-3 auxotroph with his-3+ plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained an ectopically integrated his-3+ allele. This response was specific to histidine. The reduction in prototrophic nuclei was confirmed by several criteria: inoculum size test, hyphal tip analysis, genomic Southern analysis, and by visual change in colour of the transformant incorporating genetic colour markers. Construction and analyses of three-component heterokaryons revealed that the change in nuclear ratio resulted from interaction of auxotrophic nucleus with prototrophic nucleus that contained an ectopically integrated his-3+ gene, but not with prototrophic nucleus that contained his-3+ gene at the normal chromosomal location. The growth rate of heterokaryons and the activity of histidinol dehydrogenase—the protein encoded by the his-3+ gene—remained unchanged despite prototrophic nuclei becoming very scarce. The results suggest that not all nuclei in the coenocytic fungal mycelium may be active simultaneously, the rare active nuclei being sufficient to confer the wild-type phenotype.

  6. Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae. (United States)

    Koning, Ann J; Larson, Lynnelle L; Cadera, Emily J; Parrish, Mark L; Wright, Robin L


    In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.

  7. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks. (United States)

    Nagaki, S; Yamamoto, M; Yumoto, Y; Shirakawa, H; Yoshida, M; Teraoka, H


    DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

  8. HMGA1与肿瘤的研究进展

    Institute of Scientific and Technical Information of China (English)

    杨霞; 李国华


    高迁移率族蛋白(high mobility group protein,HMG)由Goodwin等于1973年首次在牛胸腺细胞中发现,它是细胞核内一类水溶性强、在聚丙烯酰胺凝胶电泳中呈现高迁移率的小分子蛋白质。通常分为三大超家族HMG1/HMG2家族、HMG14/HMG17家族及HMGA1家族。2000年国际HMG学术机构根据其分子量大小、序列相识性和DNA结合特性予以重新命名为HMGA家族、HMGB家族和HMGN家族闭。HMGA可分为HMGA1和HMGA2,HMGA1分子又由HMGA1a.HMGA1b和HMGA1c 3种蛋白质组成.这三种蛋白质由同一个基因编码.

  9. The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein. (United States)

    Marrugo, J; Marsh, D G; Ghosh, B


    The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

  10. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions. (United States)

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard


    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.

  11. Wormlike micelles in mixed amino acid surfactant/nonionic surfactant aqueous systems and the effect of added electrolytes. (United States)

    Shrestha, Rekha Goswami; Rodriguez-Abreu, Carlos; Aramaki, Kenji


    The formation of viscoelastic wormlike micelles in mixed amino acid surfactant/nonionic surfactant aqueous systems in the presence of different counterions and salts is reported, and the effects of the different electrolytes on the rheological behavior are discussed. N-dodecanoylglutamic acid (LAD) is neutralized with biologically relevant L-lysine and L-arginine to obtain anionic surfactants (LAD-Lys2, LAD-Arg2) which form aqueous micellar solutions at 25 degrees C. Addition of a nonionic surfactant, tri-ethyleneglycol mono n-tetradecyl ether (C14EO3), to the aqueous solutions of both LAD-Lys2 and LAD-Arg2 causes the zero-shear viscosity (eta(0)) to increase with C14EO3 concentration gradually at first, and then sharply, indicating one-dimensional growth of the aggregates and eventual formation of entangled wormlike micelles. Further addition of C14EO3 ultimately leads to phase separation of liquid crystals. Such a phase separation, which limits the maximum attainable viscosity, takes place at lower C14EO3 concentrations for LAD-Lys2 compared to LAD-Arg2 systems. It was found that the rheological behavior of micellar solutions is significantly affected by the addition of Na+X(-) salts (X = Cl(-), Br(-), I(-), NO3(-)). The maximum viscosities obtained for the systems with added salt are all higher than that of the salt-free system, and the onset of wormlike micelle formation shift towards lower nonionic surfactant concentrations upon addition of electrolyte. The maximum attainable thickening effect of anions increases in the order NO3(-)>I(-)>Br(-)>Cl(-). The effect of temperature was also investigated. Phase separation takes place at certain temperature, which depends on the type of anion in the added salt, and decreases in the order I(-)>NO3(-)>Br(-) approximately equal Cl(-), in agreement with Hofmeister's series in terms of amphiphile solubility. The thermoresponsive rheological behavior was also found to be highly dependent on the type of anion, and anomalous

  12. Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production. (United States)

    Moura, Matthew; Pertusi, Dante; Lenzini, Stephen; Bhan, Namita; Broadbelt, Linda J; Tyo, Keith E J


    Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

  13. Purification of L-Lysine in Simulated Moving Bed and Fixed-Bed Chromatography%模拟移动床色谱及固定床色谱纯化L-赖氨酸

    Institute of Scientific and Technical Information of China (English)

    ROBATJAZI Seyed Mortaza; SHOJAOSADATI Seyed Abbas; KARBASY Seyed Mojtaba


    L-Lysine was produced by a microbial process utilizing a Corynebacterium glutamicum (ATCC 21799) strain. L-Lysine was purified from the cultivated medium by fixed-bed and simulated moving bed (SMB) chromatography. The separation conditions including pH, eluent concentration and Lys+ and Lys2+ adsorption isotherms were studied in batch adsorption. The column capacity, eluent flow rate and eluent concentration have been studied in fixed-bed chromatography. Maximum purification rate of lysine was obtained as 0.066 g/(g*h) (per gram resin and per hour) at an eluent flow rate of 10 mL/min in fixed-bed chromatography. The results obtained from SMB were 0.11 g/(g*h) for L-lysine purification rate and 96% for L-lysine recovery.

  14. Genetic analysis of methylotrophic yeast Candida boidinii PLD1. (United States)

    Lahtchev, K; Penkova, R; Ivanova, V; Tuneva, D


    This paper reports the initial experiments for genetic analysis of the haploid methylotrophic yeast Candida boidinii PLD1. The collection of multiply marked auxotrophic mutants was obtained after treatment with UV-light or X-rays. Protoplasts from several mutants were fused by the PEG-CA2+ technique and five prototrophic hybrids were isolated. The genetic structure of the hybrids was studied by means of spontaneous and induced mitotic segregation. Our data suggest that hybrids are diploids, heterozygous by parental auxotrophic markers. We obtained genetic linkage between mutations lys2-8-met-3 from one hand and ade-17-arg-24 from the other. The genetic maps constructed showed similar characteristics concerning both the order of the markers and their map distances.

  15. Enhanced stimulation of chromosomal translocations and sister chromatid exchanges by either HO-induced double-strand breaks or ionizing radiation in Saccharomyces cerevisiae yku70 mutants

    Energy Technology Data Exchange (ETDEWEB)

    Fasullo, Michael [Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12209 (United States)]. E-mail:; St Amour, Courtney [Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12209 (United States); Zeng Li [Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12209 (United States)


    DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MAT a and MAT{alpha} inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-{delta}5' and his3-{delta}3'::HOcs is enhanced in haploids and diploids expressing both MAT a and MAT{alpha} genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1::his3-{delta}3'::HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent.

  16. Lysozymes and lysozyme-like proteins from the fall armyworm, Spodoptera frugiperda. (United States)

    Chapelle, Michael; Girard, Pierre-Alain; Cousserans, François; Volkoff, Nathalie-Anne; Duvic, Bernard


    Lysozyme is an important component of the insect non-specific immune response against bacteria that is characterized by its ability to break down bacterial cell-walls. By searching an EST database from the fall armyworm, Spodoptera frugiperda (Negre et al., 2006), we identified five sequences encoding proteins of the lysozyme family. The deduced protein sequences corresponded to three classical c-type lysozymes Sf-Lys1, Sf-Lys2 and Sf-Lys3, and two lysozyme-like proteins, Sf-LLP1 and Sf-LLP2. Sf-Lys1 was purified from the hemolymph of Escherichia coli-challenged S. frugiperda larvae. The mature protein had a molecular mass of 13.975 Da with an isoelectric point of 8.77 and showed 98.3% and 96.7% identity with lysozymes from Spodoptera litura and Spodoptera exigua, respectively. As the other insect lysozymes, Sf-Lys1 was active against gram positive bacteria such as Micrococcus luteus but also induced a slight permeabilization of the inner membrane of E. coli. Genes encoding these five Sf-Lys or Sf-LLPs were differentially up-regulated in three immune-competent tissues (hemocytes, fat body and gut) after challenges with non-pathogenic bacteria, E. coli and M. luteus, or entomopathogenic bacterium, Photorhabdus luminescens. Sf-Lys1 and Sf-Lys2 were mainly induced in fat body in the presence of E. coli or P. luminescens. Sf-Lys3, which had an acidic isoelectric point, was found to be the most up-regulated of all five Sf-Lys or Sf-LLPs in hemocytes and gut after challenge with P. luminescens. More molecular data are now available to investigate differences in physiological functions of these different members of the lysozyme superfamily.

  17. Experiment list: SRX559605 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX559605 sacCer3 Input control Input control Yeast strain BY4741 NA 14288689,93.0,37.2,0 GSM1402300: Batch...nous cells || experiment_batch_number=Batch 1 || strain=BY4741:MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 || growth c

  18. Experiment list: SRX559609 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available SRX559609 sacCer3 Input control Input control Yeast strain BY4741 NA 17590483,92.6,38.7,0 GSM1402304: Batch...nous cells || experiment_batch_number=Batch 2 || strain=BY4741:MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 || growth c

  19. A BOX FULL OF KISSES%盛满亲吻的纸盒

    Institute of Scientific and Technical Information of China (English)



    @@ The story goes that some time ago,a man punished his 3-yearold daughter for wasting a roll of gold wrapping paper. Money was tight and he became infuriated when the child tried to decorate a box to put under the Christmas tree. Nevertheless, the little girl brought the gift to her father the next morning and said, "This is for you, Daddy."

  20. A Box Full of Kisses

    Institute of Scientific and Technical Information of China (English)



    The story goes that some time ago,a man pun- ished his 3-year-old daughter for wasting a roll of gold wrapping paper.Money was tight and he became infuriated when the child tried to decorate a box to put under the Christmas tree.Nevertheless,the little girl brought the gift to

  1. The Colletotrichum acutatum complex

    NARCIS (Netherlands)

    Damm, U.; Cannon, P.F.; Woudenberg, J.H.C.; Crous, P.W.


    Colletotrichum acutatum is known as an important anthracnose pathogen of a wide range of host plants worldwide. Numerous studies have reported subgroups within the C. acutatum species complex. Multilocus molecular phylogenetic analysis (ITS, ACT, TUB2, CHS-1, GAPDH, HIS3) of 331 strains previously

  2. A Forgotten Moment in Physiology: The Lovelace Woman in Space Program (1960-1962) (United States)


    published and were therefore lost to the scientific community, the physiological experimentation and testing ultimately were repeated more than a decade...recalled that he was seated at a desk in which he found a writing tablet; he wrote poetry in the dark during his 3-h test (6). Flight simulation phase

  3. Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination. (United States)

    Ciubotaru, Mihai; Schatz, David G


    V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

  4. Experiment list: SRX559604 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available cells || experiment_batch_number=Batch 1 || strain=BY4741:MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 || growth condi...SRX559604 sacCer3 TFs and others DNA-RNA hybrids Yeast strain BY4741 NA 16832766,91.8,56.5,0 GSM1402299: Bat...ch1 ChIP-seq Wild-type; Saccharomyces cerevisiae; ChIP-Seq source_name=asynchronous

  5. Experiment list: SRX559608 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available cells || experiment_batch_number=Batch 2 || strain=BY4741:MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 || growth condi...SRX559608 sacCer3 TFs and others DNA-RNA hybrids Yeast strain BY4741 NA 12026546,75.7,45.7,0 GSM1402303: Bat...ch2 ChIP-seq Wild-type; Saccharomyces cerevisiae; ChIP-Seq source_name=asynchronous

  6. Enthalpies of Solution of Complexes of Rare Earth Nitrate with L-α-Histidine in Water

    Institute of Scientific and Technical Information of China (English)

    刘洋; 房艳; 高胜利; 陈三平; 史启祯


    The enthalpies of solution in water of complexes of RE(NO3)3 (RE=La~Nd, Sm~Lu, Y) with L-α-Histidine (His) were measured at 298.15 K. The standard enthalpies of formation of RE(His)3+(aq) were calculated. The "tetrad effect" regularity was observed from the curve, which is the enthalpies of solution plotted against the atomic numbers of the elements in lanthanide series.

  7. EST Table: CA946258 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946258 KUE000165 10/09/28 90 %/133 aa ref|XP_001862705.1| histone H3.2 [Culex qui...niProt:P08898#protein_id:CAB05834.1 10/09/10 94 %/125 aa AGAP012709-PA Protein|UNKN:24531720:24532127:1|gene...4961|ref|XP_969677.1| PREDICTED: similar to His3:CG31613 CG31613-PA [Tribolium castaneum] FS918947 L9 ...

  8. Cellular plasticity enables adaptation to unforeseen cell-cycle rewiring challenges.

    Directory of Open Access Journals (Sweden)

    Yair Katzir

    Full Text Available The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is

  9. Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay. (United States)

    Howlett, N G; Schiestl, R H


    The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.

  10. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody. (United States)

    Chai, Ning; Swem, Lee R; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah


    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

  11. Carbohydrate analysis on hybrid poly(dimethylsiloxane)/glass chips dynamically coated with ionic complementary peptide. (United States)

    Li, Nan; Hai, Xiaoman; Yu, Xiaoling; Dang, Fuquan


    A facile and efficient dynamic coating method using an ionic complementary peptide was established for high-performance separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled carbohydrates in a hybrid poly(dimethylsiloxane) (PDMS)/glass microfluidic channel. EAK16-II with a sequence of [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] can readily self-organize into a complete coating layer tightly adsorbed on both hydrophobic PDMS and hydrophilic glass surfaces, which efficiently suppressed nonspecific analyte adsorption and minimized electroosmotic flow (EOF). Separation conditions were systematically investigated with respect to EAK16-II concentration, running buffer, buffer pH, and field strength (Esep). Under the optimal conditions, rapid and reproducible separations of maltodextrin ladder, glycans from glucosamine capsules, tablets, and pomegranate peel extracts were achieved with over 450000 theoretical plates per meter in the hybrid PDMS/glass microchannels dynamically coated with 1.0mg/mL EAK16-II-0.05% n-dodecyl β-d-maltoside (DDM), and the relative standard deviation (RSD) values were less than 3.2% (n=4) for the migration times. The present work provides a facile and efficient means to minimize EOF and nonspecific analyte adsorption in microfluidic chips fabricated in various substrates, thereby broadening the applications of microfluidic chips in complicated biological assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Decreased uv mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, L.; Hinkle, D.; Prakash, S.


    A DNA replication mutant of yeast, cdc8, was found to decrease uv-induced reversion of lys2-1, arg4-17, tryl and ural. This effect was observed with all three alleles of cdc8 tested. Survival curves obtained following uv irradiation in cdc8 rad double mutants show that cdc8 is epistatic to rad6, as well as to rad1; cdc8 rad51 double mutants seem to be more sensitive than the single mutants. Since uv-induced reversion in cdc8 rad1 and cdc8 rad51 double mutants is like that of the cdc8 single mutants, we conclude that CDC8 plays a direct role in error-prone repair. To test whether CDC8 codes for a DNA polymerase, we have purified both DNA polymerase I and DNA polymerase II from cdc8 and CDC+ cells. The purified DNA polymerases from cdc8 were no more heat labile than those from CDC+, suggesting that CDC8 is not a structural gene for either enzyme.

  13. Defects in Base Excision Repair Sensitize Cells to Manganese in S. cerevisiae

    Directory of Open Access Journals (Sweden)

    Adrienne P. Stephenson


    Full Text Available Manganese (Mn is essential for normal physiologic functioning; therefore, deficiencies and excess intake of manganese can result in disease. In humans, prolonged exposure to manganese causes neurotoxicity characterized by Parkinson-like symptoms. Mn2+ has been shown to mediate DNA damage possibly through the generation of reactive oxygen species. In a recent publication, we showed that Mn induced oxidative DNA damage and caused lesions in thymines. This study further investigates the mechanisms by which cells process Mn2+-mediated DNA damage using the yeast S. cerevisiae. The strains most sensitive to Mn2+ were those defective in base excision repair, glutathione synthesis, and superoxide dismutase mutants. Mn2+ caused a dose-dependent increase in the accumulation of mutations using the CAN1 and lys2-10A mutator assays. The spectrum of CAN1 mutants indicates that exposure to Mn results in accumulation of base substitutions and frameshift mutations. The sensitivity of cells to Mn2+ as well as its mutagenic effect was reduced by N-acetylcysteine, glutathione, and Mg2+. These data suggest that Mn2+ causes oxidative DNA damage that requires base excision repair for processing and that Mn interferes with polymerase fidelity. The status of base excision repair may provide a biomarker for the sensitivity of individuals to manganese.

  14. Toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three popular DNA intercalating fluorescent dyes. (United States)

    Sayas, Enric; García-López, Federico; Serrano, Ramón


    We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Peptide environment specifies conformation. Helicity of hydrophobic segments compared in aqueous, organic, and membrane environments. (United States)

    Li, S C; Deber, C M


    Transmembrane segments in integral membrane proteins exist characteristically as helices in lipid bilayers, yet are often rich in residues considered helix-destabilizing (Val, Ile, Gly) in soluble proteins. We propose that helicity of a transmembrane segment is likely to be affected by factors other than the "intrinsic" helical propensities of its component amino acids. This hypothesis is tested by comparing the conformation(s) in aqueous, organic, membrane-mimetic (micellar), and membrane (bilayer) environments of designed model peptides with systematically altered helical propensity and/or segmental hydrophobicity. Peptides of prototypic sequence NH2-(Ser-Lys)2-Ala5-Leu6-Ala7-Ala8-Leu9-Ala10-++ +Trp11-Ala12-Leu13-Ala14- (Lys-Ser)3-OH were synthesized, which incorporate a hydrophobic core "guest" segment (residues 5-14) into a water-soluble hydrophilic host matrix. Related peptides featured substitution of Leu6,9,13-->Gly, Leu6,9,13-->Ala, and Ala7,10,14-->Gly. Circular dichroism spectra revealed that algorithms for soluble proteins correctly predicted peptide helical proclivities in aqueous solutions, but peptide helicity in organic (trifluoroethanol) solvents, membrane-mimetic SDS micelles, and negatively charged lipid bilayer vesicles, was found to be governed almost exclusively by the segmental hydrophobicity of the peptide mid-hydrophobic core segment. In related Trp fluorescence studies, peptide-membrane association was similarly correlated with extent of hydrophobic interaction.

  16. Manipulation of peptide conformations by fine-tuning of the environment and/or the primary sequence. (United States)

    Li, S C; Kim, P K; Deber, C M


    The widely observed phenomenon that peptides are capable of adopting multiple conformations in different environments suggests that secondary structure formation in a peptide segment is a process involving not only the peptide itself but also the surrounding solvent media. The influence of the primary sequence and the molecular environment on peptide conformations are now investigated using synthetic peptides of amino acid sequence H2N-(Ser-Lys)2-Ala-X-Gly-Ala-X-Gly-Trp-Ala-X-Gly-(Lys-Ser)3-OH, where X = Ile or Val. These two peptides, namely 3I (X = Ile) and 3V (X = Val), are found to lack defined secondary structure in aqueous buffer. However, discrete conformational states, e.g., alpha-helices and beta-sheets, are readily generated and interconverted for both peptides when the buffer is modulated with the addition of methanol, sodium dodecyl sulfate (SDS) micelles, or phospholipid vesicles. The role of the primary sequence in affecting peptide conformations is manifested in that peptides 3I and 3V, which differ respectively in their content of beta-branched Ile or Val residues, differ in their secondary structures at monomeric concentrations in 2 mM SDS and in mixed lipid vesicles of phosphatidic acid and phosphatidylcholine. The overall results suggest that peptide segments can be conformationally flexible entities poised to react to minor modulation in either the molecular environment or the primary sequence, a circumstance that may be relevant to protein functioning and folding.

  17. Effect of Heat Treatment and Salt Concentration on Free Amino Acids Composition of Sudanese Braided (Muddaffara Cheese during Storage

    Directory of Open Access Journals (Sweden)

    Mohamed O. E. Altahir


    Full Text Available The aim of this study was to assess the effect of heat treatment and salt concentrations (0, 5, and 10% on the free amino acids (FAA composition of Sudanese braided cheese (BC ripened for up to 3 months at 5±2°C. Heat and salt concentration significantly affected the FAA of braided cheese. The free amino acids concentrations of BC ripened in 0%, 5%, and 10% salted whey (SW were significantly fluctuated. Under ripening conditions tested (salt level + time, braided cheese made from pasteurized milk (BCPM had consistently lower values of FAA than braided cheese made from raw milk (BCRM. In fresh cheese, the major FAA in BCRM were Glu (36.12 nmol/ml, Leu (26.77nmol/ml and Lys (14.51 nmol/ml while the major ones in BCPM were Lys (2.94 nmol/ml and Ala (2.45 nmol/ml. BCPM stored in 10% SW had shorter quality life compared to that stored in 5% salted whey.

  18. Actin filament attachments for sustained motility in vitro are maintained by filament bundling.

    Directory of Open Access Journals (Sweden)

    Xiaohua Hu

    Full Text Available We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+, Lys-Lys(2+, or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+ buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.

  19. Synthetic Cyclolipopeptides Selective against Microbial, Plant and Animal Cell Targets by Incorporation of D-Amino Acids or Histidine. (United States)

    Vilà, Sílvia; Badosa, Esther; Montesinos, Emilio; Planas, Marta; Feliu, Lidia


    Cyclolipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln) (BPC194) were prepared on solid-phase and screened against four plant pathogens. The incorporation at Lys5 of fatty acids of 4 to 9 carbon atoms led to active cyclolipopeptides. The influence on the antimicrobial activity of the Lys residue that is derivatized was also evaluated. In general, acylation of Lys1, Lys2 or Lys5 rendered the sequences with the highest activity. Incorporation of a D-amino acid maintained the antimicrobial activity while significantly reduced the hemolysis. Replacement of Phe with a His also yielded cyclolipopeptides with low hemolytic activity. Derivatives exhibiting low phytotoxicity in tobacco leaves were also found. Interestingly, sequences with or without significant activity against phytopathogenic bacteria and fungi, but with differential hemolysis and phytotoxicity were identified. Therefore, this study represents an approach to the development of bioactive peptides with selective activity against microbial, plant and animal cell targets. These selective cyclolipopeptides are candidates useful not only to combat plant pathogens but also to be applied in other fields.

  20. Synthetic Cyclolipopeptides Selective against Microbial, Plant and Animal Cell Targets by Incorporation of D-Amino Acids or Histidine.

    Directory of Open Access Journals (Sweden)

    Sílvia Vilà

    Full Text Available Cyclolipopeptides derived from the antimicrobial peptide c(Lys-Lys-Leu-Lys-Lys-Phe-Lys-Lys-Leu-Gln (BPC194 were prepared on solid-phase and screened against four plant pathogens. The incorporation at Lys5 of fatty acids of 4 to 9 carbon atoms led to active cyclolipopeptides. The influence on the antimicrobial activity of the Lys residue that is derivatized was also evaluated. In general, acylation of Lys1, Lys2 or Lys5 rendered the sequences with the highest activity. Incorporation of a D-amino acid maintained the antimicrobial activity while significantly reduced the hemolysis. Replacement of Phe with a His also yielded cyclolipopeptides with low hemolytic activity. Derivatives exhibiting low phytotoxicity in tobacco leaves were also found. Interestingly, sequences with or without significant activity against phytopathogenic bacteria and fungi, but with differential hemolysis and phytotoxicity were identified. Therefore, this study represents an approach to the development of bioactive peptides with selective activity against microbial, plant and animal cell targets. These selective cyclolipopeptides are candidates useful not only to combat plant pathogens but also to be applied in other fields.

  1. Quantitation and analysis of the formation of HO-endonuclease stimulated chromosomal translocations by single-strand annealing in Saccharomyces cerevisiae. (United States)

    Liddell, Lauren; Manthey, Glenn; Pannunzio, Nicholas; Bailis, Adam


    Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms(1-3). The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs(4). Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases(5), as well as cancer in humans(6-9). Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange(10,11). The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-Δ3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-Δ5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for


    Institute of Scientific and Technical Information of China (English)

    曾柳根; 徐灵; 王军花; 盛军庆; 辜清; 彭扣; 洪一江


    The Sox genes family comprises several transcription factors that share a highly conserved HMG (High-Mobility-Group) box and has been studied in many species, including a variety of vertebrates and several of invertebrates such as fruit fly, nematode and Portunustrituberculatus. But there are only few reports on Sox gene family of freshwater bivalve so far, which have important value in evolution and production. H. Schlegelii, which originated from Lake Biwa in Japan and was introduced into China in 1998, is one of the representative freshwater pearl mussels. It has been widely applied in the Chinese freshwater pearl industry for its high quality pearl bearing ability. In order to know the function of Sox genes in this mussel, a degenerate PCR, referred to the HMG box of human SRY gene, was used to amplify the conserved sequence of HMG domains of Sox genes. Two different HMG sequences were got from DNA and the testis cDNA, named DNA-HMG1, DNA-HMG2 and cDNA-HMG. Amino acids sequences analysis showed that those HMG sequences had high homology with the Sox1, Sox2, Sox3 and Sox 14 genes from other animals including human being. But there showed no difference between the male and female. A partial cDNA sequence of Sox2 gene (refereed as hs-Sox2) with 1774 bp including partial ORF and complete 3' untranslated region (UTR) was cloned from the testis cDNA by RACE-PCR methods. DNA sequences analysis showed that it had high homology with the SoxB gene in Patella vulgata and the Soxl gene in human being. The putative 249 amino acid sequence contained one conserved HMG box like the human SYR gene, and exhibited 98% homology with human, mouse, chicken and zebrafish. For further know the expression level of Sox2 gene, real-time PCR method was used to examine its mRNA level in different tissues and different months on testis of H. Schlegelii. Results showed that the hs-Soxl mRNA was ubiquitously expressed in all the tissues, with the highest in kidney, followed by intestine

  3. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin. (United States)

    Brophy, Megan Brunjes; Hayden, Joshua A; Nolan, Elizabeth M


    Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His(3)Asp and His(4) sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His(3)Asp and His(4) sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 affords dissociation constants of K(d1) = 133 ± 58 pM and K(d2) = 185 ± 219 nM for CP in the absence of Ca(II). These values decrease to K(d1) ≤ 10 pM and K(d2) ≤ 240 pM in the presence of excess Ca(II). The K(d1) and K(d2) values are assigned to the His(3)Asp and His(4) sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.

  4. Assessment of the effect of methionine supplementation and inclusion of hydrolyzed wheat protein in milk protein-based milk replacers on the performance of intensively fed Holstein calves. (United States)

    Castro, J J; Hwang, G H; Saito, A; Vermeire, D A; Drackley, J K


    The objectives of this study were to compare 2 milk replacers containing only milk proteins with or without supplemental Met, and to compare a milk replacer containing hydrolyzed wheat protein at 4.5% of dry matter (DM) and supplemental Lys and Met against the 2 all-milk-protein formulas, by assessing their effect on the growth performance, efficiency, and plasma urea nitrogen of pre-weaning Holstein calves. Thus, 57 Holstein calves were allotted to the following 3 treatments: (1) a skim milk plus whey protein concentrate-based milk replacer (SMWP) containing about 2.6% Lys and 0.6% Met on a DM basis; (2) SMWP + M based on skim milk and whey proteins, containing about 2.6% Lys, and supplemental Met to reach 0.9% on a DM basis; and (3) a skim milk plus whey protein concentrate plus 4.5% of the DM as hydrolyzed wheat protein based milk replacer (HWP + LM) where the wheat protein replaced 50% of the whey protein concentrate, and also contained supplemental Lys and Met to match the profile of SMWP + M (i.e., Lys 2.6 and Met 0.9% on DM basis). No difference in any of the responses was observed by supplementing the milk protein based formula with Met or when hydrolyzed wheat protein was added to the formula. Results indicate that (1) a milk replacer based on skim milk protein and whey protein with a Lys concentration of ~2.6% does not benefit from Met supplementation, and (2) milk replacer containing 4.5% of the DM as hydrolyzed wheat protein and supplemented with Lys and Met can support the same growth performance as milk protein-based formulas.

  5. Cost-effectiveness of posaconazole versus fluconazole or itraconazole in the prevention of invasive fungal infections among high-risk neutropenic patients in Spain

    Directory of Open Access Journals (Sweden)

    Grau Santiago


    Full Text Available Abstract Background We evaluated the cost-effectiveness of posaconazole compared with standard azole therapy (SAT; fluconazole or itraconazole for the prevention of invasive fungal infections (IFI and the reduction of overall mortality in high-risk neutropenic patients with acute myelogenous leukaemia (AML or myelodysplastic syndromes (MDS. The perspective was that of the Spanish National Health Service (NHS. Methods A decision-analytic model, based on a randomised phase III trial, was used to predict IFI avoided, life-years saved (LYS, total costs, and incremental cost-effectiveness ratio (ICER; incremental cost per LYS over patients' lifetime horizon. Data for the analyses included life expectancy, procedures, and costs associated with IFI and the drugs (in euros at November 2009 values which were obtained from the published literature and opinions of an expert committee. A probabilistic sensitivity analysis (PAS was performed. Results Posaconazole was associated with fewer IFI (0.05 versus 0.11, increased LYS (2.52 versus 2.43, and significantly lower costs excluding costs of the underlying condition (€6,121 versus €7,928 per patient relative to SAT. There is an 85% probability that posaconazole is a cost-saving strategy compared to SAT and a 97% probability that the ICER for posaconazole relative to SAT is below the cost per LYS threshold of €30,000 currently accepted in Spain. Conclusions Posaconazole is a cost-saving prophylactic strategy (lower costs and greater efficacy compared with fluconazole or itraconazole in high-risk neutropenic patients.

  6. Molecular Characterization and In Silico Analysis of the Pheromone-Binding Protein of the European Grapevine Moth Lobesia botrana (Denis & Schiffermüller) (Lepidoptera, Tortricidae). (United States)

    Mutis, A; Palma, R; Venthur, H; Iturriaga-Vásquez, P; Faundez-Parraguez, M; Mella-Herrera, R; Kontodimas, D; Lobos, C; Quiroz, A


    The European grapevine moth Lobesia botrana (Denis & Schiffermüller) is an economically important insect in Europe. The species invaded vineyards in Chile, Argentina, and California during 2008-2010 causing severe problems. A major component of the sex pheromone, (E,Z)-7,9-dodecadienyl acetate (E7,Z9-12:Ac), is used in a mating disruption technique when grapevine moth populations are low or to monitor pest numbers. It is thought that these sexual pheromones are blends of volatiles that typically are specific to a species and are transported in the insect antenna by pheromone-binding proteins (PBPs) across the sensillar lymph to the olfactory receptors. Currently, an increasing number of Lepidopteran PBPs are being identified and cloned. However, there are no studies of the olfactory system and of proteins involved in the olfactory perception of L. botrana at the molecular level. In the present study, we report, for the first time, the sequence of a PBP from L. botrana (LbotPBP), which was determined using reverse transcription technology. Homology modeling was used to generate the three-dimensional protein structure. The model suggests that PBP consists of six α-helices as follows: Lys2-Met23 (α1), Thr28-Phe36 (α2), Arg46-Leu59 (α3), His70-Asn80 (α4), Glu84-Asn100 (α5), and Cys108-Lys125 (α6), held together by three disulfide bridges, Cys19-Cys54, Cys50-Cys108, and Cys97-Cys117. Docking simulations based on this model suggested that Trp114 is a key residue in the recognition of acetate pheromones, such as E7,Z9-12:Ac. In silico results in this study are consistent with previous findings in which E7,Z9-12:Ac acts as the most active compound in behavioral and electroantennographic assays.

  7. Influence of glycine residues on peptide conformation in membrane environments. (United States)

    Li, S C; Deber, C M


    Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, yet their primary sequences are rich in residues known in globular proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have now designed and synthesized a series of model 20-residue peptides with "guest" hydrophobia segments embedded in "host" N- and C-terminal hydrophilic matrices. Molecular design was based on the prototypical sequence NH2-(Ser-Lys)2-Ala5-Leu6-x7-Ala8-Leu9-y10-Trp 11-Ala12-Leu13-z14-(Lys-Ser)3-OH. The 10-residue hydrophobic mid-segment 5-14 is expected to act as ca. three turns of an alpha-helix. In the present work, we compare the 20-residue peptide having three "helix-forming" Ala residues [x = y = z = Ala (peptide 3A)] to the corresponding peptide 3G (x = y = z = Gly) which contains three "helix-breaking" Gly residues. Trp was inserted to provide a measure of aromatic character typical of TM segments; Ser and Lys enhanced solubility in aqueous media. Circular dichroism studies in water, in a membrane-mimetic [sodium dodecylsulfate (SDS)], medium, and in methanol solutions, demonstrated the exquisite sensitivity of the conformations of these peptides to environment, and proved that despite its backbone flexibility, Gly can be accommodated as readily as Ala into a hydrophobic alpha-helix in a membrane. Nevertheless, the relative stability of Ala- vs. Gly-containing helices emerged in methanol solvent titration and temperature dependence experiments in SDS.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Dietary lysine imbalance affects muscle proteome in zebrafish (Danio rerio): a comparative 2D-DIGE study. (United States)

    de Vareilles, Mahaut; Conceição, Luis E C; Gómez-Requeni, Pedro; Kousoulaki, Katerina; Richard, Nadège; Rodrigues, Pedro M; Fladmark, Kari E; Rønnestad, Ivar


    Lysine (Lys) is an indispensable amino acid (AA) and generally the first limiting AA in vegetable protein sources in fish feeds. Inadequate dietary Lys availability may limit protein synthesis, accretion and growth of fish. This experiment aimed to further elucidate the role of Lys imbalance on growth by examining the myotomal muscle proteome of juvenile zebrafish (Danio rerio). Quadruplicate groups of 8 fish were fed either a low-Lys [Lys(-), 1.34 g kg(-1)], medium/control (Lys, 2.47 g kg(-1)) or high-Lys [Lys(+), 4.63 g kg(-1)] diet. Fish growth was monitored from 33 to 49 days post-fertilization (dpf) and trunk myotomal muscle proteome of Lys(-) and Lys(+) treatments were screened by 2D-DIGE and MALDI ToF tandem mass spectrometry. Growth rate was negatively affected by diet Lys(-). Out of 527 ± 11 (mean ± S.E.M.) protein spots detected (∼10-150 kDa and 4-7 pI value), 30 were over-expressed and 22 under-expressed in Lys(-) fish (|fold-change| >1.2, p value muscle protein accretion. The Lys deficiency also possibly induced a higher feeding activity, reflected in the over-expression of beta enolase and mitochondrial ATP synthase. Contrarily, in the faster growing fish [Lys(+)], over-expression of apolipoprotein A-I, F-actin capping protein and Pdlim7 point to increased energy storage as fat and enhanced muscle growth, particularly by mosaic hyperplasia. Thus using an exploratory approach, this study pinpoints interesting candidates for further elucidating the role of dietary Lys on growth of juvenile fish.

  9. In vitro and in vivo studies of the effects of halogenated histidine analogs on Plasmodium falciparum.


    Panton, L J; Rossan, R N; Escajadillo, A; Matsumoto, Y; Lee, A.T.; Labroo, V M; Kirk, K L; Cohen, L. A.; Aikawa, M.; Howard, R J


    The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by t...

  10. Hypnotic imagery rehearsal in the treatment of nightmares: a case report. (United States)

    Donatone, Brooke


    This case report discusses a patient who experienced frequent nightmares and chronic low-level anxiety during his 3 1/2 year imprisonment. He developed post traumatic stress disorder (PTSD), in part because he adamantly insisted that he had been wrongfully incarcerated. The literature supports the use of hypnotic imagery rehearsal for treating nightmares that stem from PTSD. Due to the patient's distrust of others and trauma history, it was uncertain whether hypnotic intervention would be effective. It is of note, there is no indication in the literature that hypnosis has been used with people on parole, let alone individuals who believe they were wrongly accused of committing a crime.

  11. EST Table: CA946024 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946024 KI000061 10/09/28 87 %/114 aa ref|XP_001983086.1| GG16350 [Drosophila erec.../114 aa ZK131.7#CE03253#WBGene00001887#locus:his-13#histone H3#status:Predicted#UniProt:P08898#protein_id:CA...:AGAP012709 10/09/10 87 %/114 aa gnl|Amel|GB14620-PA 10/09/10 87 %/114 aa gi|91094961|ref|XP_969677.1| PREDICTED: similar to His3:CG31613 CG31613-PA [Tribolium castaneum] FS918947 L9 ...

  12. EST Table: CA946026 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946026 KI000063 10/09/28 low homology 10/08/30 low homology 10/08/28 78 %/114 aa ...ZK131.7#CE03253#WBGene00001887#locus:his-13#histone H3#status:Predicted#UniProt:P08898#protein_id:CAB05834.1...l|Amel|GB14620-PA 10/09/10 80 %/114 aa gi|91094961|ref|XP_969677.1| PREDICTED: similar to His3:CG31613 CG31613-PA [Tribolium castaneum] FS918947 L9 ...

  13. A novel method for repair of testis rupture after gunshot trauma: repair with Tutoplast processed pericardium. (United States)

    Ciftci, Seyfettin; Ozkul, Bahattin; Ustuner, Murat; Nagihanlnan; Culha, Mustafa Melih


    Gunshot wound injury to the external genitalia is relatively uncommon. However, if a testis isaffected in such a case, early surgical exploration should be carried out. A 16-year-old boy presented with right testicular rupture. Tunica albugineal defect could not be closed primarily. We used a Tutoplast® processed bovine pericardium to repair the defect of tunica albuginea. At his 3-month follow-up visit, there was no complication. Doppler blood flow of testis was normal. In this case, preservation of testis tissue was obtained with early exploration and repair of the big tunica albugineal defect with Tutoplast® processed pericardium.

  14. Construction of Yeast Vectors with Resistance to Geneticin

    Institute of Scientific and Technical Information of China (English)

    林会兰; 张广; 周全; 陈国强


    Two Escherichia coli-Saccharomyces cerevisiae shuttle vectors containing a resistance marker to geneticin (G418) are constructed. Both vectors contain a kanamycin-resistant marker (KanMX4) module coding aminoglycoside 3'-phosphotransferase (APH) that renders E. coli resistant to kanamycin and S. cerevisiae to geneticin. These vectors overcome the shortage of the conventional yeast vectors bearing HIS3, TRP1, LEU2, and URA3 modules as selection markers, which require hosts to be auxotrophic. Green fluorescent protein (GFP) is used as the reporter to examine the functions of the vectors. The vectors are powerful tools for the convenient cloning and controlled expression of genes in most S. cerevisiae strains.

  15. Analysis of interchromosomal mitotic recombination. (United States)

    McGill, C B; Shafer, B K; Higgins, D R; Strathern, J N


    A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

  16. Inherited adaptation of genome-rewired cells in response to a challenging environment (United States)

    David, Lior; Stolovicki, Elad; Haziz, Efrat; Braun, Erez


    Despite their evolutionary significance, little is known about the adaptation dynamics of genomically rewired cells in evolution. We have confronted yeast cells carrying a rewired regulatory circuit with a severe and unforeseen challenge. The essential HIS3 gene from the histidine biosynthesis pathway was placed under the exclusive regulation of the galactose utilization system. Glucose containing medium strongly represses the GAL genes including HIS3 and these rewired cells are required to operate this essential gene. We show here that although there were no adapted cells prior to the encounter with glucose, a large fraction of cells adapted to grow in this medium and this adaptation was stably inherited. The adaptation relied on individual cells that switched into an adapted state and, thus, the adaptation was due to a response of many individual cells to the change in environment and not due to selection of rare advantageous phenotypes. The adaptation of numerous individual cells by heritable phenotypic switching in response to a challenge extends the common evolutionary framework and attests to the adaptive potential of regulatory circuits. PMID:20811567

  17. Microsatellite-based genotyping of Candida albicans isolated from patients with superficial candidiasis. (United States)

    Shimizu, Kazue; Hattori, Hisao; Adachi, Hidesada; Oshima, Ryosuke; Horii, Toshinobu; Tanaka, Reiko; Yaguchi, Takashi; Tomita, Yasushi; Akiyama, Masashi; Kawamoto, Fumihiko; Kanbe, Toshio


    This study aimed to examine the genotype distribution of Candida albicans and the major genotypes involved in superficial candidiasis. The genotypes of C. albicans isolated from the infection sites of patients with superficial candidiasis (referred to as infection isolates) were analyzed by fragment analysis using 4 microsatellite markers (HIS3, CDC3, CAI and CAIII). Genotypes of the infection isolates were compared with those of C. albicans isolated from oral mucosa of non-candidiasis patients (referred to as oral isolates). Isolates of C. albicans showed 4 major genotypes for HIS3/CAI (" a " for 148 : 148 / 23 : 23," b " for 148 : 160 / 33 : 41," c " for 148 : 164 / 32 : 41 and " d " for 152 : 152 / 18 : 27). The genotypes " a "," b " and " d " were commonly found in oral (4.7, 8.8 and 7.6%, respectively) and infection (6.6, 9.2 and 15.4%, respectively) isolates. No isolates of genotype " c " were isolated from infection sites. The genotype " a " was found in the isolates from patients with genitalia candidiasis. Genotyping of multiple isolates from an individual patient showed that C. albicans from infection sites was genetically homogenous as compared with that of oral isolates, even in the same patient with candidiasis.

  18. Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

    Energy Technology Data Exchange (ETDEWEB)

    Nikolics, K.; Szonyi, E.; Ramachandran, J.


    Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl (2,4(5)-NAPS) group into amino acid residues at position 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp/sup 3/ by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The (D-Lys(2,4-NAPS))/sup 6/ analog was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. (Orn(2,4-NAPS))/sup 8/-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, (Orn(2,5-NAPS))/sup 8/-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, (Orn(2,4-NAPS))/sup 8/GnRH is very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe/sup 6/, (Orn(2,4-NAPS))/sup 8/-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a K/sub d/ comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

  19. Effect of Lysine to Digestible Energy Ratio on Growth Performance and Carcass Characteristics in Finishing Pigs

    Directory of Open Access Journals (Sweden)

    S. B. Cho


    Full Text Available This experiment was performed to investigate the effects of lysine (Lys to DE ratio on growth performance, and carcass characterics in finishing barrows. Ninety six cross-bred finishing barrows ((Landrace×Yorkshire ×Duroc, average BW 58.25±0.48 kg were assigned as a randomized complete block design by 2 energy levels and 4 Lys:DE ratios on the basis of BW to one of 8 treatments with 3 replications with 4 animals per pen. The levels of DE and Lys:DE ratio for each treatment were i DE 3.35 Mcal/kg, 1.5 g Lys/Mcal DE, ii DE 3.35 Mcal/kg, 1.8 g Lys/Mcal DE, iii DE 3.35 Mcal/kg, 2.1 g Lys/Mcal DE, iv DE 3.35 Mcal/kg, 2.4 g Lys/Mcal DE, v DE 3.60 Mcal/kg, 1.5 g Lys/Mcal DE, vi DE 3.60 Mcal/kg, 1.8 g Lys/Mcal DE, vii DE 3.60 Mcal/kg, 2.1 g Lys/Mcal DE, viii DE 3.60 Mcal/kg, 2.4 g Lys/Mcal DE. During finishing period from 58 kg to 103 kg of BW, increased energy density in the diet increased (p<0.05 ADG and gain:feed ratio, but did not influence ADFI. As Lys:DE ratio was increased, ADG, ADFI and gain:feed ratio were improved in finishing barrows (p<0.05. There were positive interactions (p<0.05 between carcass weight, grade, and backfat thickness and energy density and Lys level (p<0.05. In conclusion, data from our current study suggest that maximum yields including ADG, gain:feed ratio, carcass weight and grade can be achieved by administrating finishing pigs with an ideal Lys:DE ratio, Lys 2.1 g/DE Mcal.

  20. Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1

    Directory of Open Access Journals (Sweden)

    Rolain Thomas


    Full Text Available Abstract Background Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the

  1. The chaperone like function of the nonhistone protein HMGB1

    Energy Technology Data Exchange (ETDEWEB)

    Osmanov, Taner; Ugrinova, Iva [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria); Pasheva, Evdokia, E-mail: [Institute of Molecular Biology, Bulgarian Academy of Sciences (Bulgaria)


    -synthetic acetylation for the chaperone function of HMGB1 protein. The presence of an acetyl groups at Lys 2 decreases strongly the stimulating effect of the protein in the stepwise salt dialysis experiment and the same tendency persisted in the dialysis free experiment.

  2. Prevention of acute exacerbations of chronic obstructive bronchitis with carbocysteine lysine salt monohydrate: a multicenter, double-blind, placebo-controlled trial. (United States)

    Allegra, L; Cordaro, C I; Grassi, C


    The efficacy and safety of carbocysteine lysine salt monohydrate (SCMC-Lys) in the prevention of acute exacerbations associated with chronic obstructive bronchitis were evaluated in a multicenter double-blind, placebo-controlled, parallel group trial in 662 outpatients. After a 1-month run-in period, patients were randomized to three groups and assigned to receive one of the following oral treatments: continuous SCMC-Lys 2.7 g once daily, intermittent SCMC-Lys at the same dosage (1-week courses alternating with 1-week intervals on placebo) or placebo. Each treatment lasted for 6 months and spanned the cooler months of the year. Evaluation was based on a daily recording of relevant clinical symptoms and signs and subsequent evaluation of the collected data by three blinded independent physicians. A total of 146 patients (23%) failed to complete the 6-month treatment (mostly due to difficulties in complying with protocol requirements), without clear-cut differences in the dropout rate in the three groups. An intention-to-treat analysis revealed that the incidence of patients without exacerbations in the group assigned to continuous SCMC-Lys treatment was significantly higher than in the placebo-treated group (p < 0.001). The total number of patients with at least one exacerbation was 66 (29.6%) in the group treated with continuous SCMC-Lys compared with 100 (45.9%) with placebo. In the former group the time between initiation of treatment and first exacerbation was significantly prolonged. The average number of days with acute respiratory illness was significantly decreased in the group receiving continuous SCMC-Lys compared with the group receiving placebo, and this was associated with a significant reduction in the antibiotic consumption during the trial period. In patients assigned to intermittent treatment, results of the assessed endpoints did not differ significantly from those observed in the placebo group. No serious adverse effects were reported. It is

  3. In vitro plasma protein binding and aqueous aggregation behavior of astaxanthin dilysinate tetrahydrochloride. (United States)

    Zsila, Ferenc; Fitos, Ilona; Bikádi, Zsolt; Simonyi, Miklós; Jackson, Henry L; Lockwood, Samuel F


    The tetrahydrochloride salt of astaxanthin di-L-lysinate (lys(2)AST) is a highly water-dispersible astaxanthin-amino acid conjugate, with an aqueous dispersibility of > or = 181.6 mg/mL. The statistical mixture of stereoisomers has been well characterized as an aqueous-phase superoxide anion scavenger, effective at micromolar (microM) concentrations. In the current study, the aqueous aggregation behavior and in vitro plasma protein binding [with fatty-acid-free human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP)] were investigated with a suite of techniques, including circular dichroism (CD) and UV-vis spectroscopy, ultrafiltration, competitive ligand displacement, and fluorescence quenching. Induced CD bands obtained in Ringer buffer solution of HSA demonstrated high affinity monomeric binding of the compound at low ligand per protein (L/P) ratios (in aqueous solution alone the carotenoid molecules formed card-pack aggregates). The binding constant ( approximately 10(6)M(-1)) and the binding stoichiometry (approximately 0.2 per albumin molecule) were calculated from CD titration data. CD displacement and ultrafiltration experiments performed with marker ligands of HSA indicated that the ligand binding occurred at a site distinct from the main drug binding sites of HSA (i.e., Sites I and II). At intermediate L/P ratios, both monomeric and aggregated ("chirally complexed") binding occurred simultaneously at distinct sites of the protein. At high L/P ratios, chiral complexation predominantly occurred on the asymmetric protein template. The tentative location of the chirally-complexed aggregation on the HSA template was identified as the large interdomain cleft of HSA, where carotenoid derivatives have been found to bind previously. Only weak binding to AGP was observed. These results suggest that parenteral use of this highly potent, water-dispersible astaxanthin-amino acid conjugate will result in plasma protein association, and plasma protein binding at

  4. Cartilage Calcification Mimics Polychondritis in Bone Scintigraphy

    Directory of Open Access Journals (Sweden)

    Hasan Atilgan


    Full Text Available 58 year-old male patient with sternal pain was referred to our Nuclear Medicine Clinic for bone scintigraphy for 2.5 months. Markedly increased activity accumulation in the first bilateral sternocostal junction and increased activity accumulations in 3rd, 4th, 5th sternocostal junctions and lateral portion of inferior part of corpus sterni were seen in late static images without increased perfusion and hyperemia. Soft tissue density and lytic lesions were seen bilaterally in bilateral first costa, sternocostal joints and in right side of xiphoid in his 3D computed tomography (CT. Sternocostal lesions that were seen in bone scintigraphy and CT, was reported as normal in biopsy.

  5. 清华励志修车哥 小人物也有春天%Bicycle Repairman Finds the Right Gear

    Institute of Scientific and Technical Information of China (English)


    On a chilly day, a man wearing a dirty glove was changing a bicycle chain in front of his repair shop at Tsinghua University. Compared to the surrounding buildings of China's top university, his 3-square-meter shop looks quite small and shabby. But the owner shows his pride, saying, "With my shop, I can repair bicycles for students. Furthermore, with the experience and money I get here, I will manufacture my own bicycles with lower prices but superior quality for them."%从试图结束自己生命的打工者,到成为备受学生尊敬的"修车哥",清华大学修车师傅任玉华在这个校园里追寻着自己的理想,也用自己的故事感动着身边的人。

  6. Dipeptide catalysed prebiotic polymerization of RNA

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Luisi, Pier Luigi; Monnard, Pierre-Alain


    be an important factor from an origin-of-life point of view. Short peptides are plausible products of prebiotic chemistry2. Consequently, they could have influenced chemical evolution on an early stage. An enzyme catalysing hydrolytic reactions can in principle be used as catalyst for condensation: the reverse...... reaction to hydrolysis. The direction of the catalysis either toward hydrolysis or condensation is determined by thermodynamic constraints. In an aqueous medium (a general requirement for prebiotically compatible reactions), hydrolysis is thermodynamically favoured over condensation. However......, the thermodynamic equilibrium towards condensation can be shifted even in this environment. For example, the reverse-proteolysis in prebiotic environment has been described for SerHis3. In this case, SerHis was able to condensate amino acids into insoluble peptides, which in turn pulled the equilibrium further...

  7. Associated rare anomalies in prune belly syndrome: A case report

    Directory of Open Access Journals (Sweden)

    Andreas Fette


    Full Text Available The triad of deficient abdominal wall musculature, undescended testes and urinary tract anomalies characterizes the Prune Belly Syndrome (PBS. PBS can be associated with other comorbid urological and non urological conditions. But the full pathogenesis and best treatment is still a matter of debate. A term newborn with a classical PBS (Woodhouse Group 2, Smith and Woodard Group 2 plus lung hypoplasia and funnel chest deformity, a megapenis with a tight phimosis and an obturated anterior urethra is presented. Unfortunately, the baby died in urosepsis and renal failure in his 3rd week of life, despite urine drainage surgery and peritoneal dialysis undertaken. According to the best of our knowledge, this is an unique combination of rare anomalies in PBS patients.

  8. The Potato ERF Transcription Factor StERF3 Negatively Regulates Resistance to Phytophthora infestans and Salt Tolerance in Potato. (United States)

    Tian, Zhendong; He, Qin; Wang, Haixia; Liu, Ying; Zhang, Ying; Shao, Fang; Xie, Conghua


    Ethylene response factors (ERFs) are unique to the plant kingdom and play crucial roles in plant response to various biotic and abiotic stresses. We show here that a potato StERF3, which contains an ERF-associated amphiphilic repression (EAR) motif in its C-terminal region, negatively regulates resistance to Phytophthora infestans and salt tolerance in potato. The StERF3 promoter responds to induction by salicylic acid, ABA ethylene and NaCl, as well as P. infestans, the causal agent of potato late blight disease. StERF3 could bind to the GCC box element of the HIS3 promoter and activate transcription of HIS3 in yeast cells. Importantly, silencing of StERF3 in potato produced an enhanced foliage resistance to P. infestans and elevated plant tolerance to NaCl stress accompanied by the activation of defense-related genes (PR1, NPR1 and WRKY1). In contrast, StERF3-overexpressing plants showed reduced expression of these defense-related genes and enhanced susceptibility to P. infestans, suggesting that StERF3 functions as a negative regulator of downstream defense- and/or stress-related genes in potato. StERF3 is localized to the nucleus. Interestingly, yeast two-hybrid assay and a bimolecular fluorescence complementation (BiFC) test clarified that StERF3 could interact with other proteins in the cytoplasm which may lead to its re-localization between the nucleus and cytoplasm, revealing a novel means of StERF3 regulation. Taken together, these data provide new insights into the mechanism underlying how StERF3 negatively regulates late blight resistance and abiotic tolerance in potato and may have a potential use in engineering late blight resistance in potato.

  9. A New Method for Genomic Non-Trace Molecular Manipulation Suitable for Kluyveromyces marxianus%一种针对马克斯克鲁维酵母(Kluyveromyces marxianus)的无痕基因组改造方法

    Institute of Scientific and Technical Information of China (English)

    郭超; 陈勇毅; 周峻岗; 余垚; 吕红


    马克斯克鲁维酵母位于酵母科克鲁维属,具有高分泌能力、高生长速率、多底物利用以及耐高温等特点,具有工业应用前景.因此,建立马克斯克鲁维酵母的基因工程改造技术十分重要.本文以马克斯克鲁维酵母URA3基因缺陷菌株为出发菌株,依据同源重组原理,选用KmURA3作为可循环使用的筛选标签基因,建立了一种针对马克斯克鲁维酵母的无残留多基因敲除技术,以满足其遗传工程改造的需求.本研究利用这种技术成功构建了Kmhis3△、Kmura3 △his3△、Kmura3 △ade2△、Kmura3△his3 △ade2△、Kmhis3 △ade2△等5株营养缺陷型菌株,为马克斯克鲁维酵母的分子遗传学研究以及工业应用研究提供了遗传材料.

  10. De novo-designed metallopeptides with type 2 copper centers: modulation of reduction potentials and nitrite reductase activities. (United States)

    Yu, Fangting; Penner-Hahn, James E; Pecoraro, Vincent L


    Enzymatic reactions involving redox processes are highly sensitive to the local electrostatic environment. Despite considerable effort, the complex interactions among different influential factors in native proteins impede progress toward complete understanding of the structure-function relationship. Of particular interest is the type 2 copper center Cu(His)3, which may act as an electron transfer center in peptidylglycine α-hydroxylating monooxygenase (PHM) or a catalytic center in copper nitrite reductase (CuNiR). A de novo design strategy is used to probe the effect of modifying charged amino acid residues around, but not directly bound to, a Cu(His)3 center embedded in three-stranded coiled coils (TRI-H)3 [TRI-H = Ac-G WKALEEK LKALEEK LKALEEK HKALEEK G-NH2]. Specifically, the peptide TRI-EH (=TRI-HK22E) alters an important lysine to glutamate just above the copper binding center. With a series of TRI-EH peptides mutated below the metal center, we use a variety of spectroscopies (EPR, UV-vis, XAS) to show a direct impact on the protonation equilibria, copper binding affinities, reduction potentials, and nitrite reductase activities of these copper-peptide complexes. The potentials at a specific pH vary by 100 mV, and the nitrite reductase activities range over a factor of 4 in rates. We also observe that the affinities, potentials, and catalytic activities are strongly influenced by the pH conditions (pH 5.8-7.4). In general, Cu(II) affinities for the peptides are diminished at low pH values. The interplay among these factors can lead to a 200 mV shift in reduction potential across these peptides, which is determined by the pH-dependent affinities of copper in both oxidation states. This study illustrates the strength of de novo protein design in elucidating the influence of ionizable residues on a particular redox system, an important step toward understanding the factors that govern the properties of this metalloenzyme with a goal of eventually improving the

  11. Bilateral ureteral obstruction revealing a benign prostatic hypertrophy: a case report and review of the literature. (United States)

    Riyach, Omar; Ahsaini, Mustapha; Kharbach, Youssef; Bounoual, Mohammed; Tazi, Mohammed Fadl; El Ammari, Jalal Eddine; Mellas, Soufiane; Fassi, Mohammed El Jamal; Khallouk, Abdelhak; Farih, Moulay Hassan


    Prostatic hyperplasia is the most frequent tumor in men older than 50 years of age. Bilateral hydronephrosis secondary to benign prostatic hypertrophy is a rare condition most often due to vesicoureteral reflux. Herein we report a case of a patient with bilateral hydronephrosis with distal ureter obstruction caused by detrusor hypertrophy due to prostatic hyperplasia, our analysis of the clinical data and a review of the relevant published literature. We report a case of a 65-year-old Berber man with clinically significant storage, bladder-emptying symptoms and bilateral low back pain with renal biologic failure and bilateral ureterohydronephrosis, distal ureteral stenosis, detrusor hypertrophy and prostate hyperplasia without significant post-void residual urine volume visualized by abdominal sonography. The patient underwent bilateral JJ stent insertion with transurethral resection of the prostate. The patient was discharged 3 days after surgery without any obvious complications. At his 3-month follow-up examination, the JJ stent was removed and the patient had comfortable urination without renal failure. This is an extremely rare condition that has important diagnostic considerations because of the possibility of comorbid severe obstructive uropathy and chronic renal failure.

  12. Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae. (United States)

    Fasullo, M; Dave, P


    Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cerevisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MATlocus. The difference in levels of stimulation between MATa/MAT alpha diploid and MAT alpha haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m2), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MAT alpha gene was introduced by DNA transformation into a MATa/mat alpha::LEU2+ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATa gene was introduced by DNA transformation into a MAT alpha haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV. The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.

  13. Identification and molecular properties of SUMO-binding proteins in arabidopsis

    KAUST Repository

    Park, Hyeongcheol


    Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes. ©2011 KSMCB.

  14. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Vidal M.S.


    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  15. Multiscale schemes for the predictive description and virtual engineering of materials.

    Energy Technology Data Exchange (ETDEWEB)

    von Lilienfeld-Toal, Otto Anatole


    This report documents research carried out by the author throughout his 3-years Truman fellowship. The overarching goal consisted of developing multiscale schemes which permit not only the predictive description but also the computational design of improved materials. Identifying new materials through changes in atomic composition and configuration requires the use of versatile first principles methods, such as density functional theory (DFT). Using DFT, its predictive reliability has been investigated with respect to pseudopotential construction, band-gap, van-der-Waals forces, and nuclear quantum effects. Continuous variation of chemical composition and derivation of accurate energy gradients in compound space has been developed within a DFT framework for free energies of solvation, reaction energetics, and frontier orbital eigenvalues. Similar variations have been leveraged within classical molecular dynamics in order to address thermal properties of molten salt candidates for heat transfer fluids used in solar thermal power facilities. Finally, a combination of DFT and statistical methods has been used to devise quantitative structure property relationships for the rapid prediction of charge mobilities in polyaromatic hydrocarbons.

  16. Jim Virdee, the new spokesperson of CMS

    CERN Multimedia


    Jim Virdee and Michel Della Negra. On 21 June Tejinder 'Jim'Virdee was elected by the CMS collaboration as its new spokesperson, his 3-year term of office beginning in January 2007. He will take over from Michel Della Negra, who has been CMS spokesperson since its formalization in 1992. Three distinguished physicists stood as candidates for this election: Dan Green from Fermilab, programme manager of the US-CMS collaboration and coordinator of the CMS Hadron Calorimeter project; Jim Virdee from Imperial College London and CERN, deputy spokesperson of CMS since 1993; Gigi Rolandi from the University of Trieste and CERN, ex-Aleph spokesperson and currently involved in the preparations of the physics analyses to be done with CMS. On the early evening of 21 June, 141 of the 142 members of the CMS collaboration board, some represented by proxies, took part in a secret ballot. After two rounds of voting Jim Virdee was elected as spokesperson with a clear majority. Jim thanked the CMS collaboration 'for putting conf...

  17. Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides. (United States)

    Parmar, Avanish S; Xu, Fei; Pike, Douglas H; Belure, Sandeep V; Hasan, Nida F; Drzewiecki, Kathryn E; Shreiber, David I; Nanda, Vikas


    We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

  18. Schwannoma of the Median Nerve at the Wrist and Palmar Regions of the Hand: A Rare Case Report

    Directory of Open Access Journals (Sweden)

    Harun Kütahya


    Full Text Available Schwannomas are also known as neurolemmas that are usually originated from Schwann cells located in the peripheric nerve sheaths. They are the most common tumours of the hand (0.8–2%. They usually present solitary swelling along the course of the nerve however multiple lesions may be present in cases of NF type 1, familial neurofibromatosis, and sporadic schwannomatosis. Schwannomas are generally represented as an asymptomatic mass; however pain, numbness and fatigue may take place with the increasing size of the tumour. EMG (electromyelography, MRI (magnetic resonance imagination, and USG (ultrasound are helpful in the diagnosis. Surgical removal is usually curative. In this paper, we present a 24-year-old male referred to our clinic for a lump located at the volar side of the left wrist and a lump located in his left palm and numbness at his 3rd and 4th fingers. Total excision was performed for both lesions. Histopathological examination of the masses revealed typical features of schwannoma. At the 6th-month followup the patient was symptom-free except for slight paresthesia of the 3rd and the 4th fingers. For our knowledge, this is the second case in the literature presenting wrist and palm involvement of the median nerve schwannoma.

  19. New spokesperson for the LHCb collaboration

    CERN Multimedia

    Katarina Anthony


    Pierluigi Campana begins his 3-year tenure as LHCb spokesperson this June. As the new voice for the collaboration, Campana will lead the experiment through what should prove to be a very exciting phase.   Pierluigi Campana, from the Istituto Nazionale di Fisica Nucleare in Frascati, has been with the collaboration since 2000 and was heavily involved in the construction of the muon chamber of the LHCb detector. He replaces Andrei Golutvin, from Imperial College London and Russia’s Institute for Theoretical and Experimental Physics. “Leading such a large collaboration is not an easy task,” says Campana. While he will rely heavily upon the work of his predecessor, he plans on leaving his own mark on the position: “One of the main goals of my job will be to enhance the spirit of collaboration between the different institutes within our experiment.” LHCb plays a key role in the search for new physics. The experiment is conducting a very precise search...

  20. From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival. (United States)

    Liu, Meng; Wang, Guang; Zhang, Shi-Yao; Zhong, Shan; Qi, Guo-Long; Wang, Chao-Jie; Chuai, Manli; Lee, Kenneth Ka Ho; Lu, Da-Xiang; Yang, Xuesong


    As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia.

  1. Thermokinetic Study on the Complexation Reaction of the First-Row Transitional Metal Chlorides with Histidine

    Institute of Scientific and Technical Information of China (English)

    CHEN,San-Ping(陈三平); GAO,Sheng-Li(高胜利); SHI,Qi-Zhen(史启祯)


    The enthalpy change of the complexation reactions of the first-row transitional metal chlorides including CrCl3,MnCl2, FeCl2, CoCl2, NiCl2 and CuCl2 with L-α-histidine in water were determined by a microcalorimeter at 298.15-323.15 K. The standard enthalpy of formation of Cr(His)3+2 (aq) and M(His)2+2 (aq) (M=Mn, Fe, Co,Ni and Cu) were calculated. Based on the thermodynamic and kinetic equations of the reactions, three thermodynamic parameters (the activation enthalpy, the activation entropy, the activation free energy), the rate constants, and three kinetic parameters (the apparent activation energy, the pre-exponential constant and the reaction order) are obtained. The solid complexes of CrCl3, MnCl2, FeCl2, CoCl2, NiCl2 and CuCl2 with histidine were prepared and acterized by IR as well. The results showed that, with the atomic number increasing, three thermodynamic parameters, △G≠(-), △H≠(-) and △S≠(-) of the complexation reaction of these metal chlorides with L-α-histidine in water present an analogy regularity.

  2. Endovascular treatment of post-laparoscopic pancreatectomy splenic arteriovenous fistula with splenic vein aneurysm. (United States)

    Ueda, Tatsuo; Murata, Satoru; Yamamoto, Akira; Tamai, Jin; Kobayashi, Yuko; Hiranuma, Chiaki; Yoshida, Hiroshi; Kumita, Shin-Ichiro


    Splenic arteriovenous fistulas (SAVFs) with splenic vein aneurysms are extremely rare entities. There have been no prior reports of SAVFs developing after laparoscopic pancreatectomy. Here, we report the first case. A 40-year-old man underwent a laparoscopic, spleen-preserving, distal pancreatectomy for an endocrine neoplasm of the pancreatic tail. Three months after surgery, a computed tomography (CT) scan demonstrated an SAVF with a dilated splenic vein. The SAVF, together with the splenic vein aneurysm, was successfully treated using percutaneous transarterial coil embolization of the splenic artery, including the SAVF and drainage vein. After the endovascular treatment, the patient's recovery was uneventful. He was discharged on postoperative day 6 and continues to be well 3 mo after discharge. An abdominal CT scan performed at his 3-mo follow-up demonstrated complete thrombosis of the splenic vein aneurysm, which had decreased to a 40 mm diameter. This is the first reported case of SAVF following a laparoscopic pancreatectomy and demonstrates the usefulness of endovascular treatment for this type of complication.

  3. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS). (United States)

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A


    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1).

  4. Engineering allosteric regulation into the hinge region of a circularly permuted TEM-1 beta-lactamase. (United States)

    Mathieu, Valéry; Fastrez, Jacques; Soumillion, Patrice


    In nature, the activity of many enzymes involved in important biochemical pathways is controlled by binding a ligand in a site remote from the active site. The allosteric sites are frequently located in hinge regulatory subunits, in which a conformational change can occur and propagate to the active site. The enzymatic activity is then enhanced or decreased depending on the type of effectors. Many artificial binding sites have been created to engineer an allosteric regulation. Generally, these sites were engineered near the active site in loops or at the surface of contiguous helices or strands but rarely in hinge regions. This work aims at exploring the possibility of regulating a monomeric enzyme whose active site is located at the interface between two domains. We anticipated that binding of a ligand in the hinge region linking the domains would modify their positioning and, consequently, modulate the activity. Here, we describe the design of two mutants in a circularly permuted TEM-1 (cpTEM-1) beta-lactamase. The first one, cpTEM-1-His(3) was created by a rational design. It shows little regulation upon metal ion binding except for a weak activation with Zn(2+). The second one, cpTEM-1-3M-His(2), was selected by a directed evolution strategy. It is allosterically down-regulated by Zn(2+), Ni(2+) and Co(2+) with binding affinities around 300 microM.


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    Patricia de Souza Bonfim-Mendonca


    Full Text Available SUMMARY Introduction: The majority of nosocomial fungal infections are caused by Candida spp. where C. albicans is the species most commonly identified. Molecular methods are important tools for assessing the origin of the yeasts isolated in hospitals. Methods: This is a study on the genetic profifiles of 39 nosocomial clinical isolates of C. albicans using two typing methods: random amplifified polymorphic DNA (RAPD and microsatellite, two different primers for each technique were used. Results: RAPD provided 10 and 11 different profiles with values for SAB of 0.84 ± 0.126 and 0.88 ± 0.08 for primers M2 and P4, respectively. Microsatellite using two markers, CDC3 and HIS3, allowed the observation of six and seven different alleles, respectively, with combined discriminatory power of 0.91. Conclusions: Although genetic variability is clear, it was possible to identify high similarity, suggesting a common origin for at least a part of isolates. It is important to emphasize that common origin was proven from yeasts isolated from colonization (urine, catheter or endotracheal secretions and blood culture from the same patient, indicating that the candidemia must have started from a site of colonization. The combination of RAPD and microsatellite provides a quick and efficient analysis for investigation of similarity among nosocomial isolates of C. albicans.

  6. Integrative modules for efficient genome engineering in yeast

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    Triana Amen


    Full Text Available We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast Saccharomyces cerevisiae. A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into HIS3, URA3, ADE2, and TRP1 loci. The desired level of expression can be achieved by using constitutive (TEF1p, GPD1p, inducible (CUP1p, GAL1/10p, and daughter-specific (DSE4p promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (TEF1p-GPD1p, TEF1p-CUP1p, and TEF1p-DSE4p allow tractable metabolic engineering.

  7. The tachykinin peptide neurokinin B binds copper(I) and silver(I) and undergoes quasi-reversible electrochemistry: towards a new function for the peptide in the brain. (United States)

    Grosas, Aidan B; Kalimuthu, Palraj; Smith, Alison C; Williams, Peter A; Millar, Thomas J; Bernhardt, Paul V; Jones, Christopher E


    The tachykinin neuropeptide family, which includes substance P and neurokinin B, is involved in a wide array of biological functions. Among these is the ability to protect against the neurotoxic processes in Alzheimer's Disease, but the mechanisms driving neuroprotection remain unclear. Dysregulation of metal ions, particularly copper, iron and zinc is a common feature of Alzheimer's Disease, and other amyloidogenic disorders. Copper is known to be released from neurons and recent work has shown that some tachykinins can bind Cu(II) ions, and that neurokinin B can inhibit copper uptake into astrocytes. We have now examined whether neurokinin B is capable of binding Cu(I), which is predicted to be available in the synapse. Using a combination of spectroscopic techniques including cyclic voltammetry and magnetic resonance we show that neurokinin B can bind Cu(I) either directly from added CuCl or by reduction of Cu(II)-bound neurokinin B. The results showed that the Cu(I) binding site differs greatly to that of Cu(II) and involves thioether coordination via Met2 and Met10 and an imidazole nitrogen ligand from His3. The Cu(I) coordination is also different to the site adopted by Ag(I). During changes in oxidation state, copper remains bound to neurokinin B despite large changes to the inner coordination sphere. We predict that neurokinin B may be involved in synaptic copper homeostasis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Suppression pheromone and cockroach rank formation (United States)

    Kou, Rong; Chang, Huan-Wen; Chen, Shu-Chun; Ho, Hsiao-Yung


    Although agonistic behaviors in the male lobster cockroach ( Nauphoeta cinerea) are well known, the formation of an unstable hierarchy has long been a puzzle. In this study, we investigate how the unstable dominance hierarchy in N. cinerea is maintained via a pheromone signaling system. In agonistic interactions, aggressive posture (AP) is an important behavioral index of aggression. This study showed that, during the formation of a governing hierarchy, thousands of nanograms of 3-hydroxy-2-butanone (3H-2B) were released by the AP-adopting dominant in the first encounter fight, then during the early domination period and that this release of 3H-2B was related to rank maintenance, but not to rank establishment. For rank maintenance, 3H-2B functioned as a suppression pheromone, which suppressed the fighting capability of rivals and kept them in a submissive state. During the period of rank maintenance, as the dominant male gradually decreased his 3H-2B release, the fighting ability of the subordinate gradually developed, as shown by the increasing odds of a subordinate adopting an AP (OSAP). The OSAP was negatively correlated with the amount of 3H-2B released by the dominant and positively correlated with the number of domination days. The same OSAP could be achieved earlier by reducing the amount of 3H-2B released by the dominant indicates that whether the subordinate adopts an offensive strategy depends on what the dominant is doing.

  9. Aerobic induction of respiro-fermentative growth by decreasing oxygen tensions in the respiratory yeast Pichia stipitis. (United States)

    Klinner, U; Fluthgraf, S; Freese, S; Passoth, V


    The fermentative and respiratory metabolism of Pichia stipitis wild-type strain CBS 5774 and the derived auxotrophic transformation recipient PJH53 trp5-10 his3-1 were examined in differentially oxygenated glucose cultures in the hermetically sealed Sensomat system. There was a good agreement of the kinetics of gas metabolism, growth, ethanol formation and glucose utilisation, proving the suitability of the Sensomat system for rapid and inexpensive investigation of strains and mutants for their respiratory and fermentative metabolism. Our study revealed respiro-fermentative growth by the wild-type strain, although the cultures were not oxygen-limited. The induction of respiro-fermentative behaviour was obviously due to the decrease in oxygen tension but not falling below a threshold of oxygen tension. The responses differed depending on the velocity of the decrease in oxygen tension. At high oxygenation (slow decrease in oxygen tension), ethanol production was induced but glucose uptake was not influenced. At low oxygenation, glucose uptake and ethanol formation increased during the first hours of cultivation. The transformation recipient PJH53 most probably carries a mutation that influences the response to a slow decrease in oxygen tension, since almost no ethanol formation was found under these conditions.

  10. Genotypes of Candida albicans involved in development of candidiasis and their distribution in oral cavity of non-candidiasis individuals. (United States)

    Takagi, Yuki; Hattori, Hisao; Adachi, Hidesada; Takakura, Shunji; Horii, Toshinobu; Chindamporn, Ariya; Kitai, Hiroki; Tanaka, Reiko; Yaguchi, Takashi; Fukano, Hideo; Kawamoto, Fumihiko; Shimozato, Kazuo; Kanbe, Toshio


    Genotype characteristics and distribution of commensal Candida albicans should be studied to predict the development of candidiasis, however, extensive genotype analysis of commensal C. albicans has not been made. In this study, 508 C. albicans isolates were collected from patients with/without candidiasis and divided into 4 isolate groups (SG-1, oral cavity of non-candidiasis patients; SG-2, patients with cutaneous candidiasis; SG-3, patients with vaginal candidiasis; SG-4, patients with candidemia). These isolates were characterized to study the relationship between genotypes and pathogenicity using microsatellite analysis. Using CDC3 and CAI, 5 genotypes (I, 111: 115/33: 41; II, 115: 119/23: 23; III, 115: 123/18: 27; IV, 115: 123/33: 40; and V, 123: 127/32: 41) were found in 4.2%, 8.9%, 7.1%, 2.2% and 3.1% of the isolates, respectively. Genotypes II and III were commonly found in all isolate groups. These genotypes were further divided into 28 types by additional HIS3 and CAIII microsatellite markers. In this analysis, C. albicans with type 6 and type 23 was widely distributed as a commensal species in the oral cavity of non-candidiasis patients and found to be related with candidiasis development. Additionally, genotypes I and IV were found in SG-2 and/or SG-4, suggesting that the fungus with those genotypes is also involved in this development. In contrast, genotype V was not identified in any infective isolates.

  11. Species of the Colletotrichum acutatum complex associated with anthracnose diseases of fruit in Brazil. (United States)

    Bragança, Carlos A D; Damm, Ulrike; Baroncelli, Riccardo; Massola Júnior, Nelson S; Crous, Pedro W


    Although Colletotrichum acutatum was recently investigated and shown to be a species complex comprising about 30 species, the name is still used in its broad sense for anthracnose pathogens of fruits in Brazil. In this study, a multilocus molecular analysis was carried out based on a dataset of ITS, HIS3, GAPDH, CHS-1, TUB2 and ACT sequences of Colletotrichum strains belonging to the C. acutatum species complex from fruits collected in different regions in Brazil combined with sequences of ex-type and other reference strains of species belonging to this complex. The strains were revealed to belong to Colletotrichum nymphaeae, Colletotrichum melonis, Colletotrichum abscissum and one new species, namely Colletotrichum paranaense, from apple and peach. Morphological descriptions of the new species and a strain closely related to but diverging from C. melonis are provided. From the data presently available, the most common species on apple fruits in Brazil is C. nymphaeae. In a pathogenicity test, strains of all four species caused lesions on detached apple, peach and guava fruits, except for strain CBS 134730 that did not infect guava fruits. Copyright © 2016 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  12. Bridging psychological barriers between the child and the father after his returning from the war: Could group art therapy help?

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    Mandić-Gajić Gordana


    Full Text Available Introduction. War veterans with chronic post-traumatic stress disorder (PTSD have poorer family and parenting functioning, but little research has focused on these impairments. Case re-port. This paper presented how the series of drawings and the group art therapy process enhanced bridging the psychological barriers of a 33-year-old male PTSD war veteran to engagement with the child. After two years of deployment he returned home and suffered mostly from PTSD numbness and avoidance symptoms. The veteran had the family readjustment difficulties and felt guilty for being detached from his 3-year-old son. He under-went integrative treatment in the Day Unit Program. The drawings series were made by free associations. Clinical observations and group discussions were recorded in the group art therapy protocols. The presented patient got gratifications and support from the group members for his illustration of popular cartoon heroes, and decided to draw Mickey Mouse at home. On the next session he shared his satisfaction for bridging the gap between him and his son, having done the same drawings with his son at home. Beck's depression inventory (BDI was used for self-rating of depression and a reduction of BDI score from 18 to 6 during the treatment course was recorded. Conclusions. Series of drawings illustrated shift from war related past toward current family life of the war veteran. Group art therapy gave him gratification and support with hope and a sense of belonging, thus facilitated his parenting readjustment.

  13. 应用酵母双杂交筛选系统从药用植物中发现Aβ聚集抑制剂%Application of a yeast two-hybrid based screening system in the identification of amyloid-beta aggregation inhibitors in pharmaceutical plants

    Institute of Scientific and Technical Information of China (English)

    王丽威; 杨雁芳; 张英涛


    研究证据表明,β淀粉样肽即Aβ的自我聚集是阿尔兹海默病(AD)重要的发病因素.因此,Aβ聚集抑制剂被认为是潜在的AD治疗候选药物.在本研究中,我们建立了一个基于酵母双杂交技术的Aβ聚集抑制剂筛选系统.通过采用拼接PCR技术(assembly PCR),克隆了人源A342的cDNA并将其插入到分别含有酵母转录因子GAL4转录激活区(GAL4AD)与DNA结合区(GAL4BD)的两个表达载体中.通过以上两个载体的共转化实现了两个融合蛋白GAL4AD-Aβ42与GAL4BD-Aβ42在AH109酵母菌株中的共表达.由于Aβ42片段在酵母中的自我相互作用使GAL4转录因子的活性在酵母中得到重建,从而激活了依赖于GAL4活性的四个报告基因HIS3,ADE2,lacZ与MELI的转录与表达.以上报告基因的正常表达使具有多种营养缺陷表型的AH109酵母获得了在缺乏组氨酸与腺嘌呤的合成选择培养基上正常生长的能力.通过采用生长抑制作为筛选标记,应用本系统对红景天属植物的Aβ聚集抑制活性进行了分析,进而发现本属植物很可能成为Aβ聚集抑制剂发现的重要资源.%The aggregation of amyloid-beta (Aβ) peptide,has been demonstrated to be critical for the development of Alzheimer's disease (AD).Aβ aggregation inhibitors are thus considered to be drug candidates for AD therapy.In the present study,we developed a novel screening tool based on the yeast two-hybrid system to screen Aβ aggregation inhibitors.The human Aβ42 peptide cDNA was cloned using assembly PCR and inserted into each of the yeast expression plasmids containing either the GAL4 activation domain (GAL4AD) or the DNA-binding domain (GAL4BD).Co-transformation of the above plasmids led to the expression of the fusion proteins GAL4AD-Aβ42 and GAL4BD-Aβ42 in the AH 109 yeast strain.The self interaction of Aβ42 fragments reconstructed the GAL4 transcriptor and thus activated the GAL4 responsive transcription of four reporter genes

  14. In vitro and in vivo studies of the effects of halogenated histidine analogs on Plasmodium falciparum. (United States)

    Panton, L J; Rossan, R N; Escajadillo, A; Matsumoto, Y; Lee, A T; Labroo, V M; Kirk, K L; Cohen, L A; Aikawa, M; Howard, R J


    The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by these two compounds, 50% inhibition resulting at 200 microM concentration. Morphological analysis of parasite development proved a sensitive assay, since development of mature trophozoites was inhibited 50% by 25 microM 2-F-HIS or 100 2-I-HIS. Electron microscopy studies suggested different mechanisms of action of 2-F-HIS and 2-I-HIS on P. falciparum. 2-F-HIS produced a decrease in knob number at the erythrocyte surface and accumulation of electron-dense material under the parasite membrane. 2-I-HIS had no obvious effect on knobs or electron-dense material but affected parasite morphology. Surprisingly, 2-chloro-L-histidine and 2-bromo-L-histidine did not inhibit P. falciparum in vitro, even though their halogen atom substituents are intermediate in size between F and I atoms. 2-F-HIS and 2-I-HIS were tested in vivo against P. falciparum in owl monkeys (Aotus sp.) but were ineffective at doses that were nontoxic.

  15. Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (Ziziphus jujuba Mill. under a Variety of Conditions.

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    Jiaodi Bu

    Full Text Available Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1, Histone-H3 (His3, and Polyadenylate-binding protein-interacting protein (PAIP were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3, Glyceraldehyde-3-phosphate dehydrogenase (GADPH, and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.

  16. Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA. (United States)

    Cho, Sung Ki; Kang, Il-Ho; Carr, Tyrell; Hannapel, David J


    Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3' untranslated region (UTR) of the RNA in mediating transport. Because the 3' UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3' UTR of StBEL5 as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3' UTR of StBEL5 using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

  17. Using the yeast three-hybrid system to identify proteins that interact with a phloem-mobile mRNA

    Directory of Open Access Journals (Sweden)

    Sung Ki eCho


    Full Text Available Heterografting and RNA transport experiments have demonstrated the long-distance mobility of StBEL5 RNA, its role in controlling tuber formation, and the function of the 503-nt 3´ untranslated region (UTR of the RNA in mediating transport. Because the 3´ UTR of StBEL5 is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3´ UTR of StBEL5 as bait in the yeast three-hybrid system to identify putative partner RNA-binding proteins (RBPs. From this screen, 116 positive cDNA clones were isolated based on nutrient selection, HIS3 activation, and lacZ induction and were sequenced and classified. Thirty-five proteins that function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3´ UTR of StBEL5 using β-galactosidase assays in the yeast three-hybrid system and RNA gel-shift assays. Among the final selections were two RNA-binding proteins, a zinc-finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the yeast three-hybrid system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RNA-binding proteins. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

  18. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3. (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert


    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  19. Structural elements of metal selectivity in metal sensor proteins. (United States)

    Pennella, Mario A; Shokes, Jacob E; Cosper, Nathaniel J; Scott, Robert A; Giedroc, David P


    Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR are homologous zinccobalt-responsive and nickelcobalt-responsive transcriptional repressors in vivo, respectively, and members of the ArsRSmtB superfamily of prokaryotic metal sensor proteins. We show here that Zn(II) is the most potent negative allosteric regulator of czr operatorpromoter binding in vitro with the trend Zn(II)>Co(II)Ni(II), whereas the opposite holds for the binding of NmtR to the nmt operatorpromoter, Ni(II)>Co(II)>Zn(II). Characterization of the metal coordination complexes of CzrA and NmtR by UVvisible and x-ray absorption spectroscopies reveals that metals that form four-coordinate tetrahedral complexes with CzrA [Zn(II) and Co(II)] are potent regulators of DNA binding, whereas metals that form five- or six-coordinate complexes with NmtR [Ni(II) and Co(II)] are the strongest allosteric regulators in this system. Strikingly, the Zn(II) coordination complexes of CzrA and NmtR cannot be distinguished from one another by x-ray absorption spectroscopy, with the best fit a His-3-carboxylate complex in both cases. Inspection of the primary structures of CzrA and NmtR, coupled with previous functional data, suggests that three conserved His and one Asp from the C-terminal alpha5 helix donate ligands to create a four-coordinate complex in both CzrA and NmtR, with NmtR uniquely capable of expanding its coordination number in the Ni(II) and Co(II) complexes by recruiting additional His ligands from a C-terminal extension of the alpha5 helix.

  20. Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain. (United States)

    Delneri, D; Gardner, D C; Bruschi, C V; Oliver, S G


    By in silicio analysis, we have discovered that there are seven open reading frames (ORFs) in Saccharomyces cerevisiae whose protein products show a high degree of amino acid sequence similarity to the aryl alcohol dehydrogenase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehydrogenase activity by degrading aromatic aldehydes to the corresponding alcohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the AAD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-generated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blot analysis. Double, triple and quadruple mutant strains were obtained by classical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyces lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested for the degradation of aromatic aldehydes using both spectrophotometry and high performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenotype and mating and sporulation efficiencies were not affected in the septuple deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility that the nutritional markers used for gene replacement are causing this effect.

  1. Cloning and functional identification of stress-resistant BeDREB genes from Bermuda grass

    Institute of Scientific and Technical Information of China (English)

    XIE Yongli; WANG Zizhang; LIU Qiang; ZHANG Shuping


    Dehydration-responsive element-binding (DREB)proteins,specifically binding to the dehydration-responsive element (DRE),have been identified as a group of important transcription activators of plants which regulate the expression of genes in response to drought,high-salt and low- temperature stresses.Two DREB-like genes from Bermuda grass that are induced by low-temperature or high-salt stresses were cloned using RT-PCR and RACE methods,and were named BeDREB1 and BeDREB2,respectively (GenBank accession No:AY462117 and AY462118).They contained an ORF of 753 bp encoding 251 amino acids,showing the typical characteristics of the DREB gene family.Interestingly,these two genes isolated from Bermuda grass induced either by low-temperature stress or high-salt stress shared 97.8% homology.Furthermore,it was demonstrated that both BeDREB1 and BeDREB2 could bind to the wild-type DRE element to activate the transcription of the reporter gene HIS3,driven by a promoter carrying DRE cis-element in yeast strain 4721,in the presence of 3-AT.RT-PCR showed that BeDREB1 and BeDREB2 genes could be greatly induced by low-temperature and high-salt stresses,respectively.Their expressions were changed following the inducible time.In conclusion,all results indicate that BeDREB1 and BeDREB2 genes isolated from treated Bermuda grass are new members'of the DREB transcription activator family,which may play very important roles in signal transduction related to stresses.

  2. Elevated mutation rate during meiosis in Saccharomyces cerevisiae. (United States)

    Rattray, Alison; Santoyo, Gustavo; Shafer, Brenda; Strathern, Jeffrey N


    Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

  3. Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Daniel S Yuan

    Full Text Available Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.

  4. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae. (United States)

    Chee, Mark K; Haase, Steven B


    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  5. Thermodynamics of binding of a sulfonamide inhibitor to metal-mutated carbonic anhydrase as studied by affinity capillary electrophoresis. (United States)

    Sato, Yosuke; Hoshino, Hitoshi; Iki, Nobuhiko


    By affinity capillary electrophoresis (ACE), the thermodynamic binding constants of a sulfonamide (SA) inhibitor to bovine carbonic anhydrase II (CA) and metal mutated variants (M-CAs) were evaluated. 1-(4-Aminosulfonylphenylazo)-2-naphthol-6,8-disulfonate was used as the SA in the electrophoretic buffer for ACE. The Scatchard analysis of the dependence of the electrophoretic mobility of native CA on the SA concentration provided the binding constant to be Kb=(2.29±0.05)×10(6) M(-1) (at pH8.4, 25°C). On the other hand, apoCA showed far smaller value [Kb=(3.76±0.14)×10(2) M(-1)], suggesting that the coordination of SA to the Zn(II) center controlled the binding thermodynamics. The ACE of M-CAs showed the same behaviors as native CA but with different Kb values. For example, Co-CA adopting the same tetrahedral coordination geometry as native CA exhibited the largest Kb value [(2.55±0.05)×10(6) M(-1)] among the M-CAs. In contrast, Mn- and Ni-CA, which adopted the octahedral coordination geometry, had Kb values that were about two orders of magnitude lower. Because the hydrophobic cavity of CA around the active center pre-organized the orientation of SA, thereby fixing the ligating NH(-) moiety to the apex of the tetrahedron supported by three basal His3 of CA, metals such as Zn and Co at the center of M-CA gave the most stable CA-SA complex. However, pre-organization was not favored for octahedral geometry. Thus, pre-organization of SA was the key to facilitating the tetrahedral coordination geometry of the Zn(II) active center of CA.

  6. 试论阿尔别宁身上的“恶魔性”因素%On the Demonic Elements of Albenin

    Institute of Scientific and Technical Information of China (English)



    “恶魔性”作为潜藏于人的心理和情感世界的内在物质构成人的内在局限性,它经常通过各种途径转换为人的外在行动。文章试图通过对《假面舞会》主人公阿尔别宁在误信妻子出轨的谣言之后行动的分析,来阐释其人身上“恶魔性因素”的三个方面:第一,往往自觉地边缘化于中心,因而极其孤独;第二,对世界持有一种全面怀疑乃至怨恨,特别看重个人意志的自由,甚至颇为自私;第三,在正常情况下,既具创造性,更有毁灭性。%Demonism, the internal substance hidden in psychological and emotional world, constitutes human's limitation and converts to human's external action through various means.The paper makes an attempt to analyze the action of Albenin, the protagonist in Masked Ball, who believed the rumor of his being betrayed by his wife, so as to illustrate his 3 demonic Elements: first, his solitude caused by his instinctive marginalization; second, his complete doubt or even hatred of the world, valuing his individual freedom or even selfishness; third, his construction as well as destruction under normal circumstances.

  7. Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Alison Rattray


    Full Text Available Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3 placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts. Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

  8. Adaptation by Plasticity of Genetic Regulatory Networks (United States)

    Brenner, Naama


    Genetic regulatory networks have an essential role in adaptation and evolution of cell populations. This role is strongly related to their dynamic properties over intermediate-to-long time scales. We have used the budding yeast as a model Eukaryote to study the long-term dynamics of the genetic regulatory system and its significance in evolution. A continuous cell growth technique (chemostat) allows us to monitor these systems over long times under controlled condition, enabling a quantitative characterization of dynamics: steady states and their stability, transients and relaxation. First, we have demonstrated adaptive dynamics in the GAL system, a classic model for a Eukaryotic genetic switch, induced and repressed by different carbon sources in the environment. We found that both induction and repression are only transient responses; over several generations, the system converges to a single robust steady state, independent of external conditions. Second, we explored the functional significance of such plasticity of the genetic regulatory network in evolution. We used genetic engineering to mimic the natural process of gene recruitment, placing the gene HIS3 under the regulation of the GAL system. Such genetic rewiring events are important in the evolution of gene regulation, but little is known about the physiological processes supporting them and the dynamics of their assimilation in a cell population. We have shown that cells carrying the rewired genome adapted to a demanding change of environment and stabilized a population, maintaining the adaptive state for hundreds of generations. Using genome-wide expression arrays we showed that underlying the observed adaptation is a global transcriptional programming that allowed tuning expression of the recruited gene to demands. Our results suggest that non-specific properties reflecting the natural plasticity of the regulatory network support adaptation of cells to novel challenges and enhance their evolvability.

  9. A strategy for constructing aneuploid yeast strains by transient nondisjunction of a target chromosome

    Directory of Open Access Journals (Sweden)

    Peck Anders T


    Full Text Available Abstract Background Most methods for constructing aneuploid yeast strains that have gained a specific chromosome rely on spontaneous failures of cell division fidelity. In Saccharomyces cerevisiae, extra chromosomes can be obtained when errors in meiosis or mitosis lead to nondisjunction, or when nuclear breakdown occurs in heterokaryons. We describe a strategy for constructing N+1 disomes that does not require such spontaneous failures. The method combines two well-characterized genetic tools: a conditional centromere that transiently blocks disjunction of one specific chromosome, and a duplication marker assay that identifies disomes among daughter cells. To test the strategy, we targeted chromosomes III, IV, and VI for duplication. Results The centromere of each chromosome was replaced by a centromere that can be blocked by growth in galactose, and ura3::HIS3, a duplication marker. Transient exposure to galactose induced the appearance of colonies carrying duplicated markers for chromosomes III or IV, but not VI. Microarray-based comparative genomic hybridization (CGH confirmed that disomic strains carrying extra chromosome III or IV were generated. Chromosome VI contains several genes that are known to be deleterious when overexpressed, including the beta-tubulin gene TUB2. To test whether a tubulin stoichiometry imbalance is necessary for the apparent lethality caused by an extra chromosome VI, we supplied the parent strain with extra copies of the alpha-tubulin gene TUB1, then induced nondisjunction. Galactose-dependent chromosome VI disomes were produced, as revealed by CGH. Some chromosome VI disomes also carried extra, unselected copies of additional chromosomes. Conclusion This method causes efficient nondisjunction of a targeted chromosome and allows resulting disomic cells to be identified and maintained. We used the method to test the role of tubulin imbalance in the apparent lethality of disomic chromosome VI. Our results indicate

  10. Isolation and identification of compounds from marine mangrove plant Avicennia marina%红树植物白骨壤化学成分的分离鉴定

    Institute of Scientific and Technical Information of China (English)

    孙昱; 丁怡; 林文翰


    目的:研究红树植物白骨壤(Avicennia marina)叶子部位次生代谢产物的结构多样性.方法:采用多种色谱方法进行分离纯化,通过波谱方法,并对照文献鉴定化合物结构.结果:分离并鉴定了21个苯丙素和萘醌类化合物,包括3个新化合物,分别是erythro-guaiacylglycerol-β-ferulic acid ether(1),marinnone A(16)和marinnone B(17);18个已知物,分别是threo-guaiacylglycerol-β-ferulic acid ether(2),eleutheroside E2(3),(+)-lirioresinol A(4),dihydroxymethyl-his(3,5-dimethoxy-4-hydroxyphenyl)tetrahydrofuran-9-O-β-glucopyranoside(5),(+)-lyonire-sinol 3α-O-β-D-glucopyranoside(6),(-)-lyoniresinol 3α-O-β-D-glucopyranoside(7),epi-pinoresinol(8),leucoseceptoside A(9),jionoside C(10),salsaside A(11),ilicifolioside A(12),acteoside(13),isoacteoside(14),ethyl ferulate(15),avicennone D(18),avicenone E(19),avicennol C(20),stenocarpoquinone B(21),其中化合物1和2互为差向异构体,16和17互为同分异构体.结论:化合物1,16,17为新化合物,13个已知化合物2~12,14,15为首次从该属植物中获得.

  11. The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain† (United States)

    Bafaro, Elizabeth M.; Antala, Sagar; Nguyen, Tuong-Vi; Dzul, Stephen P.; Doyon, Brian; Stemmler, Timothy L.; Dempski, Robert E.


    The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4. Previously, it has been postulated that this domain regulates hZIP4 levels in the plasma membrane in a zinc-dependent manner. The objective of this research was to examine the zinc binding properties of the large intracellular loop of hZIP4. Therefore, we have recombineantly expressed and purified M3M4 and showed that this domain binds two zinc ions. Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn2+ ions bind sequentially, with the first Zn2+ binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn2+ binding to a His4 site with a weaker affinity. Circular dichroism spectroscopy revealed that the M3M4 domain is intrinsically disordered, with only a small structural change induced upon Zn2+ coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn2+ concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn2+ binding. PMID:25882556

  12. Clinical and Prognostic Profiles of Cardiomyopathies Caused by Mutations in the Troponin T Gene. (United States)

    Ripoll-Vera, Tomás; Gámez, José María; Govea, Nancy; Gómez, Yolanda; Núñez, Juana; Socías, Lorenzo; Escandell, Ángela; Rosell, Jorge


    Mutations in the troponin T gene (TTNT2) have been associated in small studies with the development of hypertrophic cardiomyopathy characterized by a high risk of sudden death and mild hypertrophy. We describe the clinical course of patients carrying mutations in this gene. We analyzed the clinical characteristics and prognosis of patients with mutations in the TNNT2 gene who were seen in an inherited cardiac disease unit. Of 180 families with genetically studied cardiomyopathies, 21 families (11.7%) were identified as having mutations in TNNT2: 10 families had Arg92Gln, 5 had Arg286His, 3 had Arg278Cys, 1 had Arg92Trp, 1 had Arg94His, and 1 had Ile221Thr. Thirty-three additional genetic carriers were identified through family assessment. The study included 54 genetic carriers: 56% were male, and the mean average age was 41 ± 17 years. There were 33 cases of hypertrophic cardiomyopathy, 9 of dilated cardiomyopathy, and 1 of noncompaction cardiomyopathy, and maximal myocardial thickness was 18.5 ± 6mm. Ventricular dysfunction was present in 30% of individuals and a history of sudden death in 62%. During follow-up, 4 patients died and 14 (33%) received a defibrillator (8 probands, 6 relatives). Mean survival was 54 years. Carriers of Arg92Gln had early disease development, high penetrance, a high risk of sudden death, a high rate of defibrillator implantation, and a high frequency of mixed phenotype. Mutations in the TNNT2 gene were more common in this series than in previous studies. The clinical and prognostic profiles depended on the mutation present. Carriers of the Arg92Gln mutation developed hypertrophic or dilated cardiomyopathy and had a significantly worse prognosis than those with other mutations in TNNT2 or other sarcomeric genes. Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  13. Copper(I) nitro complex with an anionic [HB(3,5-Me2Pz)3]− ligand: a synthetic model for the copper nitrite reductase active site. (United States)

    Hsu, Sodio C N; Chang, Yu-Lun; Chuang, Wan-Jung; Chen, Hsing-Yin; Lin, I-Jung; Chiang, Michael Y; Kao, Chai-Lin; Chen, Hsuan-Ying


    The new copper(I) nitro complex [(Ph(3)P)(2)N][Cu(HB(3,5-Me(2)Pz)(3))(NO(2))] (2), containing the anionic hydrotris(3,5-dimethylpyrazolyl)borate ligand, was synthesized, and its structural features were probed using X-ray crystallography. Complex 2 was found to cocrystallize with a water molecule, and X-ray crystallographic analysis showed that the resulting molecule had the structure [(Ph(3)P)(2)N][Cu(HB(3,5-Me(2)Pz)(3))(NO(2))]·H(2)O (3), containing a water hydrogen bonded to an oxygen of the nitrite moiety. This complex represents the first example in the solid state of an analogue of the nitrous acid intermediate (CuNO(2)H). A comparison of the nitrite reduction reactivity of the electron-rich ligand containing the CuNO(2) complex 2 with that of the known neutral ligand containing the CuNO(2) complex [Cu(HC(3,5-Me(2)Pz)(3))(NO(2))] (1) shows that reactivity is significantly influenced by the electron density around the copper and nitrite centers. The detailed mechanisms of nitrite reduction reactions of 1 and 2 with acetic acid were explored by using density functional theory calculations. Overall, the results of this effort show that synthetic models, based on neutral HC(3,5-Me(2)Pz)(3) and anionic [HB(3,5-Me(2)Pz)(3)](-) ligands, mimic the electronic influence of (His)(3) ligands in the environment of the type II copper center of copper nitrite reductases (Cu-NIRs).

  14. Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Alison Rattray


    Full Text Available Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3 placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts. Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

  15. Analysis of genital Candida albicans infection by rapid microsatellite markers genotyping

    Institute of Scientific and Technical Information of China (English)

    SHI Wei-min; MEI Xing-yu; GAO Fei; HUO Ke-ke; SHEN Liang-liang; QIN Hai-hong; WU Zhou-wei; ZHENG Jie


    Background Candida albicans (C. albicans) infection, often occurring in genital candidiasis, has increased dramatically recently. Developing an efficient C. albicans typing method may contribute to understanding its epidemiological characteristics and guiding efficient treatment. We used rapid microsatellite genotyping assay for interstrain differentiation of C. albicans isolates and explored some characteristics of its spread.Methods DNA was extracted from C. albicans isolates from gentalia, recta and mouths of 39 female cases and 27 male cases of genital candidiasis. Three fluorescent primers for the microsatellite markers in conserved genes (CDC3, EF3and HIS3) of C. albicans were used to amplify the isolates DNA by PCR. Fluorescent signals were read with an automatic sequencer and analyzed with GeneScan software.Results Analysis of the three microsatellites markers showed 18 gene allelic associations in genital C. albicans infected patients: 10 allelic associations in female and 11 allelic associations in male, of which 3 allelic associations shared by both genders covered 71% of infections. The most dominant allele association of pathogenic strains for both genders was 116:124, 122:131,160:200 that covered about 50% of infection. Gentalia and recta shared the same strains in 80%of female patients, but in only 3.8% of male patients. There were 2.7% female patients, but no males, with same strain in both gentalia and mouths. Five of seven genital C. albicans infected couples had the same allelic associations of which 4were the dominant pathogenic C. albicans susceptible for both genders.Conclusions The predominant allelic association of the pathogenic strain in genital C. albicans infection is 116:124,122:131, 160:200. Vaginal pathogenic strains are probably maintained from the rectal reservoir. Pathogenic strains of male patients are probably from frequent sexual intercourse. The aggressiveness of some strains varies with gender.

  16. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts. (United States)

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah


    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.

  17. Phylogeny and variability of Colletotrichum truncatum associated with soybean anthracnose in Brazil. (United States)

    Rogério, F; Ciampi-Guillardi, M; Barbieri, M C G; Bragança, C A D; Seixas, C D S; Almeida, A M R; Massola, N S


    Fungal diseases are among the main factors limiting high yields of soybean crop. Colletotrichum isolates from soybean plants with anthracnose symptoms were studied from different regions and time periods in Brazil using molecular, morphological and pathogenic analyses. Bayesian phylogenetic inference of GAPDH, HIS3 and ITS-5.8S rDNA sequences, the morphologies of colony and conidia, and inoculation tests on seeds and seedlings were performed. All isolates clustered only with Colletotrichum truncatum species in three well-separated clusters. Intraspecific genetic diversity revealed 27 distinct haplotypes in 51 fungal isolates; some of which were identical to C. truncatum sequences from other regions around the world, while others were related to alternative hosts. Conidia were falcate, hyaline, unicellular and aseptate, formed in acervuli, with variable dimensions. Despite being pathogenic to seedlings by both inoculation methods, variation was observed in the aggressiveness of the tested isolates, which was not correlated with genetic variation. The identification of C. truncatum in the sampled isolates was evidenced as being the only causal agent of soybean anthracnose in Brazil until 2007, with relevant genetic, morphological and pathogenic variability as well as a broad geographical origin. The wide distribution of the predominant C. truncatum haplotype indicated the existence of a highly efficient mechanism of pathogen dispersal over long distances, reinforcing the role of seeds as the primary source of disease inoculum. The characterization and distribution of Colletotrichum species in soybean-producing regions in Brazil is fundamental for understanding the disease epidemiology and for ensuring effective control strategies against anthracnose. © 2016 The Society for Applied Microbiology.

  18. Reference gene selection for quantitative real-time PCR normalization in Quercus suber. (United States)

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia


    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  19. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    Directory of Open Access Journals (Sweden)

    Liliana Marum

    Full Text Available The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber, have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old or collected in different dates (active growth period versus dormancy. The three statistical methods (geNorm, NormFinder, and CV method used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  20. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

    Directory of Open Access Journals (Sweden)

    Penn Charles W


    Full Text Available Abstract Background Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. Results Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the λ-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6 × His, 3 × FLAG, 4 × ProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the λ-Red system, which can lead

  1. Lack of evidence for association of primary sclerosing cholangitis and primary biliary cirrhosis with risk alleles for Crohn's disease in Polish patients

    Directory of Open Access Journals (Sweden)

    Gaj Pawel


    Full Text Available Abstract Background Numerous papers have addressed the association of mutations and polymorphisms of susceptibility genes with autoimmune inflammatory disorders. We investigated whether polymorphisms that confer susceptibility to Crohn's disease could be classified also as predisposing factors for the development of primary sclerosing cholangitis and primary biliary cirrhosis in Polish patients. Methods The study included 60 patients with CD, 77 patients with PSC, of which 61 exhibited IBD (40 UC, 8 CD, and 13 indeterminate colitis, and 144 patients with PBC. All the patients were screened against Crohn's disease associating genetic polymorphisms. The polymorphisms were chosen according to previously confirmed evidence for association with Crohn's disease, including Pro268Ser, Arg702Trp, Gly908Arg and 1007fs in NOD2/CARD15, Leu503Phe/-207G>C in SLC22A4/OCTN1/SLC22A5/OCTN2, Arg30Gln in DLG5, Thr300Ala in ATG16L1, and Arg381Gln, His3Gln and exon-3'UTR in IL23R. Genotyping was carried out using TaqMan SNP genotyping assays. Results We confirmed a strong association between three NOD2/CARD15 gene variants (Pro268Ser, OR = 2.52, 95% CI = 1.34 – 4.75; (Arg702Trp, OR = 6.65, 95% CI = 1.99 – 22.17; (1007fs, OR = 9.59, 95% CI = 3.94 – 23.29, and a weak association between both the protective OCTN1/OCTN2 CC haplotype (OR = 0.28, 95% CI = 0.08 – 0.94, and a variant of ATG16L1 gene (Thr300Ala, OR = 0.468, 95% CI = 0.24 – 0.90 with Crohn's disease. In contrast, none of the polymorphisms exhibited association with susceptibility to primary sclerosing cholangitis and primary biliary cirrhosis, including a group of primary sclerosing cholangitis patients with concurrent IBD. Conclusion Although the clinical data indicate non-random co-occurrence of inflammatory bowel disease and primary sclerosing cholangitis, consistently with the previously published studies, no genetic association was found between the genetic variants predisposing to Crohn

  2. Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae. (United States)

    Schnier, J; Schwelberger, H G; Smit-McBride, Z; Kang, H A; Hershey, J W


    Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential

  3. Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

    Directory of Open Access Journals (Sweden)

    Hiu-Kwan Chow

    Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

  4. Evaluation of Acting Domain and Strength Mediating the Protein Self-interactions of SVP and FLC in Brassica juncea%芥菜开花负调因子SVP及FLC同源互作域筛选和作用强度分析

    Institute of Scientific and Technical Information of China (English)

    汤青林; 丁宁; 李念祖; 王志敏; 宋明



  5. 芥菜开花调控蛋白SVP与FLC酵母表达载体的构建及其相互作用研究%Determination of Interactions Between SVP and FLC in Brassica juncea Coss. by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    汤青林; 李念祖; 丁宁; 陈竹睿; 宋明; 王志敏


    为深入研究芥菜开花信号整合子的两个核心调节因子SHORTVEGETATIVEPHASE(SVP)与FLOWERINGLOCUSC(FLC)相互作用的分子机理,通过PCR扩增,从芥菜材料‘QJ’中分别克隆含EcoRI/BamHI双酶切位点的SVP和FLC编码区全长,并利用酵母双杂交体系,将FLC与GAL4报告基因DNA激活域融合(pGADT7FLC),SVP与GAL4报告基因DNA结合域融合(pGBKT7SVP)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现白激活和毒性现象。融合的二倍体酵母(pGADT7FLC×pGBKT7SVP)能在选择性固体培养基QDODUA(SD/-Ade/-His/-Leu/-Trp/X-α-GaFAbA)上生长,并且菌落呈蓝色。将诱饵质粒(pGBKT7SVP)与猎物质粒(pGADT7FLC)载体互换(pGADT7SVP、pGBKTTFLC),再次转化酵母后仍能融合成二倍体酵母(pGADT7SVP×pGBKT7FLC),并同时激活报告基因AURI-C、HIS3、ADE2、MELl,由此表明SVP与FLC蛋白能够相互结合。%The fate of the flowering signal integrators is determined by SHORT VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC) . For further study on the mechanism of the mutual recognition between SVP and FLC in Brassicajuncea Coss. (Mustard) variety' QJ', the coding sequences of SVP and FLC with digestion sites of EcoR I/BamH I were respectively amplified via PCR, and the interactions between SVP and FLC were detected by the yeast two-hybrid system. The full-length FLC was fused to the GAL4 DNA activation domain, which was designated as pGADT7FLC and then transformed into Y187 yeast stain. While SVP was fused to the GAL4 DNA binding domain, which was designated as pGBKT7SVP and then transformed into Y2HGold yeast stain. The two transformed yeaststains did not exhibit autoactivation and toxicity. The yeast stains of pGADT7FLC and pGBKT7SVP could mate into yeast diploids. The zygote diploids grew on selective agar plates QDO/X/A (SD/-Ade/-His/-Leu/-Trp/X-a-Gal/AbA) with blue stains. The results

  6. Identification of Acting Domains Mediating the Protein Interactions Between SVP and FLC in Brassicajuncea Coss.%芥菜开花调控因子SVP与FLC蛋白互作的结构域筛选与鉴定

    Institute of Scientific and Technical Information of China (English)

    汤青林; 李念祖; 宋明; 丁宁; 陈竹睿; 刘智宇; 王志敏


    为阐明芥菜开花路径核心调节子SVP与FLC相互作用的结构域,从酵母重组质粒pGADT7SVP、pGBKT7FLC分别亚克隆了5个SVP截短体(SVPl~5)和5个FLC截短体(FLCl~5)。SVPI一5与FLCl~5编码蛋白的结构域均分别为MI、MIK、K、IKC和KC。利用酵母双杂交体系,分别构建酵母猎物质粒pGADT7SVPl—5与诱饵质粒pGBKT7FLCl~5,并转化对应的酵母Y187、Y2HGold菌。酵母转化子Y187[pGADT7SVP2~5]能与Y2HGold[-pGBKT7FLC]融合,并可在选择性固体培养基QDO/X/A上长出蓝色菌落,表明FLC能与截短体蛋白SVP2~5异源结合,SVP的K域(SVP3)可独立作用于FLC蛋白。此外,Y187[pGADT7SVP]×Y2HGold[pGBKT7FLC2~5]也能同时激活报告基因AURl.C、HIS3、ADE2、施z,,表明FLC的K域(FLC3)也可独立作用于SVP。进一步研究发现:Y187pGADT7SVP3]xY2Ht30ld[pGBKT7FLC3]正向杂交以及Y187I-pGADT7FLC3]×Y2HGoldl-pGBKT7SVP3]载体互换后杂交均可相互作用,表明SVP的K域(SVP第96~173位氨基酸区域)与FLC的K域(FLC第114~167位氨基酸区域)能够异源结合,是介导SVP与FLC蛋白互作的关键结构域。%SVP and FLC were the key regulators in plant flowering pathways. For further study on the molecular mechanism of flowering control in Brassicajuncea Coss. (mustard), the protein interaction domains between SVP and FLC were screened via yeast two-hybrid system. The truncated genes of SVP1- 5 and FLC1-5 were respectively subcloned from yeast recombination plasmids pGADT7SVP and pGBKT7FLC. The proteins encoded by SVP1-5 or FLC1-5 had MI-domain, MIK-domain, K-domain,IKC-domain and KC-domain, respectively. SVP1-5 truncated forms were fused into bait plasmid pGADT7, which were designated as pGADT7SVP1- 5 and then transformed into Y187 yeast stains. FLC1 -5 truncated forms were fused into prey plasmid pGBKT7, which were designated as pGBKT7FLC1- 5 and then transformed into Y2HGold stains. All the transformed yeast

  7. 免疫组织化学Image Pro Plus图像半定量分析的参数选择%Preferences selection of immunohistochemistry semiquantitative analysis by Image Pro Plus image analysis system

    Institute of Scientific and Technical Information of China (English)

    徐洪; 杨方; 袁媛; 程华; 魏中秋; 李淑钰


    100以下的阳性显色;采用吸管取色、RGB、HIS 3种选择阳性显色的方法;编写宏文件,计算各组阳性细胞面积、平均光密度值(MD)、移分光密度值(IOD)的均值和总和,并与人工计数法测算结果进行比较.结果:在滤过面积10~100范围内,面积参数对各组阳性细胞面积总和、IOD总和统计结果无影响,与人工计数法结果一致;采用HIS选色测算的面积总和、IOD总和与人工计数法结果一致.结论:IPP图像分析能够简易、快速地进行免疫组织化学结果的半定量分析,面积参数在一定范围内对测算结果无影响,HIS选色方式可能较吸管显色和RGB选色方式好,而IOD总和是比较可靠、稳定的半定量指标.

  8. Correlation between SULT1A1 Arg213His Gene Polymorphisms and Uterine Leiomyomas%SULT1A1基因Arg213His 位点多态性与子宫肌瘤发生的关联性

    Institute of Scientific and Technical Information of China (English)

    周超; 林林; 张英姿; 徐天和; 张磊磊


    目的:探讨硫酸氨基转移酶(Sulfotransferase,SULT)1A1基因Arg213His位点多态性与鲁北地区汉族女性子宫肌瘤的关系.方法:以病例-对照的研究方法,采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法检测了123例子宫肌瘤患者和123例匹配对照者的SULT1A1基因Arg213His位点的基因型,应用条件Logistic回归等方法分析基因多态性与子宫肌瘤的关系.结果:1)SULT1A1基因Arg/Arg、Arg/His、His/His 3种基因型在子宫肌瘤与对照组中的分布存在显著性差异(P=0.011);2)与Arg/Arg基因型相比,Arg/His、His/His危险度均增加,分别为2.321倍和1.985倍(P=0.003和P=0.468);3)His等位基因可显著增加患子宫肌瘤的危险性(P=0.003,OR调整=2.296,95%CI为1.325~3.978).结论:SULT1A1基因Arg213His位点基因多态性与鲁北地区汉族女性子宫肌瘤的发生有关,增加了子宫肌瘤的患病风险.%Objective: To study the correlation between polymorphisms of SULT1A1 with the risk of uterine leiomyoma interaction among Han Chinese in Northern QiLu.Methods: Arg213His genotypes of the SULT1A1 gene were detected using polymerase chain reaction-restriction fragment length polymorphism in a case-control study.A total of 123 cases of uterine leiomyomas and 123 controls were included.Multivariate logistic regression analysis was used to estimate the risk of developing uterine leiomyomas associated with environmental exposures of the SULT1A1 genotype.Results: ( 1 ) The SULT1A1 polymorphisms of the Arg/Arg, Arg/His, and His/His genotypes in the uterine leiomyoma group and the control group were significantly different ( P = 0.011 ); ( 2 ) Comparedwith that of the Arg/Arg genotype, the risk of the Arg/His was genotype and His/His was both increased, by 2.321 times and 1.985 times ( P = 0.003 and P = 0.468 ).( 3 ) The risk was significantly higher among uterine leiomyoma patients with the His allele ( OR adjusted = 2.296, 95 % CI 1.325-3.978, P = 0

  9. Ethanol production by recombinant and natural xylose-utilising yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Eliasson, Anna


    from P. stipitis and the endogenous XKS1 gene under control of the PGKI promoter, into the HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain was stable for more than forty generations in continuous fermentation. The metabolic fluxes during xylose metabolism were quantitatively analysed and anaerobic ethanol formation from xylose in recombinant S. cerevisiae was demonstrated for the first time. The xylose uptake rate increased with increasing xylose concentration in the feed. However, with a feed of 15 g/l xylose and 5 g/l glucose, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux. The role of mitochondria in ethanol formation from xylose was investigated using cells of recombinant xylose-utilising S. cerevisiae with two different respiratory capacities and cells from P. stipitis grown under conditions of optimal ethanol formation. Different inhibitors were used either to inhibit the electron transport chain and simulate oxygen limitation, or to inhibit the tricarboxylic acid cycle while not disturbing the electron transport chain. The response to the inhibitors differed significantly for glucose and xylose and the effect was more pronounced for S. cerevisiae. The results indicate that mitochondria play a significant role in the maintenance of the cytoplasmic redox balance during xylose fermentation, through the action of cytoplasmically directed NADH dehydrogenase activity. Thus, more carbon was directed towards ethanol in chemostat cultivations of xylose/glucose mixtures by S. cerevisiae TMB 3001, in the presence of low amounts of oxygen. P. stipitis possesses a second, cyanide-insensitive terminal oxidase, the alternative oxidase, which seems to be of particular importance for efficient ethanol formation from xylose. The highest activity of cyanide-insensitive respiration (CIR), the highest ethanol productivity and lowest xylitol formation were all observed with cells grown under oxygen-limited conditions

  10. Ethanol production by recombinant and natural xylose-utilising yeasts

    Energy Technology Data Exchange (ETDEWEB)

    Eliasson, Anna


    from P. stipitis and the endogenous XKS1 gene under control of the PGKI promoter, into the HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain was stable for more than forty generations in continuous fermentation. The metabolic fluxes during xylose metabolism were quantitatively analysed and anaerobic ethanol formation from xylose in recombinant S. cerevisiae was demonstrated for the first time. The xylose uptake rate increased with increasing xylose concentration in the feed. However, with a feed of 15 g/l xylose and 5 g/l glucose, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux. The role of mitochondria in ethanol formation from xylose was investigated using cells of recombinant xylose-utilising S. cerevisiae with two different respiratory capacities and cells from P. stipitis grown under conditions of optimal ethanol formation. Different inhibitors were used either to inhibit the electron transport chain and simulate oxygen limitation, or to inhibit the tricarboxylic acid cycle while not disturbing the electron transport chain. The response to the inhibitors differed significantly for glucose and xylose and the effect was more pronounced for S. cerevisiae. The results indicate that mitochondria play a significant role in the maintenance of the cytoplasmic redox balance during xylose fermentation, through the action of cytoplasmically directed NADH dehydrogenase activity. Thus, more carbon was directed towards ethanol in chemostat cultivations of xylose/glucose mixtures by S. cerevisiae TMB 3001, in the presence of low amounts of oxygen. P. stipitis possesses a second, cyanide-insensitive terminal oxidase, the alternative oxidase, which seems to be of particular importance for efficient ethanol formation from xylose. The highest activity of cyanide-insensitive respiration (CIR), the highest ethanol productivity and lowest xylitol formation were all observed with cells grown under oxygen-limited conditions

  11. French Antilles and Guiana. (United States)


    This discussion of French Antilles and Guiana cover the following: the people, geography, history, government, political conditions, economy, and relations with the US. In 1983 the population totaled 303,000 with an annual growth rate of 0.09%. The infant mortality rate (1981) was 12.6/1000 and life expectancy 68 years. About 98% of the people of Martinique are of Afro European or Afro European Indian descent. The remainder are the old planter families and a sizable number of metropolitan French. Most of the work force are employed in agriculture or food processing and associated industries. Most permanent residents of Guadeloupe are of mixed Afro European descent. A few thousand Metropolitan French reside there. Most French Guianese live along the coast, about 1/2 of them in the capital. Martinique is the northernmost of the Windward Islands, which are part of the Lesser Antilles chain in the Caribbean Sea southeast of Puerto Rico. Guadeloupe comprises 2 of the Leeward Islands, which are also part of the Lesser Antilles chain. French Guiana is located on the northern coast of South America, a few degrees north of the Equator. Indians were the 1st known indigenous inhabitants of French Guiana and the French Antilles. Columbus sighted Guadeloupe in 1493, Martinique in 1493 or 1502, and the Guiana coast probably during his 3rd voyage in 1498. French Guiana, Guadeloupe, and Martinique, as overseas departments of France since 1946, are integral parts of the French Republic. Their relationship to Metropolitan France is somewhat similar to that of Alaska and Hawaii to the counterminous US. Each department has a general council composed of 1 representative elected by each canton. Guadeloupe and Martinique each elect 2 senators to the French Senate and 3 deputies to the National Assembly. French Guiana elects 1 senator and 1 deputy. In each of the 3 departments exist individuals and small political parties that advocate immediate independence, but their adherents form only

  12. 白桦开花抑制因子BpFLC与BpSVP蛋白同源互作域筛选与分析

    Institute of Scientific and Technical Information of China (English)

    郑唐春; 臧丽娜; 代丽娟; 杨传平; 曲冠证


    开花是植物由营养生长向生殖生长的转换点,该过程受内源基因与环境信号共同调控。FLC(Flowering locus C)和SVP(Short vegetative phase)是开花途径中关键的抑制开花因子。本文利用酵母双杂交技术深入研究白桦开花抑制因子FLC与SVP的自身聚合作用的分子机理。从重组质粒p GBKT7-BpFLC、p GBKT7-Bp SVP分别克隆出6个BpFLC截短体[BpFLC1(aa1~73)、BpFLC2(aa1~173)、BpFLC3(aa74~173)、BpFLC4(aa60~208)、BpFLC5(aa74~208)和BpFLC6(aa174~208)]和6个BpSVP截短体[BpSVP1(aa1~68)、Bp SVP2(aa1~172)、Bp SVP3(aa69~172)、BpSVP4(aa60~225)、BpSVP5(aa69~225)和Bp SVP6(aa173~225)],分别编码MADS型蛋白的MI、MIK、K、IKC、KC和C域。在酵母Y2HGold菌中,分别共转质粒pGBKT7-BpFLC1~6×pGADT7-BpFLC及p GBKT7-BpSVP×pGADT7-BpSVP1~6。酵母转化子Y2HGold[p GBKT7-BpFLC×p GADT7-BpFLC]、Y2HGold[pGBKT7-BpFLC2~5×pGADT7-BpFLC],可在选择性固体培养基TDO(SD/-Leu/-Trp/-His)、QTO/A(SD/-Leu/-Trp/-His/-Ade/Ab A)上生长,并在QDO/A/X(SD/-Leu/-Trp/-His/-Ade/AbA/X-α-gal)上长出蓝色菌落,表明BpFLC能与自身及截短体蛋白BpFLC2~5同源结合。此外酵母Y2HGold[pGBKT7-BpSVP×pGADT7-Bp SVP]、Y2HGold[pGBKT7-Bp SVP×p GADT7-BpSVP2~5]也能同时激活报告基因AUR1-C、HIS3、ADE2、MEL1,说明BpSVP能与自身及截短体蛋白BpSVP2~5同源结合。对保守区的序列结构进一步分析发现:BpFLC与BpSVP的K域是它们能够同源结合,介导BpFLC与BpSVP自身形成同源二聚体的关键。

  13. P450氧化还原酶缺陷症的临床表现和羊水穿刺产前诊断的意义%P450 oxidoreductase deficiency: clinical manifestations and prenatal diagnosis by amniocentesis

    Institute of Scientific and Technical Information of China (English)

    茅江峰; 聂敏; 高劲松; 徐洪丽; 卢双玉; 伍学焱


    目的 本研究旨在增加对P450氧化还原酶(POR)缺陷症发病机制和临床表现的认识,探讨羊水穿刺进行POR基因检测对产前诊断的重要意义.方法 对1例孕妇的既往异常妊娠史和胎儿的畸形进行描述;对孕妇及其配偶的POR基因进行检测;在本次妊娠时,对羊水细胞POR基因进行检测;对分娩胎儿的外生殖器和骨骼进行评价.结果 1)此孕妇在既往首次妊娠时出现声音低沉、痤疮增多等显著男性化表现;首次分娩双胎,均有肘关节骨融合、阴蒂肥大和脑瘫,经救治无效而死亡.2)此孕妇及其配偶的POR基因检测证实,男方存在POR基因的杂合突变(G1370A、Arg457His).3)当此孕妇再次妊娠达20周时,对羊水细胞进行POR基因检测未发现突变.4)足月顺产1女婴,外生殖器和骨骼正常.结论 羊水穿刺进行POR基因检测有助于获得此疾病相关的基因信息,指导妊娠决策.%Objective To investigate the values of P450 oxidoreductase (POR) gene test by amniocentesis for prenatal diagnosis in a pregnant woman whose husband has a heterozygous mutation in POR gene. Methods The abnormal pregnant history in mother and genitalia deformities in her fetus were described. POR gene from the woman and her husband was tested by PCR. During her second pregnancy, the cells from amniotic fluid were collected for POR gene test. The external genitalia and bone status were evaluated after birth. Results 1) the woman presented excessive virilization, such as deepen voice and facial acnes, during her first pregnancy. Her baby twins, manifesting elbow osseous fusion, clitoridauxe and cerebral palsy, died 2 weeks after birth. 2) A heterozygous mutation in POR gene (G1370A, Arg457His) was revealed in the peripheral blood cells from her husband. 3) During her second pregnancy, amniocentesis was conducted and POR gene test for cells from amniotic fluid was negative. 4) A female baby was born with normal external genitalia and

  14. Cloning of antimicrobial piscidin-like peptide and prokaryotic expressionof the putative mature peptide in Epinephelus fuscoguttatus%棕点石斑鱼Piscidin样肽基因的克隆及其成熟肽的原核表达

    Institute of Scientific and Technical Information of China (English)

    陈大玮; 邓利; 何凡; 邱聪龄; 马一见; 刘志刚


    为棕点石斑鱼Piscidin样肽(GU592793),推导氨基酸序列具有界定鱼类Piscidin的以下共同特征:(1)预测的信号肽序列与其他Piscidin的同源性较高,在60%~95%之间;(2)推测的成熟肽N端6个氨基酸残基富含Phe、Ile及His;(3)成熟肽第8个和第13个氨基酸位点均为Gly.NJ进化树分析表明,该cDNA推导的氨基酸序列与赤点石斑鱼Epinephelusakaara piscidin-like peptide (ACE78290)、斜带石斑鱼Epinephelus coioides Piscidin-like peptide(ACE78291)以高达81%的支持率聚为一支.也表明本研究成功克隆到棕点石斑鱼首条PiscidincDNA序列.推测的成熟肽为两亲性阳离子肽,等电点12.48,Schiffer-Edmunson预测其二级结构呈a螺旋构型.将推测的成熟肽序列亚克隆至pET-32a构建融合表达载体pET-32a-pis(EF),并在大肠杆菌 E.coli Origami(DE3)中成功表达出融合蛋白,以1mmol/ml IPTG于30℃进行诱导表达,4h可获得大量表达,目的蛋白以包涵体形式存在.

  15. [Figures of first laureates of the Wiktor Dega medal (XXXVII Jubilee Congress of Polish Orthopaedic and Traumatologic Society, 10-13 September 2008)]. (United States)

    Nowakowski, Andrzej; Rapała, Kazimierz


    Figures of two outstanding orthopaedists Professor Stefan Malawski and Professor Jerzy Król rewarded with the medal of the name of Wiktor Degi were described. The medal is being granted by the Chapter of the Medal as regarding for outstanding achievements for the Polish and world orthopaedics and rehabilitation. Profesor Stefan Kazimierz Malawski was born 26. 12. 1920 in the Vilnius area. In Vilnius he stated his medical studies, which he continued in Lwow and graduated in 1946 at the Marie Curie Skłodowska in Lublin. Professor Malawski's main field of interest were related to the problems related to tuberculosis of bones and joints and trauma of the lumbar and cervical spine. In the problems of bone tuberculosis he remains an unquestioned authority in Poland. His deep understanding of these clinical problems can be found in his text-book "Tuberculosis of bones and joints", which was printed in 1976. The information pertaining diagnosis and surgical treatment remain extremely valuable today. Another field of interest of Professor Malawski are pathologies of the spine. Disc disease, neoplasms of the spine, spinal stenosis and infections of the spine, spondylolisthesis are among many of his interests. This very wide field of interest can be dound in his 3 tome publication Spondyloorthopedics. His 166 papars printed in Poland and abroad bear proof of the Professors wide field of interest and deep knowledge. Professor Malawski was the first surgeon in Poland to perform surgery on the front elements of the spine in tuberculotic paraplegia. In 1958 he implemented surgical treatment of spine tumor--both primary and metastatic, by resecting them and stabilizing the spine with grafts. In the early 70's he focused on spinal stenosis. In the years 1982-1986 he was the Chairman of the Board of the Polish Orthopedic and Trauma Society. Professor Malawski introdued a modern set of Rules and Regulations, greatly simplifying the decision making process during General assemblies

  16. xTAG(R) GPP多重核酸技术在北京地区感染性腹泻诊断中的应用及流行病学研究%The application and epidemiological research of xTAG(R) GPP multiplex PCR in the diagnosis of infectious diarrhea

    Institute of Scientific and Technical Information of China (English)

    冯伟明; 顾秀丽; 隋文君; 张明新; 鲁炳怀; 王玫; 黄艳飞; 鲁辛辛


    讨xTAG(R) GPP多重核酸技术对感染性腹泻早期诊断的应用价值,了解肠道腹泻病原体的流行病学情况.方法 收集2013年10月1日至2014年9月30日于首都医科大学附属北京同仁医院门诊就诊的腹泻患者便标本592份,运用xTAG(R) GPP法进行检测,将其结果与传统方法(培养、快速酶免疫层析法、显微镜检查、实时荧光定量PCR等)比较,并进行统计学分析.结果 xTAG(R) GPP法检测592份便标本的阳性率为47.8% (283/592),男女比例1:1.02,平均发病年龄31岁.病毒检出率为18.1%,以轮状病毒A型(RotV A)为主(8.8%),主要集中在冬季,儿童多发,其次为诺如病毒G Ⅰ/GⅡ(NorV G Ⅰ/GⅡ)占8.4%,腺病毒40/41(AdeV 40/41)检出5份.细菌阳性率为35.5%,产毒型大肠埃希菌LT/ST型(ETEC)最常见(8.4%,50/592),夏季高发,青壮年易感,其他病原体依次为弯曲杆菌(Camp)7.7%、沙门菌(Salm) 7.0%、难辨梭状芽胞杆菌毒素A/B型(C.difA/B) 3.5%、志贺菌(Shig) 3.3%、0157型大肠埃希菌(E.coli 0157)3.3%及产志贺毒素大肠埃希菌stx1/stx2型(STEC-LT/ST) 1.7%,未检出小肠结肠炎耶尔森氏菌(Y.ent)与霍乱弧菌(V.cho).检出寄生虫10份(1.7%),包括隐孢子虫(Crypto)5份、痢疾阿米巴(E.his)3份及贾第虫(Gia)2份,均无明显的季节和人群分布.单一病原体感染患者占40.8%(242/592),混合感染为6.9%(41/592),其中双重感染6.1%(36/592),三重感染0.8% (5/592).结论 xTAG(R) GPP多重核酸方法操作简便、灵敏、特异,可作为感染性腹泻病原学诊断的快速方法.腹泻病原体具有显著的季节和人群优势.