WorldWideScience

Sample records for sulfolobus solfataricus p1

  1. Sugar transport in the thermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Albers, Sonja-Verena

    2001-01-01

    Summary and concluding remarks Introduction The archaeon Sulfolobus solfataricus is a thermoacidophile preferring growth at around 80oC and a pH of 2.5 to 3.5. As a thermoacidophile S. solfataricus faces two major problems: firstly, the proton permeability of membranes increases with temperature

  2. Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus

    NARCIS (Netherlands)

    Walther, J.

    2012-01-01

    The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and

  3. Harnessing hyperthermostable lactonase from Sulfolobus solfataricus for biotechnological applications.

    Science.gov (United States)

    Rémy, Benjamin; Plener, Laure; Poirier, Laetitia; Elias, Mikael; Daudé, David; Chabrière, Eric

    2016-11-23

    Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures.

  4. Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Lubelska, Joanna M.; Jonuscheit, Melanie; Schleper, Christa; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2006-01-01

    Sugar uptake in Sulfolobus solfataricus, a thermoacidophilic archaeon, occurs through high-affinity binding of protein-dependent ABC transporters. We have investigated the expression patterns of two sugar transport operons, that is, the glucose and arabinose transporters. Analysis of the araS

  5. Properties of the recombinant a-glucosidase from Sulfolobus solfataricus in relation to starch processing

    NARCIS (Netherlands)

    Martino, A.; Schiraldi, C.; Fusco, S.; Lernia, Di I.; Costabile, T.; Pellicano, T.; Marotta, M.; Generoso, M.; Oost, van der J.; Sensen, C.W.

    2001-01-01

    An -glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3) with a

  6. Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus solfataricus phosphotriesterase

    International Nuclear Information System (INIS)

    Elias, Mikael; Dupuy, Jérôme; Merone, Luigia; Lecomte, Claude; Rossi, Mosè; Masson, Patrick; Manco, Giuseppe; Chabriere, Eric

    2007-01-01

    A phosphotriesterase (PTE) from the hyperthermophilic archaeon S. solfataricus has been crystallized. Combined with biochemical and bioengineering studies, it is expected that the structure of this protein will provide insight into the natural function of the PTE family and provide important data for achieving an efficient organophosphate biodecontaminant. Organophosphates constitute the largest class of insecticides used worldwide and some of them are potent nerve agents. Consequently, organophosphate-degrading enzymes are of paramount interest as they could be used as bioscavengers and biodecontaminants. Phosphotriesterases (PTEs) are capable of hydrolyzing these toxic compounds with high efficiency. A distant and hyperthermophilic representative of the PTE family was cloned from the archeon Sulfolobus solfataricus MT4, overexpressed in Escherichia coli and crystallized; the crystals diffracted to 2.54 Å resolution. Owing to its exceptional thermostability, this PTE may be an excellent candidate for obtaining an efficient organophosphate biodecontaminant. Here, the crystallization conditions and data collection for the hyperthermophilic S. solfataricus PTE are reported

  7. Genome-scale reconstruction and analysis of the metabolic network in the hyperthermophilic archaeon Sulfolobus solfataricus.

    Directory of Open Access Journals (Sweden)

    Thomas Ulas

    Full Text Available We describe the reconstruction of a genome-scale metabolic model of the crenarchaeon Sulfolobus solfataricus, a hyperthermoacidophilic microorganism. It grows in terrestrial volcanic hot springs with growth occurring at pH 2-4 (optimum 3.5 and a temperature of 75-80°C (optimum 80°C. The genome of Sulfolobus solfataricus P2 contains 2,992,245 bp on a single circular chromosome and encodes 2,977 proteins and a number of RNAs. The network comprises 718 metabolic and 58 transport/exchange reactions and 705 unique metabolites, based on the annotated genome and available biochemical data. Using the model in conjunction with constraint-based methods, we simulated the metabolic fluxes induced by different environmental and genetic conditions. The predictions were compared to experimental measurements and phenotypes of S. solfataricus. Furthermore, the performance of the network for 35 different carbon sources known for S. solfataricus from the literature was simulated. Comparing the growth on different carbon sources revealed that glycerol is the carbon source with the highest biomass flux per imported carbon atom (75% higher than glucose. Experimental data was also used to fit the model to phenotypic observations. In addition to the commonly known heterotrophic growth of S. solfataricus, the crenarchaeon is also able to grow autotrophically using the hydroxypropionate-hydroxybutyrate cycle for bicarbonate fixation. We integrated this pathway into our model and compared bicarbonate fixation with growth on glucose as sole carbon source. Finally, we tested the robustness of the metabolism with respect to gene deletions using the method of Minimization of Metabolic Adjustment (MOMA, which predicted that 18% of all possible single gene deletions would be lethal for the organism.

  8. Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights.

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    Full Text Available A novel acylpeptide hydrolase, named APEH-3(Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss and apeh(Ss (the gene encoding the previously identified APEH(Ss from S. solfataricus, which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

  9. Metal-ion dependent catalytic properties of Sulfolobus solfataricus class II α-mannosidase

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum; Poulsen, Nina Rødtness; Johnsson, Anna Margit Susanne

    2012-01-01

    residues. Recently, evidence has accumulated that class II α-mannosidase is active in complex with a range of divalent metal ions. In the present work, with employment of the class II α-mannosidase, ManA, from the hyperthermophilic archaeon Sulfolobus solfataricus, we explored the influence of the divalent...

  10. The Dichotomy of the Poly(ADP-Ribose Polymerase-Like Thermozyme from Sulfolobus solfataricus

    Directory of Open Access Journals (Sweden)

    Maria Rosaria Faraone Mennella

    2018-01-01

    Full Text Available The first evidence of an ADP-ribosylating activity in Archaea was obtained in Sulfolobus solfataricus(strain MT-4 where a poly(ADP-ribose polymerase (PARP-like thermoprotein, defined with the acronymous PARPSso, was found. Similarly to the eukaryotic counterparts PARPSso cleaves beta-nicotinamide adenine dinucleotide to synthesize oligomers of ADP-ribose; cross-reacts with polyclonal anti-PARP-1 catalytic site antibodies; binds DNA. The main differences rely on the molecular mass (46.5 kDa and the thermophily of PARPSso which works at 80 °C. Despite the biochemical properties that allow correlating it to PARP enzymes, the N-terminal and partial amino acid sequences available suggest that PARPSso belongs to a different group of enzymes, the DING proteins, an item discussed in detail in this review.This finding makes PARPSso the first example of a DING protein in Archaea and extends the existence of DING proteins into all the biological kingdoms. PARPSsohas a cell peripheral localization, along with the edge of the cell membrane. The ADP-ribosylation reaction is reverted by a poly(ADP-ribose glycohydrolase-like activity, able to use the eukaryotic poly(ADP-ribose as a substrate too. Here we overview the research of (ADP-ribosylation in Sulfolobus solfataricus in the past thirty years and discuss the features of PARPSso common with the canonical poly(ADP-ribose polymerases, and the structure fitting with that of DING proteins.

  11. Modeling DNA Repair: Approaching In Vivo Techniques in the Hyperthermophile Sulfolobus Solfataricus

    Energy Technology Data Exchange (ETDEWEB)

    Blanton, J.; Fuss, J.; Yannone, S.M.; Tainer, J.A.; Cooper, P.K.

    2005-01-01

    Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus, isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park, has emerged as a potential model system for studying human DNA repair processes. Archaea are more closely related to Eukarya than to Eubacteria, suggesting that archaeal DNA repair machinery may model the complex human system much more closely than that of other prokaryotes. DNA repair requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, PCNA1, and a putative CSB homologue. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5α E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

  12. Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus.

    Science.gov (United States)

    De Felice, Mariarita; Esposito, Luca; Rossi, Mosè; Pisani, Francesca M

    2006-02-01

    The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.

  13. Crystallization and preliminary X-ray characterization of a PaaX-like protein from Sulfolobus solfataricus P2

    International Nuclear Information System (INIS)

    Cao, Yi; Lou, Zhiyong; Sun, Yuna; Xue, Fei; Feng, Changzeng; Gong, Xiaocui; Yang, Dongmei; Bartlam, Mark; Meng, Zhaohui; Zhang, Keqin

    2009-01-01

    In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. PaaX is a global regulator of the phenylacetyl-coenzyme A catabolon that adjusts the expression of different operons to that of the paa-encoded central pathway. In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Diffraction data were obtained to a resolution of 3.0 Å using synchrotron radiation at the Photon Factory. The crystal belonged to space group P321, with unit-cell parameters a = 86.4, b = 86.4, c = 105.5 Å

  14. Proteomic mapping of the hyperthermophilic and acidophilic archaeon Sulfolobus solfataricus P2

    Energy Technology Data Exchange (ETDEWEB)

    Barry, Richard C.; Young, Mark J.; Stedman, Kenneth M.; Dratz, Edward A.

    2006-07-14

    A proteomic map of Sulfolobus solfataricus P2, an archaeon that grows optimally at 80 C and pH 3.2, was developed using high resolution two-dimensional gel electrophoresis and peptide mass fingerprinting. A total of 867 protein spots (659 aqueous tris-soluble spots and 208 aqueous tris-insoluble) were mapped over IPG 3-10, 4-7, and 6-11, with second dimension gels made of 8-18% polyacrylamide. 324 different gene products were represented by the 867 spots, with 274 gene products being identified in the tris-soluble fractions and 100 gene products in the tris-insoluble portion. Fifty gene products were found on gels from both fractions. Additionally, an average of 1.50 + 0.12 isoforms/per protein were identified. This mapping study confirmed the expression of proteins involved in numerous metabolic, transport, energy production, nucleic acid replication, translation, and transcription pathways. Of particular interest, phosphoenolpyruvate carboxykinase (SSO2537) was detected even though the pathway for gluconeogenesis is unknown for this archaeon. Tris-soluble fractions contained many cytosolic proteins while tris-insoluble fractions contained many membrane-associated proteins, including ABC transporters and an ATP synthase. This study provides an optimized 2-DE approach for investigating the biochemical pathways and post-translational modifications employed by Sulfolobus to survive in its extreme environment.

  15. Backbone assignment of the binary complex of the full length Sulfolobus solfataricus DNA polymerase IV and DNA.

    Science.gov (United States)

    Lee, Eunjeong; Fowler, Jason D; Suo, Zucai; Wu, Zhengrong

    2017-04-01

    Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase, bypasses a wide range of DNA lesions in vitro and in vivo. In this paper, we report the backbone chemical shift assignments of the full length Dpo4 in its binary complex with a 14/14-mer DNA substrate. Upon DNA binding, several β-stranded regions in the isolated catalytic core and little finger/linker fragments of Dpo4 become more structured. This work serves as a foundation for our ongoing investigation of conformational dynamics of Dpo4 and future determination of the first solution structures of a DNA polymerase and its binary and ternary complexes.

  16. The non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) of Sulfolobus solfataricus: a key-enzyme of the semi-phosphorylative branch of the Entner-Doudoroff pathway

    NARCIS (Netherlands)

    Ettema, T.J.G.; Ahmed, H.; Geerling, A.C.M.; Oost, van der J.; Siebers, B.

    2008-01-01

    Archaea utilize a branched modification of the classical Entner¿Doudoroff (ED) pathway for sugar degradation. The semi-phosphorylative branch merges at the level of glyceraldehyde 3-phosphate (GAP) with the lower common shunt of the Emden-Meyerhof-Parnas pathway. In Sulfolobus solfataricus two

  17. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe...

  18. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    Directory of Open Access Journals (Sweden)

    Scaloni Andrea

    2007-08-01

    Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

  19. Biochemical, transcriptional and translational evidences of the phenol-meta-degradation pathway by the hyperthermophilic Sulfolobus solfataricus 98/2.

    Directory of Open Access Journals (Sweden)

    Alexia Comte

    Full Text Available Phenol is a widespread pollutant and a model molecule to study the biodegradation of monoaromatic compounds. After a first oxidation step leading to catechol in mesophilic and thermophilic microorganisms, two main routes have been identified depending on the cleavage of the aromatic ring: ortho involving a catechol 1,2 dioxygenase (C12D and meta involving a catechol 2,3 dioxygenase (C23D. Our work aimed at elucidating the phenol-degradation pathway in the hyperthermophilic archaea Sulfolobus solfataricus 98/2. For this purpose, the strain was cultivated in a fermentor under different substrate and oxygenation conditions. Indeed, reducing dissolved-oxygen concentration allowed slowing down phenol catabolism (specific growth and phenol-consumption rates dropped 55% and 39%, respectively and thus, evidencing intermediate accumulations in the broth. HPLC/Diode Array Detector and LC-MS analyses on culture samples at low dissolved-oxygen concentration (DOC  =  0.06 mg x L(-1 suggested, apart for catechol, the presence of 2-hydroxymuconic acid, 4-oxalocrotonate and 4-hydroxy-2-oxovalerate, three intermediates of the meta route. RT-PCR analysis on oxygenase-coding genes of S. solfataricus 98/2 showed that the gene coding for the C23D was expressed only on phenol. In 2D-DIGE/MALDI-TOF analysis, the C23D was found and identified only on phenol. This set of results allowed us concluding that S. solfataricus 98/2 degrade phenol through the meta route.

  20. Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes

    DEFF Research Database (Denmark)

    Sørensen, Iben Schildt; Dandanell, Gert

    1997-01-01

    From a genomic library of Sulfolobus solfataricus DSM1617 we have isolated and identified the purEK locus. Two open reading frames are identified as homologs of the purE and purK purine biosynthetic genes in Escherichia coli. The C-terminus of purE overlaps with the N-terminus of purK. When either...... of the genes is expressed from an E. coli promoter they can complement the corresponding purE and purK mutations in E. coli. PurE seems to be more closely related to eubacteria than to other archaea and to eukaryotes. Also the purK gene, which has not yet been found in other archaea, is more closely related...

  1. A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    2014-02-01

    Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

  2. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves

  3. EXPRESSION, PURIFICATION, AND SMALL ANGLE X-RAY SCATTERING OF DNA REPLICATION AND REPAIR PROTEINS FROM THE HYPERTHERMOPHILE SULFOLOBUS SOLFATARICUS

    Energy Technology Data Exchange (ETDEWEB)

    Patterson, S.M.; Hatherill, J.R.; Hammel, M.; Hura, G.L.; Tainer, J.A.; Yannone, S.M.

    2008-01-01

    Vital molecular processes such as DNA replication, transcription, translation, and maintenance occur through transient protein interactions. Elucidating the mechanisms by which these protein complexes and interactions function could lead to treatments for diseases related to DNA damage and cell division control. In the recent decades since its introduction as a third domain, Archaea have shown to be simpler models for complicated eukaryotic processes such as DNA replication, repair, transcription, and translation. Sulfolobus solfataricus is one such model organism. A hyperthermophile with an optimal growth temperature of 80°C, Sulfolobus protein-protein complexes and transient protein interactions should be more stable at moderate temperatures, providing a means to isolate and study their structure and function. Here we provide the initial steps towards characterizing three DNA-related Sulfolobus proteins with small angle X-ray scattering (SAXS): Sso0257, a cell division control and origin recognition complex homolog, Sso0768, the small subunit of the replication factor C, and Sso3167, a Mut-T like protein. SAXS analysis was performed at multiple concentrations for both short and long exposure times. The Sso0257 sample was determined to be either a mixture of monomeric and dimeric states or a population of dynamic monomers in various conformational states in solution, consistent with a fl exible winged helix domain. Sso0768 was found to be a complex mixture of multimeric states in solution. Finally, molecular envelope reconstruction from SAXS data for Sso3167 revealed a novel structural component which may function as a disordered to ordered region in the presence of its substrates and/or protein partners.

  4. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion....

  5. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer c....... solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization....

  6. Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Dennis K.; Webb, Helen; Nielsen, Jonas Willum

    2015-01-01

    +). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn2+ concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme......Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the-1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous...... enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in KM for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, KA, for the divalent metal ion (Co2+, Zn2+, Mn2+, or Cd2...

  7. High yield production and purification of two recombinant thermostable phosphotriesterase-like lactonases from Sulfolobus acidocaldarius and Sulfolobus solfataricus useful as bioremediation tools and bioscavengers.

    Science.gov (United States)

    Restaino, Odile Francesca; Borzacchiello, Maria Giovanna; Scognamiglio, Ilaria; Fedele, Luigi; Alfano, Alberto; Porzio, Elena; Manco, Giuseppe; De Rosa, Mario; Schiraldi, Chiara

    2018-03-20

    Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus. To follow this aim new induction approaches were investigated to boost the enzyme production, high cell density fermentation strategies were set-up to reach higher and higher enzyme yields up to 22-L scale, a downstream train was studied to meet the requirements of an efficient industrial purification process. Physiological studies in shake flasks demonstrated that the use of galactose as inducer increased the enzyme concentrations up to 4.5 folds, compared to the production obtained by induction with IPTG. Optimising high cell density fed-batch strategies the production and the productivity of both enzymes were further enhanced of 26 folds, up to 2300 U·L - 1 and 47.1 U·L - 1 ·h - 1 for SacPox and to 8700 U·L - 1 and 180.6 U·L - 1 ·h - 1 for SsoPox C258L/I261F/W263A, and the fermentation processes resulted scalable from 2.5 to 22.0 L. After being produced and extracted from the cells, the enzymes were first purified by a thermo-precipitation step, whose conditions were optimised by response surface methodology. A following ultra-filtration process on 100 and 5 KDa cut-off membranes drove to a final pureness and a total recovery of both enzymes of 70.0 ± 2.0%, suitable for industrial applications. In this paper, for the first time, a high yield biotechnological manufacturing process of the recombinant enzymes SacPox and SsoPox C258L/I261F/W263A was set-up. The enzyme production was

  8. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...... in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library...

  9. A systems biology approach reveals major metabolic changes in the thermoacidophilic archaeon Sulfolobus solfataricus in response to the carbon source L-fucose versus D-glucose.

    Science.gov (United States)

    Wolf, Jacqueline; Stark, Helge; Fafenrot, Katharina; Albersmeier, Andreas; Pham, Trong K; Müller, Katrin B; Meyer, Benjamin H; Hoffmann, Lena; Shen, Lu; Albaum, Stefan P; Kouril, Theresa; Schmidt-Hohagen, Kerstin; Neumann-Schaal, Meina; Bräsen, Christopher; Kalinowski, Jörn; Wright, Phillip C; Albers, Sonja-Verena; Schomburg, Dietmar; Siebers, Bettina

    2016-12-01

    Archaea are characterised by a complex metabolism with many unique enzymes that differ from their bacterial and eukaryotic counterparts. The thermoacidophilic archaeon Sulfolobus solfataricus is known for its metabolic versatility and is able to utilize a great variety of different carbon sources. However, the underlying degradation pathways and their regulation are often unknown. In this work, the growth on different carbon sources was analysed, using an integrated systems biology approach. The comparison of growth on L-fucose and D-glucose allows first insights into the genome-wide changes in response to the two carbon sources and revealed a new pathway for L-fucose degradation in S. solfataricus. During growth on L-fucose major changes in the central carbon metabolic network, as well as an increased activity of the glyoxylate bypass and the 3-hydroxypropionate/4-hydroxybutyrate cycle were observed. Within the newly discovered pathway for L-fucose degradation the following key reactions were identified: (i) L-fucose oxidation to L-fuconate via a dehydrogenase, (ii) dehydration to 2-keto-3-deoxy-L-fuconate via dehydratase, (iii) 2-keto-3-deoxy-L-fuconate cleavage to pyruvate and L-lactaldehyde via aldolase and (iv) L-lactaldehyde conversion to L-lactate via aldehyde dehydrogenase. This pathway as well as L-fucose transport shows interesting overlaps to the D-arabinose pathway, representing another example for pathway promiscuity in Sulfolobus species. © 2016 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

  10. CBD binding domain fused γ-lactamase from Sulfolobus solfataricus is an efficient catalyst for (-) γ-lactam production.

    Science.gov (United States)

    Wang, Jianjun; Zhu, Junge; Min, Cong; Wu, Sheng

    2014-05-13

    γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened. We prepared two fusion constructs of the γ-lactamase gene gla, which was from Sulfolobus solfataricus P2. These two constructs had Cbd (cellulose binding domain from Clostridium thermocellum) fused at amino or carboxyl terminus of the γ-lactamase. These two constructs were heterogeneously expressed in E. coli rosetta (DE3) as two fusion proteins. Both of them were immobilized well on Avicel (microcrystalline cellulose matrix). The apparent kinetic parameters revealed that carboxyl terminus fused protein (Gla-linker-Cbd) was a better catalyst. The V(max) and k(cat) value of Avicel immobilized Gla-linker-Cbd were 381 U mg⁻¹ and 4.7 × 10⁵ s⁻¹ respectively. And the values of the free Gla-linker-Cbd were 151 U mg⁻¹ and 1.8 × 10⁵ s⁻¹ respectively. These data indicated that the catalytic efficiency of the enzyme was upgraded after immobilization. The immobilized Gla-linker-Cbd had a 10-degree temperature optimum dropping from 80°C to 70°C but it was stable when incubated at 60°C for 48 h. It remained stable in catalyzing 20-batch reactions. After optimization, the immobilized enzyme concentration in transformation was set as 200 mg/mL. We found out that there was inhibition that occurred to the immobilized enzyme when substrate concentration exceeded 60 mM. Finally a 10 mL-volume transformation was conducted, in which 0.6 M substrate was hydrolyzed and the resolution was completed within 9 h with a 99.5% ee value. Cellulose is the most

  11. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary...... targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high...... complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  12. Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus

    International Nuclear Information System (INIS)

    Peeters, Eveline; Hoa, Bach Thi Mai; Zegers, Ingrid; Charlier, Daniel; Maes, Dominique

    2005-01-01

    The C-terminal domain of the transcriptional regulator Ss-LrpB from S. solfataricus was purified by affinity chromatography and crystallized. Crystals belong to space group P2 1 2 1 2. A complete data set was collected to a resolution of 2 Å. Ss-LrpB from Sulfolobus solfataricus P2 belongs to the bacterial/archaeal superfamily of Lrp-like (leucine-responsive regulatory protein-like) transcription regulators. The N-terminal DNA-binding domain contains a HTH motif and the C-terminal domain contains an αβ-sandwich (βαββαβ fold). The C-terminal domain was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2 1 2 1 2, with unit-cell parameters a = 59.35, b = 74.86, c = 38.08 Å and a data set was collected to 2.0 Å resolution. Structure determination using a selenomethionine derivative is under way

  13. Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the Monomeric State and in the Initial Native-like Aggregates*

    Science.gov (United States)

    Pagano, Katiuscia; Bemporad, Francesco; Fogolari, Federico; Esposito, Gennaro; Viglino, Paolo; Chiti, Fabrizio; Corazza, Alessandra

    2010-01-01

    It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined 1H and 15N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site. PMID:20223823

  14. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Carmen Sarcinelli

    2016-01-01

    Full Text Available Peroxiredoxins (Prxs are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR/thioredoxin (yTrx reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells.

  15. Functional and structural properties of the homogeneous beta-glycosidase from the extreme thermoacidophilic archaeon sulfolobus solfataricus expressed in Saccharomyces cerevisiae.

    Science.gov (United States)

    D'Auria, S; Morana, A; Febbraio, F; Vaccaro, C; De Rosa, M; Nucci, R

    1996-05-01

    A protein with beta-glycosidase activity from Sulfolobus solfataricus (S beta gly) was expressed in the yeast Saccharomyces cerevisiae. The purification procedure was made fast and easy by employing a single chromatographic step. After 5.8-fold purification, the cell extract gave a homogeneous enzyme at 166 U/mg. The recombinant enzyme was functionally and structurally similar to the wild-type enzyme. Kinetic experiments showed the same wide substrate specificity; in fact, the expressed enzyme hydrolyzed beta-D-gluco-, fuco-, and galactosides and a large number of glucoside dimers and oligomers, linked beta 1 -> 4. Moreover, the molecular mass of the enzyme was estimated to be 60 kDa by SDS-PAGE and 240 kDa by gel filtration, glycerol gradient, and ultracentrifugation analyses, indicating that the enzyme has a tetrameric structure. The N-terminal amino acid sequence, the temperature dependent activity, and content of secondary structure were similar to those of the wild-type enzyme. CD spectral and kinetic analyses showed that the only differences from the wild-type enzyme consist of the absence of lysine methylation, the presence of some glycosylated amino acid residues, and lower thermostability. Furthermore, calorimetric analyses on the expressed protein indicated values of delta dH = 5072 kJ/ mol and delta (d)C(p)= 100 kJ/mol, appreciably lower than those of the wild-type protein.

  16. Compound K Production from Red Ginseng Extract by β-Glycosidase from Sulfolobus solfataricus Supplemented with α-L-Arabinofuranosidase from Caldicellulosiruptor saccharolyticus.

    Directory of Open Access Journals (Sweden)

    Kyung-Chul Shin

    Full Text Available Ginsenoside compound K (C-K is attracting a lot of interest because of its biological and pharmaceutical activities, including hepatoprotective, antitumor, anti-wrinkling, and anti-skin aging activities. C-K has been used as the principal ingredient in skin care products. For the effective application of ginseng extracts to the manufacture of cosmetics, the PPD-type ginsenosides in ginseng extracts should be converted to C-K by enzymatic conversion. For increased yield of C-K from the protopanaxadiol (PPD-type ginsenosides in red-ginseng extract (RGE, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus (CS-abf was used along with the β-D-glucopyranoside/α-L-arabinopyranoside-hydrolyzing β-glycosidase from Sulfolobus solfataricus (SS-bgly because SS-bgly showed very low hydrolytic activity on the α-L-arabinofuranoside linkage in ginsenosides. The optimal reaction conditions for C-K production were as follows: pH 6.0, 80°C, 2 U/mL SS-bgly, 3 U/mL CS-abf, and 7.5 g/L PPD-type ginsenosides in RGE. Under these optimized conditions, SS-bgly supplemented with CS-abf produced 4.2 g/L C-K from 7.5 g/L PPD-type ginsenosides in 12 h without other ginsenosides, with a molar yield of 100% and a productivity of 348 mg/L/h. To the best of our knowledge, this is the highest concentration and productivity of C-K from ginseng extract ever published in literature.

  17. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.

    2000-01-01

    The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter...... selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

  18. Mutagenic Replication of N2-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV.

    Science.gov (United States)

    Gowda, A S Prakasha; Krzeminski, Jacek; Amin, Shantu; Suo, Zucai; Spratt, Thomas E

    2017-05-15

    Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N 2 -position of guanine to produce N 2 -BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases. The mechanisms by which mutagenic bypass occurs is not well-known. We have evaluated base pairing structures using atomic substitution of the dNTP with two stereoisomers, 2'-deoxy-N-[(7R,8S,9R,10S)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine and 2'-deoxy-N-[(7S,8R,9S,10R)-7,8,9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyren-10-yl]guanosine. We have examined the kinetics of incorporation of 1-deaza-dATP, 7-deaza-dATP, 2'-deoxyinosine triphosphate, and 7-deaza-dGTP, analogues of dATP and dGTP in which single atoms are changed. Changes in rate will occur if that atom provided a critical interaction in the transition state of the reaction. We examined two polymerases, Escherichia coli DNA polymerase I (Kf) and Sulfolobus solfataricus DNA polymerase IV (Dpo4), as models of a high fidelity and TLS polymerase, respectively. We found that with Kf, substitution of the nitrogens on the Watson-Crick face of the dNTPs resulted in decreased rate of reactions. This result is consistent with a Hoogsteen base pair in which the template N 2 -BP-dG flipped from the anti to syn conformation. With Dpo4, while the substitution did not affect the rate of reaction, the amplitude of the reaction decreased with all substitutions. This result suggests that Dpo4 bypasses N 2 -BP-dG via Hoogsteen base pairs but that the flipped nucleotide can be either the dNTP or the template.

  19. Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the C-­terminal domain of Ss-LrpB, a transcription regulator from Sulfolobus solfataricus

    OpenAIRE

    Peeters, Eveline; Hoa, Bach Thi Mai; Zegers, Ingrid; Charlier, Daniel; Maes, Dominique

    2005-01-01

    The C-terminal domain of the transcriptional regulator Ss-LrpB from S. solfataricus was purified by affinity chromatography and crystallized. Crystals belong to space group P21212. A complete data set was collected to a resolution of 2 Å.

  20. Sulfolobus acidocaldarius

    Science.gov (United States)

    Wagner, Michaela; Shen, Lu; Albersmeier, Andreas; van der Kolk, Nienke; Kim, Sujin; Cha, Jaeho; Bräsen, Christopher; Kalinowski, Jörn; Siebers, Bettina; Albers, Sonja-Verena

    2017-11-17

    Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus , only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius grown on peptides alone and peptides in presence of D-xylose allowed for the identification of the ABC transporter for D-xylose and L-arabinose transport and to gain deeper insights into pentose catabolism under the respective growth conditions. The D-xylose/L-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the Carbohydrate Uptake Transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea. Single gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for D-xylose and L-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and D-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD/KDAD; Saci_1939) was no longer able to catabolize D-xylose or L-arabinose suggesting the absence of the aldolase-dependent branch in S. acidocaldarius Importance Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications as their optimal growth conditions resemble conditions used in

  1. Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus.

    Science.gov (United States)

    Gu, Zhenyu; Zitzewitz, Jill A; Matthews, C Robert

    2007-04-27

    To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good

  2. Genome sequencing of Sulfolobus sp. A20 from Costa Rica and comparative analyses of the putative pathways of carbon, nitrogen and sulfur metabolism in various Sulfolobus strains

    Directory of Open Access Journals (Sweden)

    Xin Dai

    2016-11-01

    Full Text Available The genome of Sulfolobus sp. A20 isolated from a hot spring in Costa Rica was sequenced. This circular genome of the strain is 2,688,317 bp in size and 34.8% in G+C content, and contains 2,591 open reading frames (ORFs. Strain A20 shares ~95.6% identity at the 16S rRNA gene sequence level and less than 30% DNA-DNA hybridization (DDH values with the most closely related known Sulfolobus species (i.e., S. islandicus and S. solfataricus, suggesting that it represents a novel Sulfolobus species. Comparison of the genome of strain A20 with those of the type strains of S. solfataricus, S. acidocaldarius, S. islandicus and S. tokodaii, which were isolated from geographically separated areas, identified 1,801 genes conserved among all Sulfolobus species analyzed (core genes. Comparative genome analyses show that central carbon metabolism in Sulfolobus is highly conserved, and enzymes involved in the Entner-Doudoroff pathway, the tricarboxylic acid cycle and the CO2 fixation pathways are predominantly encoded by the core genes. All Sulfolobus species encode genes required for the conversion of ammonium into glutamate/glutamine. Some Sulfolobus strains have gained the ability to utilize additional nitrogen source such as nitrate (i.e. S. islandicus strain REY15A, LAL14/1, M14.25 and M16.27 or urea (i.e. S. islandicus HEV10/4, S. tokodaii strain7 and S. metallicus DSM 6482. The strategies for sulfur metabolism are most diverse and least understood. S. tokodaii encodes sulfur oxygenase/reductase (SOR, whereas both S. islandicus and S. solfataricus contain genes for sulfur reductase (SRE. However, neither SOR nor SRE genes exist in the genome of strain A20, raising the possibility that an unknown pathway for the utilization of elemental sulfur may be present in the strain. The ability of Sulfolobus to utilize nitrate or sulfur is encoded by a gene cluster flanked by IS elements or their remnants. These clusters appear to have become fixed at a specific

  3. Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Sani, Musa; Groeneveld, Maarten; Zolghadr, Benham; Schelert, James; Albers, Sonja-Verena; Blum, Paul; Boekema, Egbert J.; Driessen, Arnold J. M.

    Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Suffolobus soffiataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a

  4. Functional genomics of the thermo-acidophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Oost, van der J.; Walther, J.; Brouns, S.J.J.; Werken, van de H.J.G.; Snijders, A.P.L.; Wright, P.C.; Andersson, A.; Bernander, R.; Vos, de W.M.

    2006-01-01

    Archaea and bacteria that optimally grow at temperatures above 60C and 80C are referred to as thermophiles and hyperthermophiles, respectively (Stetter, 1996). Since their discovery in the late 1960s (Brock and Freeze, 1969), attempts were made to reveal the secrets of the thermal resistance of

  5. CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genus Sulfolobus

    Science.gov (United States)

    Manica, Andrea; Schleper, Christa

    2013-01-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-mediated virus defense based on small RNAs is a hallmark of archaea and also found in many bacteria. Archaeal genomes and, in particular, organisms of the extremely thermoacidophilic genus Sulfolobus, carry extensive CRISPR loci each with dozens of sequence signatures (spacers) able to mediate targeting and degradation of complementary invading nucleic acids. The diversity of CRISPR systems and their associated protein complexes indicates an extensive functional breadth and versatility of this adaptive immune system. Sulfolobus solfataricus and S. islandicus represent two of the best characterized genetic model organisms in the archaea not only with respect to the CRISPR system. Here we address and discuss in a broader context particularly recent progress made in understanding spacer recruitment from foreign DNA, production of small RNAs, in vitro activity of CRISPR-associated protein complexes and attack of viruses and plasmids in in vivo test systems. PMID:23535277

  6. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  7. Genetic engineering of sulfur-degrading Sulfolobus

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y.

    1991-01-01

    The objectives of the proposed research is to first establish a plasmid-mediated genetic transformation system for the sulfur degrading Sulfolobus, and then to clone and overexpress the genes encoding the organic-sulfur-degrading enzymes from Sulfolobus- as well as from other microorganisms, to develop a Sulfolobus-based microbial process for the removal of both organic and inorganic sulfur from coal.

  8. The architects of crenarchaeal chromatin : A biophysical characterization of chromatin proteins from Sulfolobus solfataricus

    NARCIS (Netherlands)

    Driessen, Rosalie Paula Catharina

    2014-01-01

    Understanding of chromatin organization and compaction in Archaea is currently limited. The genome of several megabasepairs long is folded by a set of small chromatin proteins to fit into the micron-sized cell. A first step in understanding archaeal chromatin organization is to study the action of

  9. Mutations and Rearrangements in the Genome of Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Redder, P.; Garrett, R. A.

    2006-01-01

    of different types of mutation and possible rearrangements that can occur in the genome, the pyrEF locus was examined for mutations that were isolated after selection with 5-fluoroorotic acid. About two-thirds of the 130 mutations resulted from insertions of mobile elements, including insertion sequence (IS...... deletions, insertions, and a duplication, were observed, and about one-fifth of the mutations occurred elsewhere in the genome, possibly in an orotate transporter gene. One mutant exhibited a 5-kb genomic rearrangement at the pyrEF locus involving a two-step IS element-dependent reaction, and its boundaries...

  10. Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography.

    Science.gov (United States)

    Veesler, David; Ng, Thiam-Seng; Sendamarai, Anoop K; Eilers, Brian J; Lawrence, C Martin; Lok, Shee-Mei; Young, Mark J; Johnson, John E; Fu, Chi-yu

    2013-04-02

    Sulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.

  11. Alterations of the transcriptome of Sulfolobus acidocaldarius by exoribonuclease aCPSF2.

    Directory of Open Access Journals (Sweden)

    Birgit Märtens

    Full Text Available Recent studies identified a 5´ to 3´ exoribonuclease termed Sso-RNase J in the crenarchaeon Sulfolobus solfataricus (Sso, which has been reclassified to the aCPSF2 (archaeal cleavage and polyadenylation specificity factor 2 group of β-CASP proteins. In this study, the Sso-aCPSF2 orthologue of Sulfolobus acidocaldarius (Saci-aCPSF2 was functionally characterized. Like Sso-aCPSF2, Saci-aCPSF2 degrades RNA with 5´ to 3´ directionality in vitro. To address the biological significance of Saci-aCPSF2, a deletion mutant was constructed, and the influence of Saci-aCPSF2 on the transcriptome profile was assessed employing high throughput RNA sequencing. This analysis revealed 560 genes with differential transcript abundance, suggesting a considerable role of this enzyme in RNA metabolism. In addition, bioinformatic analyses revealed several transcripts that are preferentially degraded at the 5´ end. This was exemplarily verified for two transcripts by Northern-blot analyses, showing for the first time that aCPSF2 proteins play a role in 5' to 3' directional mRNA decay in the crenarchaeal clade of Archaea.

  12. Identification and sequence analysis of Sulfolobus solfataricus purE and purK genes

    DEFF Research Database (Denmark)

    Sørensen, Iben Schildt; Dandanell, Gert

    1997-01-01

    of the genes is expressed from an E. coli promoter they can complement the corresponding purE and purK mutations in E. coli. PurE seems to be more closely related to eubacteria than to other archaea and to eukaryotes. Also the purK gene, which has not yet been found in other archaea, is more closely related...... to eubacteria than to eukaryotes....

  13. The chaperonin of the archaeon Sulfolobus solfataricus is an RNA-binding protein that participates in ribosomal RNA processing.

    OpenAIRE

    Ruggero, D; Ciammaruconi, A; Londei, P

    1998-01-01

    The 60 kDa molecular chaperones (chaperonins) are high molecular weight protein complexes having a characteristic double-ring toroidal shape; they are thought to aid the folding of denatured or newly synthesized polypeptides. These proteins exist as two functionally similar, but distantly related families, one comprising the bacterial and organellar chaperonins and another (the so-called CCT-TRiC family) including the chaperonins of the archaea and the eukaryotes. Although some evidence exist...

  14. A new type of didhydroorotate dehydrogenase, type 1S, from the thermoacidophilic archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Sørensen, P. G.; Dandanell, Gert

    2002-01-01

    ,6-dichlorophenolindophenol (DCIP), Q0, and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with KM values of 44.2-1.9 µM for the substrate dihydroorotate and 344-21 µM for the electron acceptor ferricyanide, as well as competitive product inhibition...

  15. Genetic engineering of sulfur-degrading Sulfolobus. Final technical report, September 1, 1990--August 31, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N.W.Y.

    1991-12-31

    The objectives of the proposed research is to first establish a plasmid-mediated genetic transformation system for the sulfur degrading Sulfolobus, and then to clone and overexpress the genes encoding the organic-sulfur-degrading enzymes from Sulfolobus- as well as from other microorganisms, to develop a Sulfolobus-based microbial process for the removal of both organic and inorganic sulfur from coal.

  16. Sulfolobus – A Potential Key Organism in Future Biotechnology

    Directory of Open Access Journals (Sweden)

    Julian Quehenberger

    2017-12-01

    Full Text Available Extremophilic organisms represent a potentially valuable resource for the development of novel bioprocesses. They can act as a source for stable enzymes and unique biomaterials. Extremophiles are capable of carrying out microbial processes and biotransformations under extremely hostile conditions. Extreme thermoacidophilic members of the well-characterized genus Sulfolobus are outstanding in their ability to thrive at both high temperatures and low pH. This review gives an overview of the biological system Sulfolobus including its central carbon metabolism and the development of tools for its genetic manipulation. We highlight findings of commercial relevance and focus on potential industrial applications. Finally, the current state of bioreactor cultivations is summarized and we discuss the use of Sulfolobus species in biorefinery applications.

  17. Studying Extrachromosomal Genetic Elements in Sulfolobus

    DEFF Research Database (Denmark)

    Guannan, Liu

    Archaea constitute a separate domain in the universal tree of life. They exhibit exceptional biological properties and provide important insights into the origin of cellular life. Rapid advances in DNA sequencing and bioinformatical methods as well as the development of versatile genetic tools have...... facilitated the characterization of viruses, plasmids and membrane vesicles. Studying the interactions between Sulfolobus and extrachromosomal genetic elements has provided many new insights into basic molecular processes. Secreted membrane vesicle seems to be a common characteristic for Sulfolobus. In order...... to gain a better understanding of the interactions between conjugative plasmids and hosts. The result also demonstrated why certain archaeal conjugative plasmids are gradually lost during continuous growth. Whereas loss of pKEF9 in S. islandicus was due to interference from the host CRISPR-Cas system...

  18. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus

    NARCIS (Netherlands)

    Wagner, Michaela; Berkner, Silvia; Ajon, Malgorzata; Driessen, Arnold J. M.; Lipps, Georg; Albers, Sonja-Verena

    Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and availability of genetic tools has lagged behind. in the present paper, we discuss the latest progress in understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus

  19. Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

    DEFF Research Database (Denmark)

    Christoffersen, Stig; Kadziola, Anders; Johansson, Eva

    2009-01-01

    . The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long ß-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two...... flexible loop residues, Tyr123 and Lys125, bind the PPi moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen......-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity....

  20. Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2

    NARCIS (Netherlands)

    Ettema, T.J.G.; Brinkman, A.B.; Lamers, P.P.; Kornet, N.; Vos, de W.M.; Oost, van der J.

    2006-01-01

    Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which

  1. Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

    DEFF Research Database (Denmark)

    Fusco, Salvatore; Liguori, Rossana; Limauro, Danila

    2015-01-01

    in a double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease...... of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the host-viral interaction in the frame of the CRISPR-response....

  2. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library...... to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms....

  3. Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of Sulfolobus solfataricus

    NARCIS (Netherlands)

    Pretz, Monika G.; Albers, Sonja-Verena; Schuurman-Wolters, Gea; Tampe, Robert; Driessen, Arnold J. M.; van der Does, Chris

    2006-01-01

    ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the

  4. Structure-Based Mutagenesis of Sulfolobus Turreted Icosahedral Virus B204 Reveals Essential Residues in the Virion-Associated DNA-Packaging ATPase.

    Science.gov (United States)

    Dellas, Nikki; Snyder, Jamie C; Dills, Michael; Nicolay, Sheena J; Kerchner, Keshia M; Brumfield, Susan K; Lawrence, C Martin; Young, Mark J

    2015-12-23

    Sulfolobus turreted icosahedral virus (STIV), an archaeal virus that infects the hyperthermoacidophile Sulfolobus solfataricus, is one of the most well-studied viruses of the domain Archaea. STIV shares structural, morphological, and sequence similarities with viruses from other domains of life, all of which are thought to belong to the same viral lineage. Several of these common features include a conserved coat protein fold, an internal lipid membrane, and a DNA-packaging ATPase. B204 is the ATPase encoded by STIV and is thought to drive packaging of viral DNA during the replication process. Here, we report the crystal structure of B204 along with the biochemical analysis of B204 mutants chosen based on structural information and sequence conservation patterns observed among members of the same viral lineage and the larger FtsK/HerA superfamily to which B204 belongs. Both in vitro ATPase activity assays and transfection assays with mutant forms of B204 confirmed the essentiality of conserved and nonconserved positions. We also have identified two distinct particle morphologies during an STIV infection that differ in the presence or absence of the B204 protein. The biochemical and structural data presented here are not only informative for the STIV replication process but also can be useful in deciphering DNA-packaging mechanisms for other viruses belonging to this lineage. STIV is a virus that infects a host from the domain Archaea that replicates in high-temperature, acidic environments. While STIV has many unique features, there exist several striking similarities between this virus and others that replicate in different environments and infect a broad range of hosts from Bacteria and Eukarya. Aside from structural features shared by viruses from this lineage, there exists a significant level of sequence similarity between the ATPase genes carried by these different viruses; this gene encodes an enzyme thought to provide energy that drives DNA packaging into

  5. Characterization of the Sulfolobus host-SSV2 virus interaction

    DEFF Research Database (Denmark)

    Contursi, P.; Jensen, S.; Aucelli, T.

    2006-01-01

    The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report the find......The Sulfolobus spindle virus, SSV2, encodes a tyrosine integrase which furthers provirus formation in host chromosomes. Consistently with the prediction made during sequence analysis, integration was found to occur in the downstream half of the tRNAGly (CCC) gene. In this paper we report...... during the growth of the natural host REY15/4, the cellular content of SSV2 DNA remains fairly low throughout the incubation of the foreign host. The accumulation of episomal DNA in the former case cannot be traced to decreased packaging activity because of a simultaneous increase in the virus titre...... in the medium. In addition, the interaction between SSV2 and its natural host is characterized by the concurrence of host growth inhibition and the induction of viral DNA replication. When this virus-host interaction was investigated using S. islandicus REY15A, a strain which is closely related to the natural...

  6. Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3.

    Science.gov (United States)

    Iverson, Eric A; Goodman, David A; Gorchels, Madeline E; Stedman, Kenneth M

    2017-05-15

    Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae , where >90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3 , allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of

  7. CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties

    DEFF Research Database (Denmark)

    Lillestøl, Reidun K; Shah, Shiraz Ali; Brügger, Kim

    2009-01-01

    Summary CRISPRs of Sulfolobus fall into three main families based on their repeats, leader regions, associated cas genes, and putative recognition sequences on viruses and plasmids. Spacer sequence matches to different viruses and plasmids of the Sulfolobales revealed some bias particularly...... for family III CRISPRs. Transcription occurs on both strands of the five repeat-clusters of Sulfolobus acidocaldarius and a repeat-cluster of the conjugative plasmid pKEF9. Leader strand transcripts cover whole repeat-clusters and are processed mainly from the 3'-end, within repeats, yielding heterogeneous...

  8. Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea.

    Science.gov (United States)

    Peng, Nan; Han, Wenyuan; Li, Yingjun; Liang, Yunxiang; She, Qunxin

    2017-04-01

    Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also possess unique features as revealed by genomics and genome comparisons and by biochemical characterization of prominent enzymes. Nevertheless, diverse genetic tools are required for in vivo experiments to verify these interesting discoveries. Considerable efforts have been devoted to the development of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing Crenarchaeota because all the remaining genera are from Euryarchaeota. Nevertheless, genetic tools developed for Sulfolobus are probably the most versatile among all archaeal models, and these include viral and plasmid shuttle vectors, conventional and novel genetic manipulation methods, CRISPR-based gene deletion and mutagenesis, and gene silencing, among which CRISPR tools have been reported only for Sulfolobus thus far. In this review, we summarize recent developments in all these useful genetic tools and discuss their possible application to research into archaeal biology by means of Sulfolobus models.

  9. Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    Wolferen, Marleen van; Ajon, Małgorzata; Driessen, Arnold J.M.; Albers, Sonja-Verena

    2013-01-01

    Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: the UV-inducible pili operon (ups operon). This operon is involved in UV-induced pili assembly, cellular aggregation, and subsequent DNA exchange

  10. Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang

    into at least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...

  11. Extraction, isolation and NMR data of the tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus metallicus harvested from a bioleaching reactor

    CSIR Research Space (South Africa)

    Bode, ML

    2008-08-01

    Full Text Available The successful extraction and isolation of the hydrolysed tetraether lipid calditoglycerocaldarchaeol (GDNT) from Sulfolobus etallicus, a key thermophilic bioleaching archaeon, is described. The archaeal biomasswas recovered directly from a...

  12. Reconstitution of the Leucine Transport System of Lactococcus lactis into Liposomes Composed of Membrane-Spanning Lipids from Sulfolobus acidocaldarius

    NARCIS (Netherlands)

    in t Veld, Geertruida; Elferink, Maria; Driessen, Arnold J.M.; Konings, Wilhelmus

    1992-01-01

    The effect of bipolar tetraether lipids, extracted from the thermophilic archaebacterium Sulfolobus acidocaldarius, on the branched-chain amino acid transport system of the mesophilic bacterium Lactococcus lactis was investigated. Liposomes were prepared from mixtures of monolayer lipids and the

  13. How a Genetically Stable Extremophile Evolves: Modes of Genome Diversification in the Archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Mao, Dominic; Grogan, Dennis W

    2017-09-01

    In order to analyze in molecular terms how Sulfolobus genomes diverge, damage-induced mutations and natural polymorphisms (PMs) were identified in laboratory constructs and wild-type isolates, respectively, of Sulfolobus acidocaldarius Among wild-type isolates drawn from one local population, pairwise nucleotide divergence averaged 4 × 10 -6 , which is about 0.15% of the corresponding divergence reported for Sulfolobus islandicus The most variable features of wild-type S. acidocaldarius genomes were homopolymer (mononucleotide) tracts and longer tandem repeats, consistent with the spontaneous mutations that occur under laboratory conditions. Natural isolates, however, also revealed large insertions/deletions and inversions, which did not occur in any of the laboratory-manipulated strains. Several of the large insertions/deletions could be attributed to the integration or excision of mobile genetic elements (MGEs), and each MGE represented a distinct system of site-specific recombination. The mode of recombination associated with one MGE, a provirus related to Sulfolobus turreted icosahedral virus , was also seen in certain chromosomal inversions. Artificially induced mutations, non-MGE insertions/deletions, and small PMs exhibited different distributions over the genome, suggesting that large-scale patterning of Sulfolobus genomes begins early in the divergence process. Unlike induced mutations, natural base pair substitutions occurred in clusters, and one cluster exhibited properties expected of nonreciprocal recombination (gene conversion) between dispersed imperfect repeats. Taken together, the results identify simple replication errors, slipped-strand events promoted by tandem repeats, homologous recombination, and rearrangements promoted by MGEs as the primary sources of genetic variation for this extremely acidophilic archaeon in its geothermal environment. IMPORTANCE The optimal growth temperatures of hyperthermophilic archaea accelerate DNA decomposition

  14. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    DEFF Research Database (Denmark)

    Xing, Xuanxuan; Zhang, Likui; Guo, Li

    2014-01-01

    Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from...... the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....

  15. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun

    2009-01-01

    , and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were...... mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the beta-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out...... constructed, which genetically complemented DeltapyrEFDeltalacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers....

  16. Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

    Science.gov (United States)

    Ouchi, Takuya; Tomita, Takeo; Horie, Akira; Yoshida, Ayako; Takahashi, Kento; Nishida, Hiromi; Lassak, Kerstin; Taka, Hikari; Mineki, Reiko; Fujimura, Tsutomu; Kosono, Saori; Nishiyama, Chiharu; Masui, Ryoji; Kuramitsu, Seiki; Albers, Sonja-Verena; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2013-04-01

    LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

  17. Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

    DEFF Research Database (Denmark)

    Greve, B.; Jensen, S.; Phan, H.

    2005-01-01

    Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plas...

  18. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen

    2015-01-01

    undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor, ADP binds like the product AMP with both the α- and β-phosphates occupying the 5’-phosphoribosyl binding site. The enzyme shows activity over a wide p......H-range and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme may be facilitated by elements in the C-terminal three-helix bundle part of the protein....

  19. CrRNA-Protospacer Recognition during CRISPR- Directed DNA Interference Sulfolobus islandicus REY 15A and Structural Studies of CRISPR Binding Proteins (CBP) of Crenarchaeon Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is one of the important known immune mechanisms in archaea and bacteria. This adaptive immune system degrades invading genetic elements and protects the cell. Amongst 3 main types I, II and III...... of CRISPR system, two types (I and III) are found in archaea. However, in Sulfolobus species, subtypes IA, I-D, and III-B, III-D and rarely III-A are found. The model organism used for interference and structural studies is S. islandicus REY15A which carries subtypes I-A and III-B (α and β). Besides CRISPR...... ribonucleoprotein complex which is involved directly in defense, there are some less- known parts of the system including CPBs (CRISPR repeat-binding proteins) which are suggested to play a role in transcription. In the first part of my thesis, I provide a brief introduction to archaea and viruses that infect...

  20. NCBI nr-aa BLAST: CBRC-XTRO-01-3737 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3737 ref|NP_343011.1| Transporter (proton symporter) [Sulfolobus solfa...taricus P2] gb|AAK41801.1| Transporter (proton symporter) [Sulfolobus solfataricus P2] NP_343011.1 0.010 25% ...

  1. Genetic technologies for extremely thermophilic microorganisms of Sulfolobus, the only genetically tractable genus of crenarchaea

    DEFF Research Database (Denmark)

    Peng, Nan; Han, Wenyuan; Li, Yingjun

    2017-01-01

    Archaea represents the third domain of life, with the information-processing machineries more closely resembling those of eukaryotes than the machineries of the bacterial counterparts but sharing metabolic pathways with organisms of Bacteria, the sister prokaryotic phylum. Archaeal organisms also...... of genetic tools for archaea ever since their discovery, and great progress has been made in the creation of archaeal genetic tools in the past decade. Versatile genetic toolboxes are now available for several archaeal models, among which Sulfolobus microorganisms are the only genus representing...

  2. The genome of Sulfolobus acidocaldarius, a model organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, L.M.; Brugger, K.; Skovgaard, Marie

    2005-01-01

    tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision...... repair system, thermopsin, and an aromatic ring dioxygenase, all of which are absent from genomes of other Sulfolobus species. However, it lacks genes for some of their sugar transporters, consistent with it growing on a more limited range of carbon sources. These results, together with the many newly...

  3. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Garrett, Roger Antony; Shah, Shiraz Ali

    2013-01-01

    Recent studies on CRISPR-based adaptive immune systems have revealed extensive structural and functional diversity of the interference complexes which often coexist intracellularly. The archaeon Sulfolobus islandicus REY15A encodes three interference modules, one of type IA and two of type IIIB...... targeting. A rationale is provided for the intracellular coexistence of the different interference systems in S.¿islandicus REY15A which cooperate functionally by sharing a single Cas6 protein for crRNA processing and utilize crRNA products from identical CRISPR spacers....

  4. Genetic analysis of the Holliday junction resolvases Hje and Hjc in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Huang, Qihong; Li, Yansheng; Zeng, Chaoning

    2015-01-01

    The in vivo functions of Hje and Hjc, two Holliday junction resolvases in Sulfolobus islandicus were investigated. We found that deletion of either hje or hjc had no effect on normal cell growth, while deletion of both hje and hjc is lethal. Although Hjc is the conserved resolvase in all archaea...

  5. Genome Analyses of Icelandic Strains of Sulfolobus islandicus, Model Organisms for Genetic and Virus-Host Interaction Studies

    DEFF Research Database (Denmark)

    Guo, Li; Brügger, Kim; Liu, Chao

    2011-01-01

    The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad h...

  6. Sulfolobus Turreted Icosahedral Virus c92 Protein Responsible for the Formation of Pyramid-Like Cellular Lysis Structures

    DEFF Research Database (Denmark)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan

    2011-01-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system...

  7. Differentiation and Structure in Sulfolobus islandicus Rod-Shaped Virus Populations

    Directory of Open Access Journals (Sweden)

    Maria A. Bautista

    2017-05-01

    Full Text Available In the past decade, molecular surveys of viral diversity have revealed that viruses are the most diverse and abundant biological entities on Earth. In culture, however, most viral isolates that infect microbes are represented by a few variants isolated on type strains, limiting our ability to study how natural variation affects virus-host interactions in the laboratory. We screened a set of 137 hot spring samples for viruses that infect a geographically diverse panel of the hyperthemophilic crenarchaeon Sulfolobus islandicus. We isolated and characterized eight SIRVs (Sulfolobus islandicus rod-shaped viruses from two different regions within Yellowstone National Park (USA. Comparative genomics revealed that all SIRV sequenced isolates share 30 core genes that represent 50–60% of the genome. The core genome phylogeny, as well as the distribution of variable genes (shared by some but not all SIRVs and the signatures of host-virus interactions recorded on the CRISPR (clustered regularly interspaced short palindromic repeats repeat-spacer arrays of S. islandicus hosts, identify different SIRV lineages, each associated with a different geographic location. Moreover, our studies reveal that SIRV core genes do not appear to be under diversifying selection and thus we predict that the abundant and diverse variable genes govern the coevolutionary arms race between SIRVs and their hosts.

  8. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

  9. Genetic Studies on CRISPR-Cas Functions in Invader Defense in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Peng, Wenfang

    Archaea and bacteria contain CRISPR-Cas (clustered regularly interspaced short palindromic repeat-CRISPR-associated) systems that protect themselves against invasion by viruses and plasmids. There are three major types of CRISPR-Cas systems, type I, II and III, that are further divided...... into at least 11 subtypes. I employed Sulfolobus islandicus Rey15A as the model to study CRISPR mechanisms. The model archaeon encodes one subtype I-A (Cascade) and two subtype III-B (Cmr-α and Cmr-β) interference systems with no apparent redundancy in cas genes or in CRISPR systems, which is ideal for genetic...... analysis of cas gene function. Furthermore, a range of genetic tools have been developed for S. islandicus Rey15A in our laboratory and a plasmid interference assay has been successfully developed for testing CRISPR-directed DNA targeting activity, which have provided a solid basis for studying...

  10. Transcriptome changes in STSV2-infected Sulfolobus islandicus REY15A undergoing continuous CRISPR spacer acquisition

    DEFF Research Database (Denmark)

    León-Sobrino, Carlos; Kot, Witold P; Garrett, Roger A

    2016-01-01

    A transcriptome study was performed on Sulfolobus islandicus REY15A actively undergoing CRISPR spacer acquisition from the crenarchaeal monocaudavirus STSV2 in rich and basal media over a 6 day period. Spacer acquisition preceded strong host growth retardation, altered transcriptional activity...... of four different CRISPR-Cas modules and changes in viral copy numbers, and with significant differences in the two media. Transcript levels of proteins involved in the cell cycle were reduced, while those of DNA replication, DNA repair, transcriptional regulation, and some antitoxin-toxin pairs...... and transposases were unchanged or enhanced. Antisense RNAs were implicated in the transcriptional regulation of adaptation and interference modules of the type I-A CRISPR-Cas system and evidence was found for the occurrence of functional coordination between the single CRISPR-Cas adaptation module...

  11. Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Brandl, Martin; Treusch, Alexander H

    2015-01-01

    The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high-resolution shotgun lipidomics...... to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high......-resolution Fourier transform mass spectrometry using an ion trap-orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub-ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we...

  12. Transcription termination in the plasmid/virus hybrid pSSVx from Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Contursi, Patrizia; Cannio, Raffaele; She, Qunxin

    2010-01-01

    The pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. A systematic study previously performed revealed the presence of nine major transcripts, the expression of which was differentially and temporally regulated over the growth cycle of S. islandicus....... In this study, two new transcripts were identified. Then, 3' termini of all the RNAs were mapped using adaptor RT-PCR and RNase protection assays, and termination/arrest positions were identified for each transcript. The majority of the identified ending positions were located in the close vicinity of a T...... and counter-transcripts might be responsible for the transcription termination at these T-track-minus loci in the closely spaced pSSVx genes....

  13. Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Dennis W. Grogan

    2012-06-01

    Full Text Available Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldarius pyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr+ clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells or conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which stretches of heteroduplex DNA formed during HR and segregated without complete resolution of inter-strand differences. Surprisingly, sectoring was also frequent in transformation with single-stranded DNAs. Oligonucleotides, for example, produced somewhat more sectored transformants when electroporated as single strands than as a duplex, although all forms (positive-strand, negative-strand, and duplex produced a diversity of genotypes from the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. These gene-conversion events exhibit little strand bias, and can occur in pre-formed heteroduplex. These properties suggest that this process does not play a central role in the fidelity of genome replication, but may generate 3’ single-strand tails, and thereby initiate the incorporation of duplex DNA into the recipient chromosome. Regardless of the molecular details of its mechanism, HR between the S. acidocaldarius chromosome and a multiply-marked DNA produces a strikingly high level of genetic diversity in a very short chromosomal interval, and suggests that HR in Sulfolobus has significant mutagenic potential if not

  14. Arithmetically Cohen-Macaulay sets of points in P^1 x P^1

    CERN Document Server

    Guardo, Elena

    2015-01-01

    This brief presents a solution to the interpolation problem for arithmetically Cohen-Macaulay (ACM) sets of points in the multiprojective space P^1 x P^1.  It collects the various current threads in the literature on this topic with the aim of providing a self-contained, unified introduction while also advancing some new ideas.  The relevant constructions related to multiprojective spaces are reviewed first, followed by the basic properties of points in P^1 x P^1, the bigraded Hilbert function, and ACM sets of points.  The authors then show how, using a combinatorial description of ACM points in P^1 x P^1, the bigraded Hilbert function can be computed and, as a result, solve the interpolation problem.  In subsequent chapters, they consider fat points and double points in P^1 x P^1 and demonstrate how to use their results to answer questions and problems of interest in commutative algebra.  Throughout the book, chapters end with a brief historical overview, citations of related results, and, where relevan...

  15. Sulfolobus islandicus meta-populations in Yellowstone National Park hot springs.

    Science.gov (United States)

    Campbell, Kate M; Kouris, Angela; England, Whitney; Anderson, Rika E; McCleskey, R Blaine; Nordstrom, D Kirk; Whitaker, Rachel J

    2017-06-01

    Abiotic and biotic forces shape the structure and evolution of microbial populations. We investigated forces that shape the spatial and temporal population structure of Sulfolobus islandicus by comparing geochemical and molecular analysis from seven hot springs in five regions sampled over 3 years in Yellowstone National Park. Through deep amplicon sequencing, we uncovered 148 unique alleles at two loci whose relative frequency provides clear evidence for independent populations in different hot springs. Although geography controls regional geochemical composition and population differentiation, temporal changes in population were not explained by corresponding variation in geochemistry. The data suggest that the influence of extinction, bottleneck events and/or selective sweeps within a spring and low migration between springs shape these populations. We suggest that hydrologic events such as storm events and surface snowmelt runoff destabilize smaller hot spring environments with smaller populations and result in high variation in the S. islandicus population over time. Therefore, physical abiotic features such as hot spring size and position in the landscape are important factors shaping the stability and diversity of the S. islandicus meta-population within Yellowstone National Park. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Biochemical characterization and helix stabilizing properties of HSNP-C' from the thermoacidophilic archaeon Sulfolobus acidocaldarius.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    2000-01-19

    Helix stabilizing nucleoid protein HSNP-C' from the thermophilic archaeon Sulfolobus acidocaldarius has been characterized with respect to its interactions with nucleic acids by gel retardation assay, affinities to immobilized matrices, electron microscopy, and fluorescence titration. The amino acids implicated in the DNA binding site of the protein have been shown by selectively modifying specific amino acyl functional groups and looking at their effects on the DNA binding properties of the protein. Lysine, arginine, tryptophan, and tyrosine residues of the protein HSNP-C' were modified with pyridoxal-5-phosphate; 2,3-butanedione; BNPS-skatole; and tetranitromethane, respectively. The modification of residues was assessed according to standard procedures. The effect of the chemical modification on the function of the protein HSNP-C' with respect to DNA protein interactions was studied and the results indicate the definite involvement of tyrosines and also the significant involvement of the flanking tryptophan residues in the DNA binding domain on the protein. Copyright 2000 Academic Press.

  17. Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

    Directory of Open Access Journals (Sweden)

    Alvaro Orell

    2013-01-01

    Full Text Available Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM, a Cu-exporting ATPase (CopA, and a transcriptional regulator (CopT. Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2 as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining.

  18. Crystal Structures of Two Isozymes of Citrate Synthase from Sulfolobus tokodaii Strain 7

    Directory of Open Access Journals (Sweden)

    Midori Murakami

    2016-01-01

    Full Text Available Thermoacidophilic archaeon Sulfolobus tokodaii strain 7 has two citrate synthase genes (ST1805-CS and ST0587-CS in the genome with 45% sequence identity. Because they exhibit similar optimal temperatures of catalytic activity and thermal inactivation profiles, we performed structural comparisons between these isozymes to elucidate adaptation mechanisms to high temperatures in thermophilic CSs. The crystal structures of ST1805-CS and ST0587-CS were determined at 2.0 Å and 2.7 Å resolutions, respectively. Structural comparison reveals that both of them are dimeric enzymes composed of two identical subunits, and these dimeric structures are quite similar to those of citrate synthases from archaea and eubacteria. ST0587-CS has, however, 55 ion pairs within whole dimer structure, while having only 36 in ST1805-CS. Although the number and distributions of ion pairs are distinct from each other, intersubunit ion pairs between two domains of each isozyme are identical especially in interterminal region. Because the location and number of ion pairs are in a trend with other CSs from thermophilic microorganisms, the factors responsible for thermal adaptation of ST-CS isozymes are characterized by ion pairs in interterminal region.

  19. Regioselective oxidation of lauric acid by CYP119, an orphan cytochrome P450 from Sulfolobus acidocaldarius.

    Science.gov (United States)

    Lim, Young-Ran; Eun, Chang-Yong; Park, Hyoung-Goo; Han, Songhee; Han, Jung-Soo; Cho, Kyoung Sang; Chun, Young-Jin; Kim, Donghak

    2010-03-01

    Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to 85 degrees C. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates. We characterized the regioselectivity of lauric acid by CYP119 using the auxiliary redox partner proteins putidaredoxin (Pd) and putidaredoxin reductase (PdR). Purified CYP119 protein showed a tight binding affinity to lauric acid (K(d)=1.1+/-0.1 microM) and dominantly hydroxylated (omega-1) position of lauric acid. We determined the steady-state kinetic parameters; k(cat) was 10.8 min(-1) and K(m) was 12 microM. The increased ratio to omega-hydroxylated production of lauric acid catalyzed by CYP119 was observed with increase in the reaction temperature. These studies suggested that the regioselectivity of CYP119 provide the critical clue for the physiological enzyme function in this thermophilic archaebacteria. In addition, regioselectivity control of CYP119 without altering its thermostability can lead to the development of novel CYP119-based catalysts through protein engineering.

  20. Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

    Directory of Open Access Journals (Sweden)

    Suzanne Wolterink-van Loo

    2009-01-01

    Full Text Available Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA displays optimal activity at 95°C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.

  1. Crystallization and preliminary X-ray diffraction analysis of the hyperthermophilic Sulfolobus islandicus lactonase

    International Nuclear Information System (INIS)

    Gotthard, Guillaume; Hiblot, Julien; Elias, Mikael; Chabrière, Eric

    2011-01-01

    A lactonase from the hyperthermophilic archaeon S. islandicus has been crystallized. In combination with biochemical and bioengineering studies, it is expected that the structure of this protein will provide insight into the natural function of the phosphotriesterase-like lactonase family. Phosphotriesterase-like lactonases (PLLs) constitute an interesting family of enzymes that are of paramount interest in biotechnology with respect to their catalytic functions. As natural lactonases, they may act against pathogens such as Pseudomonas aeruginosa by shutting down their quorum-sensing system (quorum quenching) and thus decreasing pathogen virulence. Owing to their promiscuous phosphotriesterase activity, which can inactivate toxic organophosphorus compounds such as pesticides and nerve agents, they are equally appealing as potent bioscavengers. A new representative of the PLL family has been identified (SisPox) and its gene was cloned from the hyperthermophilic archeon Sulfolobus islandicus. Owing to its hyperthermostable architecture, SisPox appears to be a good candidate for engineering studies. Here, production, purification, crystallization conditions and data collection to 2.34 Å resolution are reported for this lactonase from the hyperthermophilic S. islandicus

  2. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

    DEFF Research Database (Denmark)

    Mousaei, Marzieh; Deng, Ling; She, Qunxin

    2016-01-01

    The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions...... in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified...

  3. C68 from the Sulfolobus islandicus plasmid-virus pSSVx is a novel member of the AbrB-like transcription factor family

    DEFF Research Database (Denmark)

    Contursi, Patrizia; D'Ambrosio, Katia; Pirone, Luciano

    2011-01-01

    that does not show significant sequence homology with any protein with known three-dimensional structure. EMSA (electrophoretic mobility-shift assay) experiments, DNA footprinting and CD analyses indicate that recombinant C68, purified from Escherichia coli, binds to two different operator sites......The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein...

  4. The Genome of Sulfolobus acidocaldarius, a Model Organism of the Crenarchaeota

    DEFF Research Database (Denmark)

    Chen, Lanming; Brügger, Kim; Skovgaard, M.

    2005-01-01

    tokodaii. The S. acidocaldarius genome contains an integrated, and probably encaptured, pARN-type conjugative plasmid which may facilitate intercellular chromosomal gene exchange in S. acidocaldarius. Moreover, it contains genes for a characteristic restriction modification system, a UV damage excision...... repair system, thermopsin, and an aromatic ring dioxygenase, all of which are absent from genomes of other Sulfolobus species. However, it lacks genes for some of their sugar transporters, consistent with it growing on a more limited range of carbon sources. These results, together with the many newly...

  5. Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Silke eJachlewski

    2015-08-01

    Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

  6. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Science.gov (United States)

    Kanichay, Roby; Boni, Lawrence T.; Cooke, Peter H.; Khan, Tapan K.; Chong, Parkson Lee-Gau

    2003-01-01

    Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization. PMID:15803663

  7. Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Roby Kanichay

    2003-01-01

    Full Text Available Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs composed of polar lipid fraction E (PLFE from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998. In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr for vesicle aggregation at 25 and 40 °C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 °C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was –22.0 and –13.2 mV at 25 and 40 °C, respectively, at pH 6.6, as determined by 2-(p-toluidinylnaphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.

  8. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Science.gov (United States)

    Pinnapireddy, Shashank Reddy; Baghdan, Elias; Jedelská, Jarmila

    2017-01-01

    Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE) and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT) differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc) at certain nitrogen-to-phosphorus (N/P) ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3). Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS), atomic force microscopy (AFM), and scanning electron microscopy (Cryo-SEM), respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA) and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI). PMID:28239294

  9. Transfection Studies with Colloidal Systems Containing Highly Purified Bipolar Tetraether Lipids from Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Konrad H. Engelhardt

    2017-01-01

    Full Text Available Lipid vectors are commonly used to facilitate the transfer of nucleic acids into mammalian cells. In this study, two fractions of tetraether lipids from the archaea Sulfolobus acidocaldarius were extracted and purified using different methods. The purified lipid fractions polar lipid fraction E (PLFE and hydrolysed glycerol-dialkyl-nonitol tetraether (hGDNT differ in their structures, charge, size, and miscibility from conventional lipids. Liposomes were prepared by mixing tetraether lipids with cholesterol (CH and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP resulting in stable vectors for gene delivery. Lipoplexes were prepared by complexation of liposomes with a luciferase expressing plasmid (pCMV-luc at certain nitrogen-to-phosphorus (N/P ratios and optimised for the transient transfection of ovarian adenocarcinoma cells (SK-OV-3. Complexation efficacy was investigated by gel-red fluorescence assay. Biophysical properties, like size, surface charge, and morphology, were investigated by differential light scattering (DLS, atomic force microscopy (AFM, and scanning electron microscopy (Cryo-SEM, respectively, revealing structural differences between liposomes and lipoplexes. A range of stable transfecting agents containing tetraether lipids were obtained by incorporating 5 mol% of tetraether lipids. Lipoplexes showed a decrease in free gel-red with increasing N/P ratios indicating efficient incorporation of plasmid DNA (pDNA and remarkable stability. Transfection experiments of the lipoplexes revealed successful and superior transfection of SK-OV-3 cell line compared to the commercially available DOTAP and branched polyethyleneimine (25 kDa bPEI.

  10. Deletion of the topoisomerase III gene in the hyperthermophilic archaeon Sulfolobus islandicus results in slow growth and defects in cell cycle control

    DEFF Research Database (Denmark)

    Li, Xiyang; Guo, Li; Deng, Ling

    2011-01-01

    Topoisomerase III (topo III), a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was viable but grew more slowly t...

  11. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

    Science.gov (United States)

    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  12. Biochemical and immunological characterization of recombinant allergen Lol p 1.

    Science.gov (United States)

    Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

    1997-11-01

    Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

  13. Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

    DEFF Research Database (Denmark)

    Linde, Lise; Jensen, Kaj Frank

    1996-01-01

    and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20......-fold without much effect on Km for the substrates. The concentration of GTP required for half-maximal activation was about 80 µM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase......Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl a-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity...

  14. Uracil phosphoribosyltransferase from the extreme thermoacidophilic archaebacterium Sulfolobus shibatae is an allosteric enzyme, activated by GTP and inhibited by CTP

    DEFF Research Database (Denmark)

    Linde, Lise; Jensen, Kaj Frank

    1996-01-01

    -fold without much effect on Km for the substrates. The concentration of GTP required for half-maximal activation was about 80 µM. CTP was a strong inhibitor and acted by raising the concentration of GTP needed for half-maximal activation of the enzyme. We conclude that uracil phosphoribosyltransferase......Uracil phosphoribosyltransferase, which catalyses the formation of UMP and pyrophosphate from uracil and 5-phosphoribosyl a-1-pyrophosphate (PRPP), was partly purified from the extreme thermophilic archaebacterium Sulfolobus shibatae. The enzyme required divalent metal ions for activity...... and it showed the highest activity at pH 6.4. The specific activity of the enzyme was 50-times higher at 95°C than at 37°C, but the functional half-life was short at 95°C. The activity of uracil phosphoribosyltransferase was strongly activated by GTP, which increased Vmax of the reaction by approximately 20...

  15. Immunological and biological properties of recombinant Lol p 1.

    Science.gov (United States)

    Boutin, Y; Lamontagne, P; Boulanger, J; Brunet, C; Hébert, J

    1997-03-01

    Current forms of allergy diagnosis and therapies are based on the use of natural allergenic extracts. Despite strong evidence that higher therapeutic efficacy may be achieved with purified allergens, the purification of multiple allergic components from extracts is a fastidious and sometimes an impossible task. However, the use of recombinant allergens may be an alternative to overcome this problem. In this study, we compared the immunological properties of recombinant (r) Lol p 1 with those of the natural protein. We cloned directly the gene encoding Lol p 1 from genomic DNA of ryegrass pollen. This gene was subcloned into the expression vector pMAL-c and expressed as fusion protein. Subsequently, rLol p 1 was cleaved from maltose-binding protein using factor Xa. Using binding inhibition and proliferative assays, we assessed the immunological properties of the recombinant allergens. The capacity of rLol p 1 to trigger basophil histamine release and to elicit a skin reaction was also assessed and compared to those of its natural counterpart. We found that the Lol p 1 gene has no introns since we amplified this gene directly from genomic DNA. We demonstrated that the binding sites of anti-Lol p 1 monoclonal antibody, specific human IgG and IgE antibody are well conserved on rLol p 1 as no difference in the binding inhibition profile was observed when using either natural or recombinant protein. At the T-cell level, rLol p 1 elicited a T-cell response in mice comparable to that observed with the natural protein. In addition, we demonstrated that the biological characteristics of rLol p 1 were comparable to those of the natural counterpart, in that rLol p 1 elicited a skin wheal reaction and induced basophil histamine release in grass-allergic patients only. The data indicate that natural Lol p 1 and rLol p 1 shared identical immunological and biological properties.

  16. SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12.

    Science.gov (United States)

    Martin, A; Yeats, S; Janekovic, D; Reiter, W D; Aicher, W; Zillig, W

    1984-09-01

    Sulfolobus acidocaldarius, strain B12, which harbours a double-stranded DNA species both as a plasmid and in a linear form, which is integrated at a specific site of the chromosome, produces virus-like particles upon u.v. irradiation. These particles contain the same circular DNA and a number of coat proteins and are probably surrounded by a lipid membrane. They are lemon shaped, 100 x 60 nm in size and carry tail structures at one pole. The host cell recovers and remains lysogenic after virus production. Though a large fraction of liberated particles is found attached to structures derived from the cells, neither adsorption nor infection of a number of Sulfolobus isolates has so far been observed.

  17. ORF Alignment: NC_002754 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

  18. ORF Alignment: NC_006348 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

  19. ORF Alignment: NC_006350 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available ture Of Glcv, The ... Abc-Atpase Of The Glucose Abc Transporter From ... ... ... Sulfolobus Solfataricus pdb|1OXV|B Chain B, Crystal ... Structure Of Glcv, The Abc-Atpase Of Th...folobus Solfataricus ... pdb|1OXU|C Chain C, Crystal Structure Of Glcv, The ... Abc-Atpase Of ...tructure Of Glcv, The Abc-Atpase Of The Glucose Abc ... Transporter From S...ulfolobus Solfataricus pdb|1OXU|A ... Chain A, Crystal Structure Of Glcv, The Abc-Atpase Of ...

  20. Adjoint P1 equations solution for neutron slowing down

    International Nuclear Information System (INIS)

    Cardoso, Carlos Eduardo Santos; Martinez, Aquilino Senra; Silva, Fernando Carvalho da

    2002-01-01

    In some applications of perturbation theory, it is necessary know the adjoint neutron flux, which is obtained by the solution of adjoint neutron diffusion equation. However, the multigroup constants used for this are weighted in only the direct neutron flux, from the solution of direct P1 equations. In this work, the adjoint P1 equations are derived by the neutron transport equation, the reversion operators rules and analogies between direct and adjoint parameters. The direct and adjoint neutron fluxes resulting from the solution of P 1 equations were used to three different weighting processes, to obtain the macrogroup macroscopic cross sections. It was found out noticeable differences among them. (author)

  1. P1 peptidase–a mysterious protein of family Potyviridae

    Indian Academy of Sciences (India)

    The coding region for P1 peptidase is located at the very beginning of the viral genome of the family Potyviridae. Until recently P1 was thought of as serine peptidase with ... Jana Rohožková1 Milan Navrátil1. Faculty of Science, Palacký University in Olomouc, Šlechtitelů 11, 783 71, Olomouc-Holice, Czech Republic ...

  2. System of adjoint P1 equations for neutron moderation

    International Nuclear Information System (INIS)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos

    2000-01-01

    In some applications of perturbation theory, it is necessary know the adjoint neutron flux, which is obtained by the solution of adjoint neutron diffusion equation. However, the multigroup constants used for this are weighted in only the direct neutron flux, from the solution of direct P1 equations. In this work, this procedure is questioned and the adjoint P1 equations are derived by the neutron transport equation, the reversion operators rules and analogies between direct and adjoint parameters. (author)

  3. System of adjoint P1 equations for neutron moderation; Sistema de equacoes P1 adjuntas para a moderacao de neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos [Universidade Federal, Rio de Janeiro, RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia. Programa de Engenharia Nuclear

    2000-07-01

    In some applications of perturbation theory, it is necessary know the adjoint neutron flux, which is obtained by the solution of adjoint neutron diffusion equation. However, the multigroup constants used for this are weighted in only the direct neutron flux, from the solution of direct P1 equations. In this work, this procedure is questioned and the adjoint P1 equations are derived by the neutron transport equation, the reversion operators rules and analogies between direct and adjoint parameters. (author)

  4. An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion.

    Science.gov (United States)

    Xue, H; Guo, R; Wen, Y; Liu, D; Huang, L

    2000-07-01

    The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25 degrees C, it was enhanced substantially at 45 degrees C or higher temperatures (up to 80 degrees C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

  5. Characterization and mechanism insight of accelerated catalytic promiscuity of Sulfolobus tokodaii (ST0779) peptidase for aldol addition reaction.

    Science.gov (United States)

    Li, Rong; Perez, Bianca; Jian, Hui; Jensen, Mads Mørk; Gao, Renjun; Dong, Mingdong; Glasius, Marianne; Guo, Zheng

    2015-11-01

    A novel peptidase from thermophilic archaea Sulfolobus tokodaii (ST0779) is examined for its catalytic promiscuity of aldol addition, which shows comparable activity as porcine pancreatic lipase (PPL, one of the best enzymes identified for biocatalytic aldol addition) at 30 °C but much accelerated activity at elevated temperature. The molecular catalytic efficiency kcat/Km (M(-1) s(-1)) of this thermostable enzyme at 55 °C adds up to 140 times higher than that of PPL at its optimum temperature 37 °C. The fluorescence quenching analysis depicts that the binding constants of PPL are significantly higher than those of ST0779, and their numbers of binding sites show opposite temperature dependency. Thermodynamic parameters estimated by fluorescence quenching analysis unveil distinctly different substrate-binding modes between PPL and ST0779: the governing binding interaction between PPL and substrates is hydrophobic force, while the dominating substrate-binding forces for ST0779 are van der Waals and H-bonds interactions. A reasonable mechanism for ST0779-catalyzed aldol reaction is proposed based on kinetic study, spectroscopic analysis, and molecular stereostructure simulation. This work represents a successful example to identify a new enzyme for catalytic promiscuity, which demonstrates a huge potential to discover and exploit novel biocatalyst from thermophile microorganism sources.

  6. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Shoji Suzuki

    2017-01-01

    Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

  7. Doubling Power Output of Starch Biobattery Treated by the Most Thermostable Isoamylase from an Archaeon Sulfolobus tokodaii.

    Science.gov (United States)

    Cheng, Kun; Zhang, Fei; Sun, Fangfang; Chen, Hongge; Percival Zhang, Y-H

    2015-08-20

    Biobattery, a kind of enzymatic fuel cells, can convert organic compounds (e.g., glucose, starch) to electricity in a closed system without moving parts. Inspired by natural starch metabolism catalyzed by starch phosphorylase, isoamylase is essential to debranch alpha-1,6-glycosidic bonds of starch, yielding linear amylodextrin - the best fuel for sugar-powered biobattery. However, there is no thermostable isoamylase stable enough for simultaneous starch gelatinization and enzymatic hydrolysis, different from the case of thermostable alpha-amylase. A putative isoamylase gene was mined from megagenomic database. The open reading frame ST0928 from a hyperthermophilic archaeron Sulfolobus tokodaii was cloned and expressed in E. coli. The recombinant protein was easily purified by heat precipitation at 80 (o)C for 30 min. This enzyme was characterized and required Mg(2+) as an activator. This enzyme was the most stable isoamylase reported with a half lifetime of 200 min at 90 (o)C in the presence of 0.5 mM MgCl2, suitable for simultaneous starch gelatinization and isoamylase hydrolysis. The cuvett-based air-breathing biobattery powered by isoamylase-treated starch exhibited nearly doubled power outputs than that powered by the same concentration starch solution, suggesting more glucose 1-phosphate generated.

  8. Geographic divergence of "Sulfolobus islandicus" strains assessed by genomic analyses including electronic DNA hybridization confirms they are geovars.

    Science.gov (United States)

    Zuo, Guanghong; Hao, Bailin; Staley, James T

    2014-02-01

    Ten well-annotated genomes of "Sulfolobus islandicus" strains from different geographic locations have been released at the NCBI database. Whole genome based composition vector trees indicate that these strains show the same branching patterns as originally reported by multi-locus sequence analysis. To determine whether the ten strains meet the criteria for separate species, DNA-DNA hybridization (DDH) was performed in silico. DDH values of strains from the same geographic location, i.e., Iceland, Kamchatka and North America, ranged from 82.4 to 95.4 %, clearly qualifying them as members of the same species. The lowest DDH values found between locations ranged from 75.5 to 76.6 %, which exceed the 70 % DDH threshold for a species thereby indicating they are all members of the same species based on the currently accepted definition. The clear divergences of strains from the different geographic locations are sufficiently great to consider them as separate geovars. "S. islandicus" has not yet been validly named and a type strain has not been deposited in culture collections. We urgently recommend that those who study the organism fulfill the criteria of the International Code of Nomenclature of Bacteria in order to designate a type strain and to identify and deposit related strains of this species to make them available to the broader scientific community.

  9. Transcriptional Auto-Regulation of RUNX1 P1 Promoter

    Science.gov (United States)

    Martinez, Milka; Hinojosa, Marcela; Trombly, Daniel; Morin, Violeta; Stein, Janet; Stein, Gary; Javed, Amjad; Gutierrez, Soraya E.

    2016-01-01

    RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5’UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription. PMID:26901859

  10. Transcriptional Auto-Regulation of RUNX1 P1 Promoter.

    Directory of Open Access Journals (Sweden)

    Milka Martinez

    Full Text Available RUNX1 a member of the family of runt related transcription factors (RUNX, is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5'UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.

  11. Conjugation of nitrated acetaminophen to Der p1 amplifies peripheral blood monocyte response to Der p1.

    Directory of Open Access Journals (Sweden)

    Ryan G Thomas

    Full Text Available An association of acetaminophen use and asthma was observed in the International Study of Asthma and Allergies in Childhood study. However there are no clear mechanisms to explain an association between acetaminophen use and immunologic pathology. In acidic conditions like those in the stomach and inflamed airway, tyrosine residues are nitrated by nitrous and peroxynitrous acids. The resulting nitrotyrosine is structurally similar to 2,4-dinitrophenol and 2,4-dinitrochlorobenzene, known haptens that enhance immune responses by covalently binding proteins. Nitrated acetaminophen shares similar molecular structure.We hypothesized the acetaminophen phenol ring undergoes nitration under acidic conditions, producing 3-nitro-acetaminophen which augments allergic responses by acting as a hapten for environmental allergens.3-nitro-acetaminophen was formed from acetaminophen in the presence of acidified nitrite, purified by high performance liquid chromatography, and assayed by gas-chromatography mass spectrometry. Purified 3-nitro-acetaminophen was reacted with Dermatophagoides pteronyssinus (Der p1 and analyzed by mass spectrometry to identify the modification site. Human peripheral blood mononuclear cells proliferation response was measured in response to 3-nitro-acetaminophen and to 3-nitro-acetaminophen-modified Der p1.Acetaminophen was modified by nitrous acid forming 3-nitro-acetaminophen over a range of different acidic conditions consistent with airway inflammation and stomach acidity. The Der p1 protein-hapten adduct creation was confirmed by liquid chromatography-mass spectrometry proteomics modifying cysteine 132. Peripheral blood mononuclear cells exposed to 3-nitro-acetaminophen-modified Der p1 had increased proliferation and cytokine production compared to acetaminophen and Der p1 alone (n = 7; p < 0.05.These data suggests 3-nitro-acetaminophen formation and reaction with Der p1 provides a mechanism by which stomach acid or infection

  12. P1 peptidase–a mysterious protein of family Potyviridae

    Indian Academy of Sciences (India)

    direct evidence of having some role in host range definition; however, this is yet to be demonstrated. 3. ..... tion supports the suggestion that CVYV P1b uses a strategy of siRNA sequestration, with the interference of ..... Zucchini yellow mosaic virus for strains differentiation and phylogenetic analysis with other potyviruses.

  13. Adjoint P1 equations solution for neutron slowing down; Solucao das equacoes P1 adjuntas para moderacao de neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Cardoso, Carlos Eduardo Santos; Martinez, Aquilino Senra; Silva, Fernando Carvalho da [Universidade Federal, Rio de Janeiro, RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia. Programa de Engenharia Nuclear

    2002-07-01

    In some applications of perturbation theory, it is necessary know the adjoint neutron flux, which is obtained by the solution of adjoint neutron diffusion equation. However, the multigroup constants used for this are weighted in only the direct neutron flux, from the solution of direct P1 equations. In this work, the adjoint P1 equations are derived by the neutron transport equation, the reversion operators rules and analogies between direct and adjoint parameters. The direct and adjoint neutron fluxes resulting from the solution of P{sub 1} equations were used to three different weighting processes, to obtain the macrogroup macroscopic cross sections. It was found out noticeable differences among them. (author)

  14. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  15. The P1approximation in the transport of beta rays

    International Nuclear Information System (INIS)

    Legarda, F.; Idoeta, R.; Herranz, M.

    1994-01-01

    A validation test for the p1 approximation to the linear transport of electrons in planar geometry has been performed. The p1 approximation is shown to be a good option for the description of the transport of beta rays with endpoint energies between 400kev and 3.5Mev through aluminium foils . This approximation together with the use of only elastic interactions of electrons with atoms has found good agreement with experimental results . A calculation has been made of the fraction of transmitted electrons through foils, solving the transport equation for planar geometry in the p1 approximation and assuming that only elastic scattering processes take place. The boundary condition at the entrance of the foil was a collimated beta source, while at the end of the foil has been adopted a vaccum boundary condition.Sources considered are those for which experimental and calculated spectrum shapes are known to agree. The calculated fractional transmission through different absorber thicknesses is found to have an exponential shape . Besides this fact the attenuation coefficients found ,when compared with those empirically obtained, agree to within 5%. 1 fig.; 4 refs. (author)

  16. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  17. Catalytic activity of nuclease P1: Experiment and theory

    International Nuclear Information System (INIS)

    Miller, J.H.; Falcone, J.M.; Shibata, M.; Box, H.C.

    1994-10-01

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates

  18. IgE-binding potencies of three peach Pru p 1 isoforms

    NARCIS (Netherlands)

    Gao, Zhong-Shan; Zhou, Xiang; Yang, Zhao-Wei; Versteeg, Serge A.; Gao, Ling; Fu, Wan-Yi; Wang, Hui-Ying; Zhou, Jian-Ying; Akkerdaas, Jaap H.; van Ree, Ronald

    2016-01-01

    Pru p 1, the Bet v 1 homologue from peach, has been identified as a clinically relevant allergen. Three isoforms have been described, two in peach fruit (Pru p 1.0101 and Pru p 1.0201) and one in pollen (Pru p 1.0301). The present study aimed to compare their IgE-binding potencies. Three Pru p 1

  19. Crystallization and heavy-atom derivatization of StHsp14.0, a small heat-shock protein from Sulfolobus tokodaii

    Science.gov (United States)

    Hayashi, Takuro; Abe, Tetsuya; Takeda, Kazuki; Akiyama, Nobuhiko; Yohda, Masafumi; Miki, Kunio

    2009-01-01

    Small heat-shock proteins (sHsps) bind and stabilize proteins denatured by heat or other stresses in order to prevent unfavourable protein aggregation. StHsp14.0 is an sHsp found in the acidothermophilic archaeon Sulfolobus tokodaii. A variant of StHsp14.0 was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted X-rays to 1.85 Å resolution and belonged to space group P21212, with unit-cell parameters a = 40.4, b = 61.1, c = 96.1 Å. The V M value was estimated to be 2.1 Å3 Da−1, assuming the presence of two molecules in the asymmetric unit. Heavy-atom derivative crystals were prepared successfully by the cocrystallization method and are isomorphic to native crystals. PMID:19851008

  20. General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

    Science.gov (United States)

    Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

    2018-02-19

    DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

    Science.gov (United States)

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N; Crowley, Paula J; Brady, L Jeannine; Long, Joanna R

    2016-02-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  2. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    Tang, Wenxing; Bhatt, Avni; Smith, Adam N.; Crowley, Paula J.; Brady, L. Jeannine; Long, Joanna R.

    2016-01-01

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin

  3. Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Wenxing; Bhatt, Avni [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States); Smith, Adam N. [University of Florida, Department of Chemistry, College of Liberal Arts and Sciences (United States); Crowley, Paula J.; Brady, L. Jeannine, E-mail: jbrady@dental.ufl.edu [University of Florida, Department of Oral Biology, College of Dentistry (United States); Long, Joanna R., E-mail: jrlong@ufl.edu [University of Florida, Department of Biochemistry and Molecular Biology, College of Medicine (United States)

    2016-02-15

    The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

  4. Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

    2002-01-01

    Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit...

  5. Sulfolobus acidocaldarius UDG Can Remove dU from the RNA Backbone: Insight into the Specific Recognition of Uracil Linked with Deoxyribose

    Directory of Open Access Journals (Sweden)

    Gang-Shun Yi

    2017-01-01

    Full Text Available Sulfolobus acidocaldarius encodes family 4 and 5 uracil-DNA glycosylase (UDG. Two recombinant S. acidocaldarius UDGs (SacUDG were prepared and biochemically characterized using oligonucleotides carrying a deaminated base. Both SacUDGs can remove deoxyuracil (dU base from both double-stranded DNA and single-stranded DNA. Interestingly, they can remove U linked with deoxyribose from single-stranded RNA backbone, suggesting that the riboses on the backbone have less effect on the recognition of dU and hydrolysis of the C-N glycosidic bond. However, the removal of rU from DNA backbone is inefficient, suggesting strong steric hindrance comes from the 2′ hydroxyl of ribose linked to uracil. Both SacUDGs cannot remove 2,2′-anhydro uridine, hypoxanthine, and 7-deazaxanthine from single-stranded DNA and single-stranded DNA. Compared with the family 2 MUG, other family UDGs have an extra N-terminal structure consisting of about 50 residues. Removal of the 46 N-terminal residues of family 5 SacUDG resulted in only a 40% decrease in activity, indicating that the [4Fe-4S] cluster and truncated secondary structure are not the key elements in hydrolyzing the glycosidic bond. Combining our biochemical and structural results with those of other groups, we discussed the UDGs’ catalytic mechanism and the possible repair reactions of deaminated bases in prokaryotes.

  6. The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules.

    Science.gov (United States)

    Notomista, Eugenio; Falanga, Annarita; Fusco, Salvatore; Pirone, Luciano; Zanfardino, Anna; Galdiero, Stefania; Varcamonti, Mario; Pedone, Emilia; Contursi, Patrizia

    2015-09-04

    Pathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa. A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets. VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.

  7. NQO-Induced DNA-Less Cell Formation Is Associated with Chromatin Protein Degradation and Dependent on A0A1-ATPase in Sulfolobus

    Directory of Open Access Journals (Sweden)

    Wenyuan Han

    2017-08-01

    Full Text Available To investigate DNA damage response in the model crenarchaeon Sulfolobus islandicus, four different DNA damage agents were tested for their effects on cell death of this archaeon, including UV irradiation, methyl methanesulfonate, cisplatin, and 4-nitroquinoline 1-oxide (NQO. Cell death featured with DNA-less cell formation was revealed in DNA damage treatment with each agent. Cellular responses upon NQO treatment were characterized in details, and following sequential events were revealed, including: a modest accumulation of G1/S phase cells, membrane depolarization, proteolytic degradation of chromatin proteins, and chromosomal DNA degradation. Further insights into the process were gained from studying drugs that affect the archaeal ATP synthase, including a proton gradient uncoupler and an ATP synthase inhibitor. Whereas the proton uncoupler-mediated excess proton influx yielded cell death as observed for the NQO treatment, inhibition of ATP synthase attenuated NQO-induced membrane depolarization and DNA-less cell formation. In conclusion, the NQO-induced cell death in S. islandicus is characterized by proteolytic degradation of chromatin protein, and chromosomal DNA degradation, which probably represents a common feature for the cell death induced by different DNA damage agents.

  8. The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

    Directory of Open Access Journals (Sweden)

    Elham Peyfoon

    2010-01-01

    Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

  9. Expression of human protamine P1 in sperm of transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Wyrobek, A.J.; Keith, C.; Stilwell, J.; Lowe, X. [Lawrence Livermore National Laboratory, CA (United States); Anderson, G. [Univ. of California, Davis, CA (United States)

    1994-12-31

    Transgenic mice were produced by pronuclear injection with DNA constructs containing human protamine P1 cDNA recombined with a murine protamine P1 promoter, and were identified by PCR. Expression of human P1 was investigated using huplm, a monoclonal antibody specific for human P1, applied to murine testicular cells, smears of epididymal sperm, and smears of detergent-isolated sperm nuclei. Various antibodies and nontransgenic littermates were used as controls. Two male founders (T3 and T7) sired more than five generations of transgenic offspring each with continued expression of human P1 in their sperm. Transgenic animals appear of normal fertility with sperm of typical nuclear morphology. The human P1 transgene was expressed postmeioticly in both lines, as expected. Nearly 100% of sperm of T3 and T7 hemizygotes labeled with huplm, consistent with complete diffusion of human P1 protein through the intercellular bridge of spermatogenic cells. Human P1 labeling of sperm nuclei was not visibly affected by sonication or by treatment with the detergent MATAB or the reducing agent DTT. A third founder female (T5) showed a transmission pattern consistent with insertion of the transgene into an X chromosome; her transgenic offspring expressed human P1 in only a small fraction of sperm. Human P1 transgenes may serve as efficient targets for germinal mutations and transgenicmice may provide promising models for investigating the DNA complexes.

  10. Porcine Circovirus-Like Virus P1 Inhibits Wnt Signaling Pathway in Vivo and in Vitro.

    Science.gov (United States)

    Zhu, Xuejiao; Wen, Libin; Sheng, Shaoyang; Wang, Wei; Xiao, Qi; Qu, Meng; Hu, Yiyi; Liu, Chuanmin; He, Kongwang

    2018-01-01

    Porcine circovirus-like virus P1 is an important pathogen of the current pig industry, the infection mechanism is not entirely clear. Wnt signaling pathway plays an important role in the growth of young animals and infection of some viruses. This study was designed to demonstrate the effects of P1 infection on the Wnt signaling pathway. In vivo experiments, we demonstrated the down-regulatory effects of P1 infection in piglets and mice on the downstream components expression levels of Wnt signaling pathway, and the effects of Wnt signaling pathway activation on the pathogenesis of P1. In vitro studies, we found P1 infection down-regulated protein level of β-catenin and mRNA level of mmp2, prevented the β-catenin from entering into nucleus, abolished the TCF/LEF promoter activity, proved that P1 could inhibit the activation of Wnt signaling pathway in vitro . Finally, we found that VP1 of P1 virus also had the inhibitory effects on Wnt signaling pathway in vitro , elucidated the mechanism of P1's inhibitory effects on the Wnt signaling pathway and offered the possibility that the suppression of Wnt signaling pathway was involved in the post-weaning multisystemic wasting syndrome (PMWS), laying a foundation for elucidating the pathogenesis of P1.

  11. Porcine Circovirus-Like Virus P1 Inhibits Wnt Signaling Pathway in Vivo and in Vitro

    Directory of Open Access Journals (Sweden)

    Xuejiao Zhu

    2018-03-01

    Full Text Available Porcine circovirus-like virus P1 is an important pathogen of the current pig industry, the infection mechanism is not entirely clear. Wnt signaling pathway plays an important role in the growth of young animals and infection of some viruses. This study was designed to demonstrate the effects of P1 infection on the Wnt signaling pathway. In vivo experiments, we demonstrated the down-regulatory effects of P1 infection in piglets and mice on the downstream components expression levels of Wnt signaling pathway, and the effects of Wnt signaling pathway activation on the pathogenesis of P1. In vitro studies, we found P1 infection down-regulated protein level of β-catenin and mRNA level of mmp2, prevented the β-catenin from entering into nucleus, abolished the TCF/LEF promoter activity, proved that P1 could inhibit the activation of Wnt signaling pathway in vitro. Finally, we found that VP1 of P1 virus also had the inhibitory effects on Wnt signaling pathway in vitro, elucidated the mechanism of P1’s inhibitory effects on the Wnt signaling pathway and offered the possibility that the suppression of Wnt signaling pathway was involved in the post-weaning multisystemic wasting syndrome (PMWS, laying a foundation for elucidating the pathogenesis of P1.

  12. Identification and Characterization of Novel Catalytic Bioscavengers of Organophosphorus Nerve Agents

    Science.gov (United States)

    2013-01-01

    Shewanella algae Sulfate reducer Streptosporangium roseum BLAST Sulfolobus solfataricus P2 BLAST Thermomonospora curvata BLAST Tsukamurella paurometabola...M. aurantiaca is an aerobic, gram-positive filamentous bacteria normally found in soil. This strain has the capability to form spores and branched

  13. Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

    OpenAIRE

    Ruggero, D; Londei, P

    1996-01-01

    Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.

  14. Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment

    NARCIS (Netherlands)

    Brouns, S.J.J.; Walther, J.; Snijders, A.P.; Werken, van de H.J.G.; Willemen, H.L.D.M.; Worm, P.; Vos, de M.G.; Andersson, A.; Lundgren, M.; Mazon, H.F.; Heuvel, van den R.H.H.; Nilsson, P.; Salmon, L.; Vos, de W.M.; Wright, P.C.; Bernander, R.; Oost, van der J.

    2006-01-01

    The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to

  15. Archaeal homolog of bacterial type IV prepilin signal peptidases with broad substrate specificity

    NARCIS (Netherlands)

    Albers, Sonja-Verena; Szabó, Zalán; Driessen, Arnold J.M.

    A large number of secretory proteins in the thermoacidophile Sulfolobus solfataricus are synthesized as a precursor with an unusual leader peptide that resembles bacterial type IV prepilin signal sequences. This set of proteins includes the flagellin subunit but also various solute binding proteins.

  16. Purification and structural stability of the peach allergens Pru p 1 and Pru p 3

    NARCIS (Netherlands)

    Gaier, Sonja; Marsh, Justin; Oberhuber, Christina; Rigby, Neil M.; Lovegrove, Alison; Alessandri, Stefano; Briza, Peter; Radauer, Christian; Zuidmeer, Laurian; van Ree, Ronald; Hemmer, Wolfgang; Sancho, Ana I.; Mills, Clare; Hoffmann-Sommergruber, Karin; Shewry, Peter R.

    2008-01-01

    Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia

  17. Including an Exam P/1 Prep Course in a Growing Actuarial Science Program

    Science.gov (United States)

    Wakefield, Thomas P.

    2014-01-01

    The purpose of this article is to describe the actuarial science program at our university and the development of a course to enhance students' problem solving skills while preparing them for Exam P/1 of the Society of Actuaries (SOA) and the Casualty Actuary Society (CAS). The Exam P/1 prep course, formally titled Mathematical Foundations of…

  18. Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

    DEFF Research Database (Denmark)

    Drasbek, Mette; Christiansen, Gunna; Drasbek, Kim Ryun

    2007-01-01

    The human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant...... protein (rP1-II) covering amino acids 1107-1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes...... in this region of P1 also reduce M. pneumoniae adhesion. Therefore, peptides covering these epitopes, of 8 and 13 aa, respectively, were synthesized to further investigate the adhesion region. None of these synthetic peptides reduced the binding of M. pneumoniae to the receptors on the host cells. Instead, 10...

  19. Compensatory mutations at the HIV cleavage sites p7/p1 and p1/p6-gag in therapy-naive and therapy-experienced patients.

    Science.gov (United States)

    Verheyen, Jens; Litau, Elena; Sing, Tobias; Däumer, Martin; Balduin, Melanie; Oette, Mark; Fätkenheuer, Gerd; Rockstroh, Jürgen K; Schuldenzucker, Ulrike; Hoffmann, Daniel; Pfister, Herbert; Kaiser, Rolf

    2006-01-01

    Mutations in the genome of HIV conferring drug resistance are a major reason for the failure of antiretroviral therapy, but they often compromise viral fitness. Protease (PR) cleavage site (CS) mutations could compensate for impaired replication capacity of drug-resistant viruses. We analysed the cleavage sites p1/p7 and p1/p6-gag of 500 HIV-1 subtype B infected patients. The collective consists of 275 therapy-naive and 225 therapy-experienced patients with at least one primary PR mutation, from whom eight underwent therapy-interruption in different clinical settings. Multiple mutations within the CS p7/p1 and p1/p6-gag accumulated in therapy-experienced isolates (p7/p1: A431V-K436R-I437V and p1/p6-gag: L449F/V-P452S-P453L/A). Further rare CS mutations were totally absent in therapy-naive viruses. Sixty percent of all therapy-experienced viruses exhibited at least one therapy-associated CS mutation, but so did 10% of therapy-naive viruses. The analysis of CS and PR mutations in therapy-experienced viruses revealed several positive correlations--A431V with L24I-M46I/L-I54V-V82A; I437V with I54V-V82F/T/S; L449V with I54M/L/S/T/A; and L449F/R452S/P453L: with D30N-I84V--whereas P453L and V82A were negatively correlated. Mutagenetic trees constructed form this cross-sectional data showed an ordered accumulation of the most prominent CS mutations along two pathways L90M-I84V-P453L and I54-V82-A431V followed by either M46L or L24I. Furthermore, eight viruses with at least one therapy-associated mutation at each CS displayed an outstanding maintenance of major PR mutations during therapy interruption. These findings emphasize the relevance of CS mutations in the evolution of HIV resistance to PR inhibitors. Therefore, therapy-associated CS mutations should be considered in HIV resistance tests to estimate viral fitness in different clinical settings.

  20. Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

    Directory of Open Access Journals (Sweden)

    Yoomi Choi

    Full Text Available Cucumber mosaic virus (CMV is a destructive pathogen affecting Capsicum annuum (pepper production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase. Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP. Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

  1. Expression and tissue localization of collectin placenta 1 (CL-P1, SRCL) in human tissues

    DEFF Research Database (Denmark)

    Sellman, Lana; Skjødt, Karsten; Nielsen, Ole

    2008-01-01

    Collectin placenta-1 (CL-P1), also known as scavenger receptor with C-type lectin (SRCL), is a type II membrane glycoprotein that shares structural features with both collectins and type A scavenger receptors. CL-P1 was originally cloned from the placenta and found to be associated with endothelial....... Three monoclonal antibodies were characterized and used in immunohistochemical analyses of a panel of cryo- and formalin-fixed sections. We find that CL-P1 mainly associates with cytotrophoblasts and syncytiotrophoblasts of the placenta, alveolar macrophages and to a less degree with macrophage...

  2. Solutions of system of P1 equations without use of auxiliary differential equations coupled

    International Nuclear Information System (INIS)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos

    2000-01-01

    The system of P1 equations is composed by two equations coupled itself one for the neutron flux and other for the current. Usually this system is solved by definitions of two integrals parameters, which are named slowing down densities of the flux and the current. Hence, the system P1 can be change from integral to only two differential equations. However, there are two new differentials equations that may be solved with the initial system. The present work analyzes this procedure and studies a method, which solve the P1 equations directly, without definitions of slowing down densities. (author)

  3. Continuous wave protocol for simultaneous polarization and optical detection of P1-center electron spin resonance

    Science.gov (United States)

    Kamp, E. J.; Carvajal, B.; Samarth, N.

    2018-01-01

    The ready optical detection and manipulation of bright nitrogen vacancy center spins in diamond plays a key role in contemporary quantum information science and quantum metrology. Other optically dark defects such as substitutional nitrogen atoms (`P1 centers') could also become potentially useful in this context if they could be as easily optically detected and manipulated. We develop a relatively straightforward continuous wave protocol that takes advantage of the dipolar coupling between nitrogen vacancy and P1 centers in type 1b diamond to detect and polarize the dark P1 spins. By combining mutual spin flip transitions with radio frequency driving, we demonstrate the simultaneous optical polarization and detection of the electron spin resonance of the P1 center. This technique should be applicable to detecting and manipulating a broad range of dark spin populations that couple to the nitrogen vacancy center via dipolar fields, allowing for quantum metrology using these spin populations.

  4. EPOXI C/GARRADD (2009 P1) - MRI CALIBRATED IMAGES V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains calibrated clear-filter, C2, CN, OH and dust continuum images of comet C/Garradd (2009 P1) acquired by the Medium Resolution Visible CCD (MRI)...

  5. EPOXI C/GARRADD (2009 P1) - MRI RAW IMAGES V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains Raw clear-filter, C2, CN, OH and dust continuum images of comet C/Garradd (2009 P1) acquired by the Medium Resolution Visible CCD (MRI) from 20...

  6. EPOXI C/GARRADD (2009 P1) - HRII CALIBRATED SPECTRA V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains calibrated, 1.05- to 4.8-micron spectral images of comet C/Garradd (2009 P1) acquired by the High Resolution Infrared Spectrometer on 26 March...

  7. EPOXI C/GARRADD (2009 P1) - HRII RAW SPECTRA V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains raw, 1.05- to 4.8-micron spectral images of comet C/Garradd (2009 P1) acquired by the High Resolution Infrared Spectrometer on 26 March and...

  8. EPOXI C/GARRADD (2009 P1) - HRIV CALIBRATED IMAGES V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains calibrated clear-filter images of comet C/Garradd (2009 P1) acquired by the High Resolution Visible CCD (HRIV) from 20 February through 09...

  9. EPOXI C/GARRADD (2009 P1) - HRIV RAW IMAGES V1.0

    Data.gov (United States)

    National Aeronautics and Space Administration — This dataset contains raw clear-filter images of comet C/Garradd (2009 P1) acquired by the High Resolution Visible CCD (HRIV) from 20 February through 09 April 2012...

  10. Suppression of Zeeman relaxation in cold collisions of 2P1/2 atoms

    International Nuclear Information System (INIS)

    Tscherbul, T. V.; Dalgarno, A.; Buchachenko, A. A.; Lu, M.-J.; Weinstein, J. D.

    2009-01-01

    We present a combined experimental and theoretical study of angular momentum depolarization in cold collisions of 2 P atoms in the presence of an external magnetic field. We show that collision-induced Zeeman relaxation of Ga( 2 P 1/2 ) and In( 2 P 1/2 ) atoms in cold 4 He gas is dramatically suppressed compared to atoms in 2 P 3/2 states. Using rigorous quantum-scattering calculations based on ab initio interaction potentials, we demonstrate that Zeeman transitions in collisions of atoms in 2 P 1/2 electronic states occur via couplings to the 2 P 3/2 state induced by the anisotropy of the interaction potential. Our results suggest the feasibility of sympathetic cooling and magnetic trapping of 2 P 1/2 -state atoms, such as halogens, thereby opening up exciting areas of research in precision spectroscopy and cold-controlled chemistry.

  11. Association of glutathione-S-transferase P1 (GSTP1)-313 A> G gene ...

    African Journals Online (AJOL)

    Association of glutathione-S-transferase P1 (GSTP1)-313 A> G gene polymorphism and susceptibility to endometrial hyperplasia among Egyptian women. Afaf Elsaid, Wfaa Al-Kholy, Rana Ramadan, Rami Elshazli ...

  12. Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

    Science.gov (United States)

    Shan, Hongying; Pasin, Fabio; Tzanetakis, Ioannis E; Simón-Mateo, Carmen; García, Juan Antonio; Rodamilans, Bernardo

    2017-11-08

    The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  13. Comparison of buffers for extraction of mite allergen der p 1 from dust.

    Science.gov (United States)

    Prester, Ljerka; Kovačić, Jelena; Macan, Jelena

    2012-09-01

    Der p 1 is the main allergen of house dust mite Dermatophagoides pteronyssinus, which has routinely been detected in residential dust. However, the procedure for extracting Der p 1 from reservoir dust has not been well defined. The aim of this study was to compare Der p 1 mass fractions in dust extracts prepared using the following extraction buffers: phosphate (pH 7.4), borate (pH 8.0), and ammonium bicarbonate (pH 8.0), all with 0.05 % Tween 20. Twenty-eight dust samples were divided into three aliquots and each portion was extracted with one of the three buffers at room temperature. Der p 1 mass fractions were measured in a total of 84 dust extracts using the enzyme immunoassay (range: 0.1 μg g-1 to 7.53 μg g-1). Statistical methods including intraclass correlation showed a high agreement between Der p 1 mass fractions irrespective of the extracting medium. Our results suggest that all three buffers are suitable for the extraction of mite allergens and routine Der p 1 analysis in dust.

  14. Only Acyl Carrier Protein 1 (AcpP1 Functions in Pseudomonas aeruginosa Fatty Acid Synthesis

    Directory of Open Access Journals (Sweden)

    Jin-Cheng Ma

    2017-11-01

    Full Text Available The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS, only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coliacpP mutant strain CY1877. And P. aeruginosaacpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL, but replacement of P. aeruginosaacpP1 with E. coliacpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coliacpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.

  15. Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

    International Nuclear Information System (INIS)

    Liang Xinle; Shou Qian; Zhang Hong; Chen Min; Liu Xuan

    2009-01-01

    Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with 60 Co γ-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6( nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

  16. IGFBP-rP1, a potential molecule associated with colon cancer differentiation

    Directory of Open Access Journals (Sweden)

    Deng Hong

    2010-10-01

    Full Text Available Abstract Background In our previous studies, we have demonstrated that insulin-like growth factor binding protein-related protein1 (IGFBP-rP1 played its potential tumor suppressor role in colon cancer cells through apoptosis and senescence induction. In this study, we will further uncover the role of IGFBP-rP1 in colon cancer differentiation and a possible mechanism by revealing responsible genes. Results In normal colon epithelium, immunohistochemistry staining detected a gradient IGFBP-rP1 expression along the axis of the crypt. IGFBP-rP1 strongly expressed in the differentiated cells at the surface of the colon epithelium, while weakly expressed at the crypt base. In colon cancer tissues, the expression of IGFBP-rP1 correlated positively with the differentiation status. IGFBP-rP1 strongly expressed in low grade colorectal carcinoma and weakly expressed in high grade colorectal carcinoma. In vitro, transfection of PcDNA3.1(IGFBP-rP1 into RKO, SW620 and CW2 cells induced a more pronounced anterior-posterior polarity morphology, accompanied by upregulation with alkaline phosphatase (AKP activity. Upregulation of carcino-embryonic antigen (CEA was also observed in SW620 and CW2 transfectants. The addition of IGFBP-rP1 protein into the medium could mimic most but not all effects of IGFBP-rP1 cDNA transfection. Seventy-eight reproducibly differentially expressed genes were detected in PcDNA3.1(IGFBP-rP1-RKO transfectants, using Affymetrix 133 plus 2.0 expression chip platform. Directed Acyclic Graph (DAG of the enriched GO categories demonstrated that differential expression of the enzyme regulator activity genes together with cytoskeleton and actin binding genes were significant. IGFBP-rP1 could upreguate Transgelin (TAGLN, downregulate SRY (sex determining region Y-box 9(campomelic dysplasia, autosomal sex-reversal (SOX9, insulin receptor substrate 1(IRS1, cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4 (CDKN2B, amphiregulin

  17. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2

    Science.gov (United States)

    Wheatley, Joshua L.; Holyoak, Todd

    2007-01-01

    The high-resolution crystal structure of kexin (Kex2) in complex with a peptidyl-chloromethylketone inhibitor containing a noncognate lysine at the P1 position provides the structural basis for the differential lysine/arginine selectivity that defines the prohormone (proprotein) convertase (PC) family. By comparison with the previous structures of Kex2 and furin, this structure of the acylated enzyme provides a basis for the observed decrease in the acylation rate with substrates containing a lysine at P1 and the absence of an effect on the deacylation rate without involving mobility of the S1 lid. The structure of the complex shows that a secondary subsite in the S1 pocket is present, and that this site recognizes and binds the P1 lysine in a more shallow fashion than arginine. This results in a displacement of the bound peptide away from the S385 nucleophile relative to substrates containing a P1 arginine. It is concluded that this alternate binding site and resultant displacement of the scissile bond in the active site results in the observed decrease in the acylation rate. Studies of the inactivation kinetics of Kex2 by two peptidyl chloromethylketone inhibitors demonstrates that the selectivity between lysine and arginine at the P1 position arises at the acylation step, consistent with what was observed with peptidyl substrates [Rockwell NC, Fuller RS (2001) J Biol Chem 276:38394–38399]. PMID:17426142

  18. A P1 Transcription Factors in Epidermal Differentiation and Skin Cancer

    International Nuclear Information System (INIS)

    Eckert, R. L.; Adhikary, G.; Young, C. A.; Jans, R.; Xu, W.; Eckert, R. L.; Eckert, R. L.; Crish, J. F.; Rorke, E.L.

    2013-01-01

    A P1 (jun/fos) transcription factors (c-jun, jun B, jun D, c-fos, Fos B, Fr a-1, and Fr a-2) are key regulators of epidermal keratinocyte survival and differentiation and important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because A P1 transcription factors regulate competing processes (i.e., proliferation, apoptosis, and differentiation). Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating A P1 transcription factors in epidermis. Taken together, these studies suggest that individual A P1 transcription factors have different functions in the epidermis and in cancer development and that altering A P1 transcription factor function in the basal versus supra basal layers differentially influences the epidermal differentiation response and disease and cancer development.

  19. P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila

    Science.gov (United States)

    Hoopfer, Eric D; Jung, Yonil; Inagaki, Hidehiko K; Rubin, Gerald M; Anderson, David J

    2015-01-01

    How brains are hardwired to produce aggressive behavior, and how aggression circuits are related to those that mediate courtship, is not well understood. A large-scale screen for aggression-promoting neurons in Drosophila identified several independent hits that enhanced both inter-male aggression and courtship. Genetic intersections revealed that 8-10 P1 interneurons, previously thought to exclusively control male courtship, were sufficient to promote fighting. Optogenetic experiments indicated that P1 activation could promote aggression at a threshold below that required for wing extension. P1 activation in the absence of wing extension triggered persistent aggression via an internal state that could endure for minutes. High-frequency P1 activation promoted wing extension and suppressed aggression during photostimulation, whereas aggression resumed and wing extension was inhibited following photostimulation offset. Thus, P1 neuron activation promotes a latent, internal state that facilitates aggression and courtship, and controls the overt expression of these social behaviors in a threshold-dependent, inverse manner. DOI: http://dx.doi.org/10.7554/eLife.11346.001 PMID:26714106

  20. An unusual feature associated with LEE1 P1 promoters in enteropathogenic Escherichia coli (EPEC).

    Science.gov (United States)

    Jeong, Jae-Ho; Kim, Hyun-Ju; Kim, Kun-Hee; Shin, Minsang; Hong, Yeongjin; Rhee, Joon Haeng; Schneider, Thomas D; Choy, Hyon E

    2012-02-01

    Transcription start points in bacteria are influenced by the nature of the RNA polymerase·promoter interaction. For Escherichia coli RNA polymerase holoenzyme containing σ70, it is presumed that specific sequence in one or more of the -10, extended -10 and -35 elements of the promoter guides the RNAP to select the cognate start point. Here, we investigated the promoter driving expression of the LEE1 operon in enteropathogenic E. coli and found two promoters separated by 10 bp, LEE1 P1A (+1) and LEE1 P1B (+10) using various in vitro biochemical tools. A unique feature of P1B was the presence of multiple transcription starts from five neighbouring As at the initial transcribed region. The multiple products did not arise from stuttering synthesis. Analytical software based on information theory was employed to determine promoter elements. The concentration of the NTP pool altered the preferred transcription start points, albeit the underlying mechanism is elusive. Under in vivo conditions, dominant P1B, but not P1A, was subject to regulation by IHF. © 2012 Blackwell Publishing Ltd.

  1. Metastable Cadmium (53P1) production in the Cd+-Na charge-exchange collision

    International Nuclear Information System (INIS)

    Coelho, L.F.

    1987-01-01

    The 326.1 nm emission cross section for the collision process Cd + +Na -> Cd(5 3 P 1 ) + Na + was measured in the energy range 1.5-4.9 KeV. It decreases monotonically from 5 to 2x10 -15 cm 2 . Due to a near degeneracy in energy, this channel presents a much larges cross section than all others (except 5 3 P 0 and 5 3 P 2 , which are highly metastable). This justifies the neglect of the cascade mechanism for populating the 5 3 P 1 , state and, consequently, allows the identification of the 326.1 nm emission cross section with the 5 3 P 1 charge-exchanger excitation cross section. The measured values agree well with theoretical estimates obtained with Olson's model for near-resonant charge exchange. (author) [pt

  2. Comparative analysis between P1 and B1 equations for neutron moderation

    International Nuclear Information System (INIS)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos

    2000-01-01

    In order to calculate the neutron flux in nuclear reactors, B1 or P1 equations are solved by numerical methods for several groups of energy. The neutron fluxes obtained from the solutions of the B1 and P1 equations are similar when they are applied to large nuclear power reactors. However, an important difference between the two fluxes is that the system of P1 equations uses one more approximation than the B1 system and then, its flux is less precise. The present work shows the relations between both equations and analyzes for what conditions the two equations systems are equivalent. Furthermore, this equations are numerically solved in 54 groups of energy for a quadrangular arrange. (author)

  3. Nature of the fundamental band gap in GaNxP1-x alloys

    International Nuclear Information System (INIS)

    Shan, W.; Walukiewicz, W.; Yu, K. M.; Wu, J.; Ager, J. W. III; Haller, E. E.; Xin, H. P.; Tu, C. W.

    2000-01-01

    The optical properties of GaN x P 1-x alloys (0.007≤x≤0.031) grown by gas-source molecular-beam epitaxy have been studied. An absorption edge appears in GaN x P 1-x at energy below the indirect Γ V -X C transition in GaP, and the absorption edge shifts to lower energy with increasing N concentration. Strong photomodulation signals associated with the absorption edges in GaN x P 1-x indicate that a direct fundamental optical transition is taking place, revealing that the fundamental band gap has changed from indirect to direct. This N-induced transformation from indirect to direct band gap is explained in terms of an interaction between the highly localized nitrogen states and the extended states at the Γ conduction-band minimum. (c) 2000 American Institute of Physics

  4. Fourier analysis of a new P1 synthetic acceleration for Sn transport equations

    International Nuclear Information System (INIS)

    Turcksin, B.; Ragusa, J. C.

    2010-10-01

    In this work, is derived a new P1 synthetic acceleration scheme (P1SA) for the S N transport equation and analyze its convergence properties through the means of a Fourier analysis. The Fourier analysis is carried out for both continuous (i.e., not spatially discretized) S N equations and linear discontinuous Fem discretization. We show, thanks to the continuous analysis, that the scheme is unstable when the anisotropy is important (μ - >0.5). However, the discrete analysis shows that when cells are large in comparison to the mean free path, the spectral radius decreases and the acceleration scheme becomes effective, even for highly anisotropic scattering. In charged particles transport, scattering is highly anisotropic and mean free paths are very small and, thus, this scheme could be of interest. To use the P1SA when cells are small and anisotropy is important, the scheme is modified by altering the update of the accelerated flux or by using either K transport sweeps before the application of P1SA. The update scheme performs well as long as μ - - ≥0.9, the modified update scheme is unstable. The multiple transport sweeps scheme is convergent with an arbitrary μ - but the spectral radius increases when scattering is isotropic. When anisotropic increases, the frequency of use of the acceleration scheme needs to be decreased. Even if the P1SA is used less often, the spectral radius is significantly smaller when compared with a method that does not use it for high anisotropy (μ - ≥0.5). It is interesting to notice that using P1SA every two iterations gives the same spectral radius than the update method when μ - ≥0.5 but it is much less efficient when μ - <0.5. (Author)

  5. Direct Band Gap Gallium Antimony Phosphide (GaSbxP1−x) Alloys

    OpenAIRE

    H. B. Russell; A. N. Andriotis; M. Menon; J. B. Jasinski; A. Martinez-Garcia; M. K. Sunkara

    2016-01-01

    Here, we report direct band gap transition for Gallium Phosphide (GaP) when alloyed with just 1?2 at% antimony (Sb) utilizing both density functional theory based computations and experiments. First principles density functional theory calculations of GaSbxP1?x alloys in a 216 atom supercell configuration indicate that an indirect to direct band gap transition occurs at x?=?0.0092 or higher Sb incorporation into GaSbxP1?x. Furthermore, these calculations indicate band edge straddling of the h...

  6. The DoF of the 3-user ($p,p+1$) MIMO Interference Channel

    OpenAIRE

    Torrellas, Marc; Agustin, Adrian; Vidal, Josep; Muñoz, Olga

    2014-01-01

    The degrees of freedom (DoF) of the 3-user multiple-input multiple-output (MIMO) interference channel (IC) with full channel state information and constant channel coefficients are investigated when (p, p + 1) antennas are deployed at the transmitters and receivers, respectively. The point of departure of this paper is the work of Wang et al., which conjectured but did not prove the DoF for the antenna settings with p > 1. Here the achievability of the DoF outer bound is formally proved using...

  7. Airborne study of grass allergen (Lol p 1) in different-sized particles.

    Science.gov (United States)

    De Linares, C; Díaz de la Guardia, C; Nieto Lugilde, D; Alba, F

    2010-01-01

    The Poaceae family is considered one of the main causes of pollen allergy in industrialized countries. The aim of this study is to establish the dynamics of the Poaceae allergens and determine their distribution in the different-sized particles in the atmosphere. The air of Granada (southern Spain) was sampled during the pollination period of Poaceae using a cascade impactor and a Hirst-type volumetric collector simultaneously. The sampled airborne allergens were analyzed by indirect ELISA and field emission scanning electron microscopy. Airborne pollen was evaluated with the Spanish Aerobiological Network methodology. Poaceae pollen and allergenic activity have parallel dynamics during the period of maximum pollination, which is reflected in the positive correlations between the 2 variables. In addition, the highest Lol p 1 concentrations were recorded in particle sizes lower than 3.3 mum (stage 4-F). The Spearman correlation test showed that airborne allergens are not dependent on meteorological factors, such as humidity, wind direction or sunshine, however, Lol p 1 allergen correlated positively with Poaceae pollen. The results of the present study confirm that the Lol p 1 allergen is detected more frequently with pollutants than with coarse particles with similar dynamics and a positive correlation between airborne pollen and aeroallergens. Moreover, Lol p 1 is released in stable weather conditions without large changes in humidity or temperature. Copyright 2009 S. Karger AG, Basel.

  8. 1P1 heavy quarkonia production in hadron-hadron collisions

    International Nuclear Information System (INIS)

    Alanakyan, R.A.; Dul'yan, L.S.; Grigoryan, S.G.; Matinyan, S.G.

    1987-01-01

    The production of 1 P 1 -quarkonia (charmonium, bottomonium and toponium) with large transverse momenta is studied in collisions of ultrahigh-energy pp and pp-bar beams. The analysis was carried out in the framework of gluon-gluon fusion mechanism and nonrelativistic quarkonium model

  9. Mutation analysis of the Mycobacterium leprae folP1 gene and dapsone resistance.

    Science.gov (United States)

    Nakata, Noboru; Kai, Masanori; Makino, Masahiko

    2011-02-01

    Diaminodiphenylsulfone (dapsone) has long been used as a first-line drug worldwide for the treatment of leprosy. Diagnosis for dapsone resistance of Mycobacterium leprae by DNA tests would be of great clinical value, but the relationship between the nucleotide substitutions and susceptibility to dapsone must be clarified before use. In this study, we constructed recombinant strains of cultivable Mycobacterium smegmatis carrying the M. leprae folP1 gene with or without a point mutation, disrupting their own folP gene on the chromosome. Dapsone susceptibilities of the recombinant bacteria were measured to examine influence of the mutations. Dapsone MICs for most of the strains with mutations at codon 53 or 55 of M. leprae folP1 were 2 to 16 times as high as the MIC for the strain with the wild-type folP1 sequence, but mutations that changed Thr to Ser at codon 53 showed somewhat lower MIC values than the wild-type sequence. Strains with mutations at codon 48 or 54 showed levels of susceptibility to dapsone comparable to the susceptibility of the strain with the wild-type sequence. This study confirmed that point mutations at codon 53 or 55 of the M. leprae folP1 gene result in dapsone resistance.

  10. Mutation Analysis of the Mycobacterium leprae folP1 Gene and Dapsone Resistance▿

    Science.gov (United States)

    Nakata, Noboru; Kai, Masanori; Makino, Masahiko

    2011-01-01

    Diaminodiphenylsulfone (dapsone) has long been used as a first-line drug worldwide for the treatment of leprosy. Diagnosis for dapsone resistance of Mycobacterium leprae by DNA tests would be of great clinical value, but the relationship between the nucleotide substitutions and susceptibility to dapsone must be clarified before use. In this study, we constructed recombinant strains of cultivable Mycobacterium smegmatis carrying the M. leprae folP1 gene with or without a point mutation, disrupting their own folP gene on the chromosome. Dapsone susceptibilities of the recombinant bacteria were measured to examine influence of the mutations. Dapsone MICs for most of the strains with mutations at codon 53 or 55 of M. leprae folP1 were 2 to 16 times as high as the MIC for the strain with the wild-type folP1 sequence, but mutations that changed Thr to Ser at codon 53 showed somewhat lower MIC values than the wild-type sequence. Strains with mutations at codon 48 or 54 showed levels of susceptibility to dapsone comparable to the susceptibility of the strain with the wild-type sequence. This study confirmed that point mutations at codon 53 or 55 of the M. leprae folP1 gene result in dapsone resistance. PMID:21115799

  11. Cotnparison between the Acarex R test and a Der p 1 ELISA for the ...

    African Journals Online (AJOL)

    low Acarex R scores and the Der p 1 allergen levels detennined by EUSA. Acarex R scores of 2 and 3 ... House-dust mites are important allergens in the pathogenesis of eczema, asthma and allergic rhinitis.I .... exposure to house-dust mite allergens is associated ~th an increase in bronchial hyperreactivity and renders the ...

  12. Accumulation of house- dust mite (Der-p-1) levels on mattress covers

    African Journals Online (AJOL)

    Mattresses serve as a large reservoir for house-dust mite antigens and harbour the highest mite levels within the household. Mite reduction measures have previously been shown to be unsuccessful. The effect of mattress covers and acaracides on Der-p-1 levels in the mattresses of 60 patients with mite-allergic asthma was ...

  13. Choline Modulation of the Aβ P1-40 Channel Reconstituted into a Model Lipid Membrane

    Directory of Open Access Journals (Sweden)

    Daniela Meleleo

    2010-01-01

    Full Text Available Nicotinic acetylcholine receptors (AChRs, implicated in memory and learning, in subjects affected by Alzheimer's disease result altered. Stimulation of α7-nAChRs inhibits amyloid plaques and increases ACh release. β-amyloid peptide (AβP forms ion channels in the cell and model phospholipid membranes that are retained responsible in Alzheimer disease. We tested if choline, precursor of ACh, could affect the AβP1-40 channels in oxidized cholesterol (OxCh and in palmitoyl-oleoyl-phosphatidylcholine (POPC:Ch lipid bilayers. Choline concentrations of 5 × 10−11 M–1.5 × 10−8 M added to the cis- or trans-side of membrane quickly increased AβP1-40 ion channel frequency (events/min and ion conductance in OxCh membranes, but not in POPC:Ch membranes. Circular Dichroism (CD spectroscopy shows that after 24 and 48 hours of incubation with AβP1-40, choline stabilizes the random coil conformation of the peptide, making it less prone to fibrillate. These actions seem to be specific in that ACh is ineffective either in solution or on AβP1-40 channel incorporated into PLMs.

  14. Analysis of glutathione S-transferase (M1, T1 and P1) gene ...

    African Journals Online (AJOL)

    transferase M1, T1 and P1 genetic polymorphisms with the risk of prostate cancer in various populations. The current study was done with Iranian subjects to evaluate the association of the polymorphism of glutathione S-transferase subtypes (T, M and ...

  15. Mechanical response of FFTF reference and P1 cladding tubes under transient heating

    International Nuclear Information System (INIS)

    Youngahl, C.A.; Ariman, T.; Lepacek, B.E.

    1977-01-01

    Burst tests of Type 316 stainless steel cladding tube samples subjected to increasing temperature and relatively constant internal pressure were conducted to assist in the pretest analysis of the P1 experiment performed in the Sodium Loop Safety Facility. This paper reports and analyzes the burst test results and those of subsequent transient heating work. The use of a modified extensometer in obtaining mechanical response data for stainless steel in the high temperature range is illustrated, some of such data is provided, and the potential of further experiments and analysis is indicated. Tubing of the same design as Fast Flux Test Facility (FFTF) cladding (20% cold worked, 0.230 in. OD, 15 mil wall) was tested as-received and after annealing or electrolytic thinning. P1 tubing (38% cold worked, 0.230 in. OD, 10 mil wall) was tested before and after aging under conditions anticipated in the P1 reactor experiment. The P1 cladding was designed to simulate FFTF tubing that had experienced irradiation embrittlement and attack by cesium oxide and sodium impurities

  16. Complexity and diversity of the NKR-P1:Clr (Klrb1:Clec2 recognition systems

    Directory of Open Access Journals (Sweden)

    Christina L Kirkham

    2014-06-01

    Full Text Available The NKR-P1 receptors were identified as prototypical natural killer (NK cell surface antigens and later shown to be conserved from rodents to humans on NK cells and subsets of T cells. C-type lectin-like in nature, they were originally shown to be capable of activating NK cell function and to recognize ligands on tumour cells. However, certain family members have subsequently been shown to be capable of inhibiting NK cell activity, and to recognize proteins encoded by a family of genetically linked C-type lectin-related (Clr ligands. Some of these ligands are expressed by normal, healthy cells, and modulated during transformation, infection, and cellular stress, while other ligands are upregulated during the immune response and during pathological circumstances. Here, we discuss historical and recent developments in NKR-P1 biology that demonstrate this NK receptor-ligand system to be far more complex and diverse than originally anticipated.

  17. Development of Quasi-3DOF upper limb rehabilitation system using ER brake: PLEMO-P1

    Science.gov (United States)

    Kikuchi, T.; Fukushima, K.; Furusho, J.; Ozawa, T.

    2009-02-01

    In recent years, many researchers have studied the potential of using robotics technology to assist and quantify the motor functions for neuron-rehabilitation. Some kinds of haptic devices have been developed and evaluated its efficiency with clinical tests, for example, upper limb training for patients with spasticity after stroke. However, almost all the devices are active-type (motor-driven) haptic devices and they basically require high-cost safety system compared to passive-type (brake-based) devices. In this study, we developed a new practical haptic device 'PLEMO-P1'; this system adopted ER brakes as its force generators. In this paper, the mechanism of PLEMO-P1 and its software for a reaching rehabilitation are described.

  18. Development of Quasi-3DOF upper limb rehabilitation system using ER brake: PLEMO-P1

    International Nuclear Information System (INIS)

    Kikuchi, T; Fukushima, K; Furusho, J; Ozawa, T

    2009-01-01

    In recent years, many researchers have studied the potential of using robotics technology to assist and quantify the motor functions for neuron-rehabilitation. Some kinds of haptic devices have been developed and evaluated its efficiency with clinical tests, for example, upper limb training for patients with spasticity after stroke. However, almost all the devices are active-type (motor-driven) haptic devices and they basically require high-cost safety system compared to passive-type (brake-based) devices. In this study, we developed a new practical haptic device 'PLEMO-P1'; this system adopted ER brakes as its force generators. In this paper, the mechanism of PLEMO-P1 and its software for a reaching rehabilitation are described.

  19. Development of Quasi-3DOF upper limb rehabilitation system using ER brake: PLEMO-P1

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, T; Fukushima, K; Furusho, J; Ozawa, T [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)], E-mail: kikuchi@mech.eng.osaka-u.ac.jp

    2009-02-01

    In recent years, many researchers have studied the potential of using robotics technology to assist and quantify the motor functions for neuron-rehabilitation. Some kinds of haptic devices have been developed and evaluated its efficiency with clinical tests, for example, upper limb training for patients with spasticity after stroke. However, almost all the devices are active-type (motor-driven) haptic devices and they basically require high-cost safety system compared to passive-type (brake-based) devices. In this study, we developed a new practical haptic device 'PLEMO-P1'; this system adopted ER brakes as its force generators. In this paper, the mechanism of PLEMO-P1 and its software for a reaching rehabilitation are described.

  20. Electron-impact coherence parameters for 41 P 1 excitation of zinc

    Science.gov (United States)

    Piwiński, Mariusz; Kłosowski, Łukasz; Chwirot, Stanisław; Fursa, Dmitry V.; Bray, Igor; Das, Tapasi; Srivastava, Rajesh

    2018-04-01

    We present electron-impact coherence parameters (EICP) for electron-impact excitation of 41 P 1 state of zinc atoms for collision energies 40 eV and 60 eV. The experimental results are presented together with convergent close-coupling and relativistic distorted-wave approximation theoretical predictions. The results are compared and discussed with EICP data for collision energies 80 eV and 100 eV.

  1. Hydrothermal conversion of South African coal fly ash into pure phase Zeolite Na-P1

    CSIR Research Space (South Africa)

    Gitari, MW

    2016-08-01

    Full Text Available and 140°C, respectively. Pure-phase zeolite Na-P1 was obtained with a molar regime of 1 SiO(sub2):0.36 Al(sub2)O(sub3):0.59 NaOH:0.49 H(sub2)O at an aging temperature of 47°C for 48 h. The optimized conditions were applied to two fly ashes from two coa...

  2. Lateral supraorbital approach to ipsilateral PCA-P1 and ICA-PCoA aneurysms.

    Science.gov (United States)

    Goehre, Felix; Jahromi, Behnam Rezai; Elsharkawy, Ahmed; Lehto, Hanna; Shekhtman, Oleg; Andrade-Barazarte, Hugo; Munoz, Francisco; Hijazy, Ferzat; Makhkamov, Makhkam; Hernesniemi, Juha

    2015-01-01

    Aneurysms of the posterior cerebral artery (PCA) are rare and often associated with anterior circulation aneurysms. The lateral supraorbital approach allows for a very fast and safe approach to the ipsilateral lesions Circle of Willis. A technical note on the successful clip occlusion of two aneurysms in the anterior and posterior Circle of Willis via this less invasive approach has not been published before. The objective of this technical note is to describe the simultaneous microsurgical clip occlusion of an ipsilateral PCA-P1 and an internal carotid artery - posterior communicating artery (ICA-PCoA) aneurysm via the lateral supraorbital approach. The authors present a technical report of successful clip occlusions of ipsilateral located PCA-P1 and ICA-PCoA aneurysms. A 59-year-old female patient was diagnosed with a PCA-P1 and an ipsilateral ICA-PCoA aneurysm by computed tomography angiography (CTA) after an ischemic stroke secondary to a contralateral ICA dissection. The patient underwent microsurgical clipping after a lateral supraorbital craniotomy. The intraoperative indocyanine green (ICG) videoangiography and the postoperative CTA showed a complete occlusion of both aneurysms; the parent vessels (ICA and PCA) were patent. The patient presents postoperative no new neurologic deficit. The lateral supraorbital approach is suitable for the simultaneous microsurgical treatment of proximal anterior circulation and ipsilateral proximal PCA aneurysms. Compared to endovascular treatment, direct visual control of brainstem perforators is possible.

  3. Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

    NARCIS (Netherlands)

    Heijden, van der M.W.; Carette, J.E.; Reinhoud, P.J.; Haegi, A.; Bol, J.F.

    2001-01-01

    Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and

  4. Identification of a host cell protein impurity in therapeutic protein, P1.

    Science.gov (United States)

    Ahluwalia, Deepti; Dhillon, Harbhajan; Slaney, Thomas; Song, Hangtian; Boux, Heather; Mehta, Sheetal; Zhang, Lei; Valdez, Anulfo; Krishnamurthy, Girija

    2017-07-15

    Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Particle production in the model with intersept αsub(P)>1

    International Nuclear Information System (INIS)

    Mandzhavidze, I.D.

    1980-01-01

    The behaviour of the particle multiple production cross section σsub(n) is studied in the model with the pomeron intersept αsub(p) > 1. It is shown that the eikonal (or quasieikonal) approximation used to describe σsub(n) can be applied in a limited range; the interaction between the pomerons should be taken into account in the multiplicity asymptotics. An equation is written whose solution allows to obtain σsub(n) in the multiplicity asymptotics. Solving the equation one finds that σsub(n) decreases faster than exp[-an] with n >> anti n

  6. WQGAS - a RSYST module to calculate the P1 scattering nuclei of an ideal, monoatomic gas

    International Nuclear Information System (INIS)

    Keinert, J.; Gulden, W.

    1977-05-01

    Using the RSYST module WQGAS, complete P 1 cross section blocks up to P 3 are provided for reactor calculations. These multigroup data are based on the neutronic nuclear scattering dynamics in terms of interaction with a system of an ideal, monoatomic gas. The group constants may be calculated for any nuclear mass A, any temperature T, and energy structures in the thermal energy region. All data are based on the usual multigroup approximations for this region. When using WQGAS, the validity of these criteria should therefore be checked. (orig.) [de

  7. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.

    OpenAIRE

    Pal, S K; Chattoraj, D K

    1988-01-01

    The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

  8. U P1, an example for advanced techniques applied to high level activity dismantling

    International Nuclear Information System (INIS)

    Michel-Noel, M.; Calixte, O.; Blanchard, S.; Bani, J.; Girones, P.; Moitrier, C.; Terry, G.; Bourdy, R.

    2014-01-01

    The U P1 plant on the CEA Marcoule site was dedicated to the processing of spend fuels from the G1, G2 and G3 plutonium-producing reactors. This plant represents 20.000 m 2 of workshops housing about 1000 hot cells. In 1998, a huge program for the dismantling and cleaning-up of the UP1 plant was launched. CEA has developed new techniques to face the complexity of the dismantling operations. These techniques include immersive virtual reality, laser cutting, a specific manipulator arm called MAESTRO and remote handling. (A.C.)

  9. Unassisted Water Splitting Using a GaSbxP(1-x) Photoanode

    Energy Technology Data Exchange (ETDEWEB)

    Deutsch, Todd G [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Young, James [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Martinez-Garcia, Alejandro [University of Louisville; Russell, Barry B. [University of Louisville; Paxton, William [University of Louisville; Ravipati, Srikanth [University of Louisville; Calero-Barney, Sonia [University of Louisville; Menon, Madhu [University of Kentucky; Richter, Ernst [Daimler AG, RD/EKB; Sunkara, Mahendra K. [University of Louisville

    2018-02-21

    Here, unbiased water splitting with 2% solar-to-hydrogen efficiency under AM 1.5 G illumination using new materials based on GaSb0.03P0.97 alloy is reported. Freestanding GaSbxP1-x is grown using halide vapor phase epitaxy. The native conductivity type of the alloy is modified by silicon doping, resulting in an open-circuit potential (OCP) of 750 mV, photocurrents of 7 mA cm-2 at 10 sun illumination, and corrosion resistance in an aqueous acidic environment. Alloying GaP with Sb at 3 at% improves the absorption of high-energy photons above 2.68 eV compared to pure GaP material. Electrochemical Impedance Spectroscopy and illuminated OCP measurements show that the conduction band of GaSbxP1-x is at -0.55 V versus RHE irrespective of the Sb concentration, while photocurrent spectroscopy indicates that only radiation with photon energies greater than 2.68 eV generate mobile and extractable charges, thus suggesting that the higher-laying conduction bands in the ..gamma..1 valley of the alloys are responsible for exciton generation.

  10. FURTHER CONSTRAINTS ON THE OPTICAL TRANSMISSION SPECTRUM OF HAT-P-1b

    Energy Technology Data Exchange (ETDEWEB)

    Montalto, M.; Santos, N. C.; Martins, J. H. C.; Figueira, P.; Alonso, R. [Instituto de Astrofísica e Ciências do Espaço, Universidade do Porto, CAUP, Rua das Estrelas, PT4150-762 Porto (Portugal); Iro, N. [Theoretical Meteorology Group Klimacampus, University of Hamburg Grindelberg 5, D-20144, Hamburg (Germany); Desidera, S., E-mail: Marco.Montalto@astro.up.pt [INAF—Osservatorio Astronomico di Padova, Vicolo dellOsservatorio 5, Padova, I-35122 (Italy)

    2015-09-20

    We report on novel observations of HAT-P-1 aimed at constraining the optical transmission spectrum of the atmosphere of its transiting hot-Jupiter exoplanet. Ground-based differential spectrophotometry was performed over two transit windows using the DOLORES spectrograph at the Telescopio Nazionale Galileo. Our measurements imply an average planet to star radius ratio equal to R{sub p}/R{sub *} = (0.1159 ± 0.0005). This result is consistent with the value obtained from recent near-infrared measurements of this object, but differs from previously reported optical measurements, being lower by around 4.4 exoplanet scale heights. Analyzing the data over five different spectral bins of ∼600 Å wide, we observed a single peaked spectrum (3.7 σ level) with a blue cutoff corresponding to the blue edge of the broad absorption wing of sodium and an increased absorption in the region in-between 6180 and 7400 Å. We also infer that the width of the broad absorption wings due to alkali metals is likely narrower than the one implied by solar abundance clear atmospheric models. We interpret the result as evidence that HAT-P-1b has a partially clear atmosphere at optical wavelengths with a more modest contribution from an optical absorber than previously reported.

  11. Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

    DEFF Research Database (Denmark)

    Garcia-Pino, Abel; Christensen-Dalsgaard, Mikkel; Wyns, Lode

    2008-01-01

    Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation....... This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible...... evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin...

  12. Neutron wave reflexions in interface media with transport equation P1 approximation

    International Nuclear Information System (INIS)

    Oliveira Vellozo, S. de.

    1977-01-01

    The propagation of neutron waves in non multiplying media is investigated employing the Telegrapher's equation obtained from the P 1 approximation of the time, space and energy dependent Boltzmann equation. Solution of the problem of propagation of sinusoidally modulated source incident on one face of the medium is obtained by analysing the Fourier component of a pulsed source introduced, for the corresponding frequency. The amplitude and the phase of the flux are computed as a function of frequency in media consisting of one, two and three regions in order to study the effects of reflection at the interfaces. The results are compared with those from the Diffusion approximation obtained by neglecting the term involving the second order time derivative. (author)

  13. Comment on the search for the charmonium 1P1 and its theoretical interpretation

    Science.gov (United States)

    Porter, F. C.

    1984-10-01

    We comment on a recent paper which discusses the theoretical implications of the Crystal Ball search for the 1P1 state of charmonium. On behalf of the Crystal Ball collaboration: California Institute of Technology - C. Edwards, R. Partridge, C. Peck, F. Porter; Harvard University - D. Antreasyan, Y.F. Gu, K. Strauch, K. Wacker, A. Weinstein; Princeton University - D. Aschman, M. Cavalli-Sforza, D. Coyne, C. Newman-Holmes, H. Sadrozinski; Stanford Linear Accelerator Center - E. Bloom, F. Bulos, G. Godfrey, C. Kiesling, W. Lockman, M. Oreglia; Stanford University - D. Gelphman, R. Hofstadter, R. Horisberger, I. Kirkbride, H. Kolanoski, K. Königsmann, R. Lee, A. Osterheld, B. Pollock and J. Tompkins.

  14. Evidence for the horizontal transfer of an integrase gene from a fusellovirus to a pRN-like plasmid within a single strain of Sulfolobus and the implications for plasmid survival

    DEFF Research Database (Denmark)

    Peng, Xu

    2008-01-01

    A fusellovirus SSV4 and a pRN-like plasmid pXZ1 were co-isolated from a single strain of Sulfolobus. In contrast to the previously characterized virus-plasmid hybrids pSSVx and pSSVi, which can coexist intracellulary with a fusellovirus, pXZ1 is not packaged into viral particles and shows no viral...... infectivity. The virus and plasmid carry genomes of 15 135 and 6970 bp, respectively. For SSV4, 33 predicted ORFs are compactly organized with a strong preference for UGA stop codons, three-quarters of which overlap with either the Shine-Dalgarno motif or the start codon of the following gene. pXZ1 carries...... seven ORFs, three of which encode an atypical RepA, a PlrA and a CopG protein. A fourth ORF exhibits a high nucleotide sequence identity to the SSV4 integrase gene, which suggests that it has been transferred to the plasmid from SSV4. A single point mutation within an otherwise identical 500 bp region...

  15. Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom.

    Science.gov (United States)

    Fernandes, I; Assumpção, G G; Silveira, C R F; Faquim-Mauro, E L; Tanjoni, I; Carmona, A K; Alves, M F M; Takehara, H A; Rucavado, A; Ramos, O H P; Moura-da-Silva, A M; Gutiérrez, J M

    2010-11-01

    BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation. (c) 2010 Elsevier Ltd. All rights reserved.

  16. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    NARCIS (Netherlands)

    N.M. van Maarseveen (Noortje); D. Andersson (Dan); M. Lepšík (Martin); A. Fun (Axel); P.J. Schipper (Pauline); D. de Jong (Dorien); C.A.B. Boucher (Charles); M. Nijhuis (Monique)

    2012-01-01

    textabstractBackground: Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes

  17. Understanding the Biocompatibility of Sintered Calcium Phosphate with Ratio of [Ca]/[P] = 1.50

    Directory of Open Access Journals (Sweden)

    Feng-Lin Yen

    2012-01-01

    Full Text Available Biocompatibility of sintered calcium phosphate pellets with [Ca]/[P] = 1.50 was determined in this study. Calcium pyrophosphate (CPP phase formed on the sintered pellets immersed in a normal saline solution for 14 d at 37∘C. The intensities of hydroxyapatite (HA reflections in the X-ray diffraction (XRD patterns of the pellets were retrieved to as-sintered state. The pellet surface morphology shows that CPP crystallites were clearly present and make an amorphous calcium phosphate (ACP to discriminate against become to the area of slice join together. In addition, the intensities of the CPP reflections in the XRD patterns were the highest when the pellets were immersed for 28 d. When the CPP powders were extracted from the pellets after immersion in the solution for 14 d, the viability of 3T3 cells remained above 90% for culture times from 1 to 4 d. The pellet surface morphology observed using optical microscopy showed that the cells did not adhere to the bottom of the sintered pellets when cultured for 4 d; however, some CPP phase precipitates were formed, as confirmed by XRD. In consequence, the results suggest that the sintered HA powders are good materials for use in biomedical applications because of their good biocompatibility.

  18. Variational P1 approximations of general-geometry multigroup transport problems

    International Nuclear Information System (INIS)

    Rulko, R.P.; Tomasevic, D.; Larsen, E.W.

    1995-01-01

    A variational approximation is developed for general-geometry multigroup transport problems with arbitrary anisotropic scattering. The variational principle is based on a functional that approximates a reaction rate in a subdomain of the system. In principle, approximations that result from this functional ''optimally'' determine such reaction rates. The functional contains an arbitrary parameter α and requires the approximate solutions of a forward and an adjoint transport problem. If the basis functions for the forward and adjoint solutions are chosen to be linear functions of the angular variable Ω, the functional yields the familiar multigroup P 1 equations for all values of α. However, the boundary conditions that result from the functional depend on α. In particular, for problems with vacuum boundaries, one obtains the conventional mixed boundary condition, but with an extrapolation distance that depends continuously on α. The choice α = 0 yields a generalization of boundary conditions derived earlier by Federighi and Pomraning for a more limited class of problems. The choice α = 1 yields a generalization of boundary conditions derived previously by Davis for monoenergetic problems. Other boundary conditions are obtained by choosing different values of α. The authors discuss this indeterminancy of α in conjunction with numerical experiments

  19. HLA-DM focuses on conformational flexibility around P1 pocket to catalyze peptide exchange

    Directory of Open Access Journals (Sweden)

    Liusong eYin

    2013-10-01

    Full Text Available Peptides presented by major histocompatibility complex class II (MHCII molecules to CD4+ T cells play a central role in the initiation of adaptive immunity. This antigen presentation process is characterized by the proteolytic cleavage of foreign and self proteins, and loading of the resultant peptides onto MHCII molecules. Loading and exchange of antigenic peptides is catalyzed by a non-classical MHCII molecule, HLA-DM. The impact of HLA-DM on epitope selection has been appreciated for a long time. However, the molecular mechanism by which HLA-DM mediates peptide exchange remains elusive. Here, we review recent efforts in elucidating how HLA-DM works, highlighted by two recently solved co-structures of HLA-DM bound to HLA-DO (a natural inhibitor of HLA-DM, or to HLA-DR1 (a common MHCII. In light of these efforts, a model for HLA-DM action in which HLA-DM utilizes conformational flexibility around the P1 pocket of the MHCII-peptide complex to catalyze peptide exchange is proposed.

  20. Population pharmacokinetics and pharmacodynamics of ponesimod, a selective S1P1 receptor modulator.

    Science.gov (United States)

    Krause, Andreas; Brossard, Patrick; D'Ambrosio, Daniele; Dingemanse, Jasper

    2014-06-01

    Ponesimod (ACT-128800), a reversible, orally active, selective S1P1 receptor modulator, prevents the egress of lymphocytes from the lymph node into the systemic circulation. It is currently in clinical development for the treatment of relapsing multiple sclerosis. Modulation of circulating lymphocytes serves as biomarker of efficacy and safety, such that the quantitative characterization of the pharmacokinetic/pharmacodynamic (PK/PD) relationship guides the clinical development of the compound. The availability of a variety of doses, dosing regimens, and treatment durations permitted estimation of the pharmacokinetics characterized by an absorption lag time followed by a sequential zero/first-order absorption and two compartments with first-order elimination. The PD are modeled as an indirect-effect model with rates of appearance and disappearance of lymphocytes in blood with a circadian rhythm and a drug effect on the rate of appearance. The model suggests a circadian variation of 9% and a maximum inhibition of 86% of total lymphocyte count with high doses at steady state. It was instrumental for the selection of doses for subsequent studies that confirmed the effect plateau in total lymphocyte count at approximately 0.5 × 10(9) counts/L.

  1. Solutions of system of P1 equations without use of auxiliary differential equations coupled; Solucoes do sistema de equacoes P1 sem o uso de equacoes diferenciais auxiliares acopladas

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos [Universidade Federal, Rio de Janeiro, RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia. Programa de Engenharia Nuclear

    2000-07-01

    The system of P1 equations is composed by two equations coupled itself one for the neutron flux and other for the current. Usually this system is solved by definitions of two integrals parameters, which are named slowing down densities of the flux and the current. Hence, the system P1 can be change from integral to only two differential equations. However, there are two new differentials equations that may be solved with the initial system. The present work analyzes this procedure and studies a method, which solve the P1 equations directly, without definitions of slowing down densities. (author)

  2. FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

    Science.gov (United States)

    Musdal, Yaman; Hegazy, Usama M; Aksoy, Yasemin; Mannervik, Bengt

    2013-09-05

    Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Bactericide, Immunomodulating, and Wound Healing Properties of Transgenic Kalanchoe pinnata Synergize with Antimicrobial Peptide Cecropin P1 In Vivo

    Directory of Open Access Journals (Sweden)

    A. A. Lebedeva

    2017-01-01

    Full Text Available Procedure of manufacturing K. pinnata water extracts containing cecropin P1 (CecP1 from the formerly described transgenic plants is established. It included incubation of leaves at +4°C for 7 days, mechanical homogenization of leaves using water as extraction solvent, and heating at +70°C for inactivating plant enzymes. Yield of CecP1 (after heating and sterilizing filtration was 0.3% of total protein in the extract. The water extract of K. pinnata + CecP1 exhibits favorable effect on healing of wounds infected with S. aureus (equal to Cefazolin and with a combination of S. aureus with P. aeruginosa (better than Cefazolin. Wild-type K. pinnata extract exhibited evident microbicide activity against S. aureus with P. aeruginosa but it was substantially strengthened in K. pinnata + CecP1 extract. K. pinnata extracts (both wild-type and transgenic did not exhibit general toxicity and accelerated wound recovery. Due to immunomodulating activity, wild-type K. pinnata extract accelerated granulation of the wound bed and marginal epithelialization even better than K. pinnata + CecP1 extract. Immunomodulating and microbicide activity of K. pinnata synergizes with microbicide activity of CecP1 accelerating elimination of bacteria.

  4. Immunomodulating and Revascularizing Activity of Kalanchoe pinnata Synergize with Fungicide Activity of Biogenic Peptide Cecropin P1

    Directory of Open Access Journals (Sweden)

    N. S. Zakharchenko

    2017-01-01

    Full Text Available Previously transgenic Kalanchoe pinnata plants producing an antimicrobial peptide cecropin P1 (CecP1 have been reported. Now we report biological testing K. pinnata extracts containing CecP1 as a candidate drug for treatment of wounds infected with Candida albicans. The drug constitutes the whole juice from K. pinnata leaves (not ethanol extract sterilized with nanofiltration. A microbicide activity of CecP1 against an animal fungal pathogen in vivo was demonstrated for the first time. However, a favorable therapeutic effect of the transgenic K. pinnata extract was attributed to a synergism between the fungicide activity of CecP1 and wound healing (antiscar, revascularizing, and immunomodulating effect of natural biologically active components of K. pinnata. A commercial fungicide preparation clotrimazole eliminated C. albicans cells within infected wounds in rats with efficiency comparable to CecP1-enriched K. pinnata extract. But in contrast to K. pinnata extract, clotrimazole did not exhibit neither wound healing activity nor remodeling of the scar matrix. Taken together, our results allow assumption that CecP1-enriched K. pinnata extracts should be considered as a candidate drug for treatment of dermatomycoses, wounds infected with fungi, and bedsores.

  5. Bactericide, Immunomodulating, and Wound Healing Properties of Transgenic Kalanchoe pinnata Synergize with Antimicrobial Peptide Cecropin P1 In Vivo.

    Science.gov (United States)

    Lebedeva, A A; Zakharchenko, N S; Trubnikova, E V; Medvedeva, O A; Kuznetsova, T V; Masgutova, G A; Zylkova, M V; Buryanov, Y I; Belous, A S

    2017-01-01

    Procedure of manufacturing K. pinnata water extracts containing cecropin P1 (CecP1) from the formerly described transgenic plants is established. It included incubation of leaves at +4°C for 7 days, mechanical homogenization of leaves using water as extraction solvent, and heating at +70°C for inactivating plant enzymes. Yield of CecP1 (after heating and sterilizing filtration) was 0.3% of total protein in the extract. The water extract of K. pinnata + CecP1 exhibits favorable effect on healing of wounds infected with S. aureus (equal to Cefazolin) and with a combination of S. aureus with P. aeruginosa (better than Cefazolin). Wild-type K. pinnata extract exhibited evident microbicide activity against S. aureus with P. aeruginosa but it was substantially strengthened in K. pinnata + CecP1 extract. K. pinnata extracts (both wild-type and transgenic) did not exhibit general toxicity and accelerated wound recovery. Due to immunomodulating activity, wild-type K. pinnata extract accelerated granulation of the wound bed and marginal epithelialization even better than K. pinnata + CecP1 extract. Immunomodulating and microbicide activity of K. pinnata synergizes with microbicide activity of CecP1 accelerating elimination of bacteria.

  6. Immunomodulating and Revascularizing Activity of Kalanchoe pinnata Synergize with Fungicide Activity of Biogenic Peptide Cecropin P1.

    Science.gov (United States)

    Zakharchenko, N S; Belous, A S; Biryukova, Y K; Medvedeva, O A; Belyakova, A V; Masgutova, G A; Trubnikova, E V; Buryanov, Y I; Lebedeva, A A

    2017-01-01

    Previously transgenic Kalanchoe pinnata plants producing an antimicrobial peptide cecropin P1 (CecP1) have been reported. Now we report biological testing K. pinnata extracts containing CecP1 as a candidate drug for treatment of wounds infected with Candida albicans. The drug constitutes the whole juice from K. pinnata leaves (not ethanol extract) sterilized with nanofiltration. A microbicide activity of CecP1 against an animal fungal pathogen in vivo was demonstrated for the first time. However, a favorable therapeutic effect of the transgenic K. pinnata extract was attributed to a synergism between the fungicide activity of CecP1 and wound healing (antiscar), revascularizing, and immunomodulating effect of natural biologically active components of K. pinnata . A commercial fungicide preparation clotrimazole eliminated C. albicans cells within infected wounds in rats with efficiency comparable to CecP1-enriched K. pinnata extract. But in contrast to K. pinnata extract, clotrimazole did not exhibit neither wound healing activity nor remodeling of the scar matrix. Taken together, our results allow assumption that CecP1-enriched K. pinnata extracts should be considered as a candidate drug for treatment of dermatomycoses, wounds infected with fungi, and bedsores.

  7. LyP-1-conjugated Fe3O4nanoparticles suppress tumor growth by magnetic induction hyperthermia.

    Science.gov (United States)

    Teo, Peishan; Wang, Xiaowen; Zhang, Jieying; Zhang, Han; Yang, Xin; Huang, Yun; Tang, Jintian

    2018-02-01

    To find a promising drug carrier to suppress tumor using magnetic induction hyperthermia (MIH) and targeted therapy, two superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylene glycol (PEG) and LyP-1, respectively, were prepared and compared. The particle size ranges of PEG-SPIONs and LyP-1-SPIONs were 10-15 nm, and 15-20 nm, respectively. In FTIR spectra, PEG-SPIONs and LyP-1-SPIONs had strong peaks between 575 and 1630 cm -1 . Specifically, the PEG-SPIONs mainly has peaks in 581 and 1630 cm -1 . The LyP-1-SPIONs mainly had peaks in 575, 1050 and 1625 cm -1 . The contents of Fe 3 O 4 in the PEG-SPIONs and LyP-1-SPIONs were about 94.24 and 89.26%, respectively. The iron contents in the MCF-7 and CT-26 cells were 33.1 ± 1.8 and 27.9 ± 0.95 pg, respectively, after co-incubation with LyP-1-SPIONs for 8 h. The LyP-1-SPIONs accumulated in the nucleus of MCF-7 cells while PEG-SPIONs in cytoplasma. In vitro, after 30 days we can found the tumor almost stopped to grow in Group LyP-1-SPIONs. LyP-1-SPIONs are promising in treating cancer as they accumulated in the nucleus of MCF-7 cells which expressed p32 and almost stopped tumor growth by combined MIH and targeted therapy.

  8. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska

    2013-03-01

    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  9. Expression of flavonoid 3’-hydroxylase is controlled by P1, the regulator of 3-deoxyflavonoid biosynthesis in maize

    Directory of Open Access Journals (Sweden)

    Sharma Mandeep

    2012-11-01

    Full Text Available Abstract Background The maize (Zea mays red aleurone1 (pr1 encodes a CYP450-dependent flavonoid 3’-hydroxylase (ZmF3’H1 required for the biosynthesis of purple and red anthocyanin pigments. We previously showed that Zmf3’h1 is regulated by C1 (Colorless1 and R1 (Red1 transcription factors. The current study demonstrates that, in addition to its role in anthocyanin biosynthesis, the Zmf3’h1 gene also participates in the biosynthesis of 3-deoxyflavonoids and phlobaphenes that accumulate in maize pericarps, cob glumes, and silks. Biosynthesis of 3-deoxyflavonoids is regulated by P1 (Pericarp color1 and is independent from the action of C1 and R1 transcription factors. Results In maize, apiforol and luteoforol are the precursors of condensed phlobaphenes. Maize lines with functional alleles of pr1 and p1 (Pr1;P1 accumulate luteoforol, while null pr1 lines with a functional or non-functional p1 allele (pr1;P1 or pr1;p1 accumulate apiforol. Apiforol lacks a hydroxyl group at the 3’-position of the flavylium B-ring, while luteoforol has this hydroxyl group. Our biochemical analysis of accumulated compounds in different pr1 genotypes showed that the pr1 encoded ZmF3’H1 has a role in the conversion of mono-hydroxylated to bi-hydroxylated compounds in the B-ring. Steady state RNA analyses demonstrated that Zmf3’h1 mRNA accumulation requires a functional p1 allele. Using a combination of EMSA and ChIP experiments, we established that the Zmf3’h1 gene is a direct target of P1. Highlighting the significance of the Zmf3’h1 gene for resistance against biotic stress, we also show here that the p1 controlled 3-deoxyanthocyanidin and C-glycosyl flavone (maysin defence compounds accumulate at significantly higher levels in Pr1 silks as compared to pr1 silks. By virtue of increased maysin synthesis in Pr1 plants, corn ear worm larvae fed on Pr1; P1 silks showed slower growth as compared to pr1; P1 silks. Conclusions Our results show that the Zmf3

  10. Extremophiles Survival to Simulated Space Conditions: An Astrobiology Model Study

    OpenAIRE

    Mastascusa, V.; Romano, I.; Di Donato, P.; Poli, A.; Della Corte, V.; Rotundi, A.; Bussoletti, E.; Quarto, M.; Pugliese, M.; Nicolaus, B.

    2015-01-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars? conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temp...

  11. The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

    DEFF Research Database (Denmark)

    Meno, Kåre; Thorsted, Peter B; Ipsen, Henrik

    2005-01-01

    . Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.......Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant...... molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft...

  12. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    Directory of Open Access Journals (Sweden)

    van Maarseveen Noortje M

    2012-04-01

    Full Text Available Abstract Background Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI resistance and also compensate for defects in viral replicative capacity (RC due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V. Results Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one. The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T could (partially be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant. Conclusions The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate.

  13. On the mechanism of loading the PCNA sliding clamp by RFC.

    Science.gov (United States)

    Dionne, Isabelle; Brown, Nicola J; Woodgate, Roger; Bell, Stephen D

    2008-04-01

    Sliding clamps play central roles in a broad range of DNA replication and repair processes. The clamps form circular molecules that must be opened and resealed around DNA by the clamp loader complex to fulfil their function. While most eukaryotes and many archea possess a homo-trimeric PCNA, the PCNA of Sulfolobus solfataricus is a heterotrimer. Here, we exploit the asymmetry of S. solfataricus PCNA to create a series of circularly permuted PCNA subunit fusions, thereby covalently closing defined interfaces within the heterotrimer. Using these concatamers, we investigate the requirements for loading the clamp onto DNA and reveal that a single defined interface within the heterotrimer is opened during the loading process. Subunit-specific interactions between S. solfataricus RFC clamp loader and PCNA permit us to superimpose our data upon the structure of yeast RFC-PCNA complex, thereby presenting a general model for PCNA loading by RFC in archaea and eukaryotes.

  14. Direct regulatory immune activity of lactic acid bacteria on Der p 1-pulsed dendritic cells from allergic patients.

    Science.gov (United States)

    Pochard, Pierre; Hammad, Hamida; Ratajczak, Céline; Charbonnier-Hatzfeld, Anne-Sophie; Just, Nicolas; Tonnel, André-Bernard; Pestel, Joël

    2005-07-01

    Lactic acid bacteria (LAB) are suggested to play a regulatory role in the development of allergic reactions. However, their potential effects on dendritic cells (DCs) directing the immune polarization remain unclear. The immunologic effect of Lactobacillus plantarum NCIMB 8826 (LAB1) on monocyte-derived dendritic cells (MD-DCs) from patients allergic to house dust mite was evaluated. MD-DCs were stimulated for 24 hours with the related allergen Der p 1 in the presence or absence of LAB1. Cell-surface markers were assessed by means of FACS analysis, and the key polarizing cytokines IL-12 and IL-10 were quantified. The subsequent regulatory effect of pulsed MD-DCs on naive or memory T cells was evaluated by determining the T-cell cytokine profile. LAB1 induced the maturation of MD-DCs, even if pulsed with Der p 1. Interestingly, after incubation with LAB1 and Der p 1, MD-DCs produced higher amounts of IL-12 than Der p 1-pulsed DCs. Indeed, the T H 2 cytokine (IL-4 and IL-5) production observed when naive or memory autologous T cells were cocultured with Der p 1-pulsed MD-DCs was highly reduced in the presence of LAB1. Finally, in contrast to naive or memory T cells exposed once to Der p 1-pulsed DCs, T cells stimulated by MD-DCs pulsed with Der p 1 and LAB1 failed to produce T H 2 cytokines in response to a new stimulation with Der p 1-pulsed DCs. Thus in the presence of LAB1, MD-DCs from allergic patients tend to reorientate the T-cell response toward a beneficial T H 1 profile.

  15. Closed expressions for $\\int_{0}^{1} t^{-1} log^{n-1}t log^{p}(1 - t) dt$

    CERN Document Server

    Kölbig, Kurt Siegfried

    1982-01-01

    Closed expressions for the integral integral /sub 0//sup 1/ t/sup -1/ log/sup n-1/t log/sup p/(1-t)dt, whose general form is given elsewhere, are listed for n=1(1)9, p=1(1)9. A formula is derived which allows an easy evaluation of these expressions by formula manipulation on a computer. The majority of the above expressions are given in a microfiche supplement to the paper.

  16. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Amr [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619 (Egypt); Hutchens, Heather M. [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Howard Berg, R. [Integrated Microscopy Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132 (United States); Sue Loesch-Fries, L., E-mail: loeschfr@purdue.edu [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States)

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  17. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    International Nuclear Information System (INIS)

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-01-01

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  18. Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

    Science.gov (United States)

    Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

    2015-01-01

    P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

  19. Comparative analysis between P1 and B1 equations for neutron moderation; Analise comparativa entre os metodos de obtencao e das solucoes das equacoes P1 e B1 para moderacao de neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Aquilino Senra; Silva, Fernando Carvalho da; Cardoso, Carlos Eduardo Santos [Universidade Federal, Rio de Janeiro, RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia. Programa de Engenharia Nuclear

    2000-07-01

    In order to calculate the neutron flux in nuclear reactors, B1 or P1 equations are solved by numerical methods for several groups of energy. The neutron fluxes obtained from the solutions of the B1 and P1 equations are similar when they are applied to large nuclear power reactors. However, an important difference between the two fluxes is that the system of P1 equations uses one more approximation than the B1 system and then, its flux is less precise. The present work shows the relations between both equations and analyzes for what conditions the two equations systems are equivalent. Furthermore, this equations are numerically solved in 54 groups of energy for a quadrangular arrange. (author)

  20. The deexcitation of the Ar (3P2, 3p1 and 1P1) states as measured by absorption both in pure argon and in the presence of additives

    International Nuclear Information System (INIS)

    Dutuit, Odile

    1974-01-01

    The de-excitation of the 3 P 2 , 3 p 1 and 1 P 1 states of argon was studied in pure argon between 10 and 200 torr and in Ar + CO and Ar + H 2 mixtures. These states are populated after excitation of the gas by a short (20 ns) pulse of 500 keV electrons (FEBETRON). Under our experimental conditions, the relation between the measured optical density of the lines studied and the concentration of absorbing species was found to be: DO = log I 0 /I ∝ (lC) n with n = 0,4. The three body rate constants k 2 were measured for the two resonant states 3 p 1 (k 2 = (1,65 ± 0,3) x 10 -32 cm 6 s -1 ) and 1 P 1 (k 2 = (1,0 ± 0,2) x 10 -32 cm 6 s -1 ); they had not been considered in previous low pressure studies. For the metastable state 3 P 2 , the measured value of k 2 ((1,6 ± 0,3) x 10 -32 cm 6 s -1 ) is in good agreement with those found in the literature. However, our two body rate constant k 1 is about ten times higher than that found in measurements at low pressure. This difference could be due to a collision-induced emission process at high pressure. The rate constants for the quenching by CO and H 2 were measured for the metastable state 3 P 2 (1,85 and 10,5 x 10 -11 cm 3 s -1 ) and for the resonant states 3 P 1 (4,5 and 20 x 10 -11 cm 3 s -1 ) and 1 P 1 (8,5 and 33 X 10 -11 cm 3 s -1 ). Comparison of the de-excitation cross sections of resonant and metastable states should lead to a better understanding of energy transfer processes from these latter. (author) [fr

  1. Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

    Directory of Open Access Journals (Sweden)

    S.F. Avram

    2013-01-01

    Full Text Available The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

  2. Photodynamics of blue-light-regulated phosphodiesterase BlrP1 protein from Klebsiella pneumoniae and its photoreceptor BLUF domain

    Science.gov (United States)

    Tyagi, A.; Penzkofer, A.; Griese, J.; Schlichting, I.; Kirienko, Natalia V.; Gomelsky, Mark

    2008-12-01

    The BlrP1 protein from the enteric bacterium Klebsiella pneumoniae consists of a BLUF and an EAL domain and may activate c-di-GMP phosphodiesterase by blue-light. The full-length protein, BlrP1, and its BLUF domain, BlrP1_BLUF, are characterized by optical absorption and emission spectroscopy. The cofactor FAD in its oxidized redox state (FAD ox) is brought from the dark-adapted receptor state to the 10-nm red-shifted putative signalling state by violet light exposure. The recovery to the receptor state occurs with a time constant of about 1 min. The quantum yield of signalling state formation is about 0.17 for BlrP1_BLUF and about 0.08 for BlrP1. The fluorescence efficiency of the FAD ox cofactor is small due to photo-induced reductive electron transfer. Prolonged light exposure converts FAD ox in the signalling state to the fully reduced hydroquinone form FAD redH - and causes low-efficient chromophore release with subsequent photo-degradation. The photo-cycle and photo-reduction dynamics in the receptor state and in the signalling state are discussed.

  3. Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution.

    Science.gov (United States)

    Knox, R B; Suphioglu, C; Taylor, P; Desai, R; Watson, H C; Peng, J L; Bursill, L A

    1997-03-01

    Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 microns in diameter) to smaller size fractions Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron-microscopic imaging technique. DECP are visualized as small carbon spheres, each 30-60 nm in diameter, forming fractal aggregates about 1-2 microns in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p 1, binds to one defined class of fine particles, DECP. DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p 1 under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

  4. First reported cases of human adenovirus serotype 14p1 infection, Ireland, October 2009 to July 2010.

    LENUS (Irish Health Repository)

    O'Flanagan, D

    2011-02-01

    We report the first nine confirmed cases of human adenovirus 14p1 infection (HAdV-14p1), identified at different locations in Ireland between October 2009 and July 2010. These were the first notifications in Ireland and all were sporadic cases. Following these notifications, the Health Protection Surveillance Centre set up an enhanced surveillance system for HAdV-14p1 infection. Seven cases were male and five were aged less than one year. Three patients died, giving a case fatality rate of 33%. It should be noted that cases presented here were diagnosed on presentation to hospital and may represent the severe end of the spectrum of HAdV 14 disease in Ireland.

  5. Evaluation of the Eological Management and Enhancement Alernative for Remediation of the K1007-P1 Pond

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, M.J.

    2005-10-31

    An evaluation of the human and ecological risks associated with the P1 Pond and surrounding environs was conducted as part of the ETTP Site-Wide Remedial Investigation. The RI provides the basis for the focus on PCBs as the most important unacceptable risk to human and ecological health in the pond. Other P1 contaminants, media, or pathways of risk to receptors are identified in the RI, but are not addressed as a major risk reduction goal for the ETTP Site-Wide Feasibility Study. Therefore, the goal of the Ecological Management alternative is to reduce unacceptable risks associated with PCBs in fish. Many of the actions proposed for this alternative, however, are likely to reduce risks associated with other contaminants and their pathways. The high PCB concentrations in fish from the P1 Pond are most certainly due in part to the current ecological condition of the pond that maximizes PCB biomagnification. This basic assumption and the factors contributing to it were evaluated by conducting an intensive field study of the P1 Pond in the summer of 2004 (for a thorough presentation of current P1 Pond biological conditions, see Peterson et al. 2005). Major hypotheses regarding the P1 Pond's current fish community, PCB fate and transport processes, pond vegetation, and limnological conditions that contribute to the high PCB levels in fish were validated by the study (Appendix A), The results of the 2004 ecological assessment, in concert with long-term datasets obtained as part of the ETTP Biological Monitoring and Abatement Program (BMAP) and recent abiotic sampling for the RI, provide the basis for the assessment of current conditions.

  6. Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors.

    Science.gov (United States)

    Shreder, Kevin R; Wong, Melissa S; Corral, Sergio; Yu, Zhizhou; Winn, David T; Wu, Min; Hu, Yi; Nomanbhoy, Tyzoon; Alemayehu, Senaiet; Fuller, Stacy R; Rosenblum, Jonathan S; Kozarich, John W

    2005-10-01

    Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.

  7. The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

    DEFF Research Database (Denmark)

    Meno, Kåre; Thorsted, Peter B; Ipsen, Henrik

    2005-01-01

    Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant....... Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1....

  8. [The obtainment and characteristics of Kalanchoe pinnata L. plants expressing the artificial gene of the cecropin P1 antimicrobial peptide].

    Science.gov (United States)

    Zakharchenko, N S; Rukavtsova, E B; Shevchuk, T V; Furs, O V; Pigoleva, S V; Lebedeva, A A; Chulina, I A; Baidakova, L K; Bur'yanov, Ya I

    2016-01-01

    Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.

  9. Immune response to Mycoplasma pneumoniae P1 and P116 in patients with atypical pneumonia analyzed by ELISA

    Directory of Open Access Journals (Sweden)

    Birkelund Svend

    2004-02-01

    Full Text Available Abstract Background Serology is often used for the diagnosis of Mycoplasma pneumoniae. It is important to identify specific antigens that can distinguish between the presence or absence of antibodies against M. pneumoniae. The two proteins, P116 and P1, are found to be immunogenic. By using these in ELISA it is possible to identify an immune response against M. pneumoniae in serum samples. Results A recombinant protein derived from the P116 protein and one from the P1 protein were used in two ELISA tests, rP116-ELISA and rP1-ELISA. Human serum samples from patients with atypical pneumonia were tested and compared to the results of the complement fixation test. There was a good agreement between the two tests but the rP1-ELISA showed the best discrimination between positive and negative samples. Conclusion Two ELISA tests based on recombinant proteins have been analysed and compared to the complement fixation test results. The two ELISA tests were found suitable for use in serodiagnostics of M. pneumoniae infections. The use of specific antigens eliminates the risk of cross reaction to an immune response against other bacteria.

  10. Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1 epsilon subgroup

    Czech Academy of Sciences Publication Activity Database

    Wolters, B.; Kyselková, Martina; Krögerrecklenfort, E.; Kreuzig, R.; Smalla, K.

    2015-01-01

    Roč. 5, January (2015), Article 765 ISSN 1664-302X R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : IncP-1 epsilon plasmid * class 1 integrons * biogas plant digestate * antibiotic resistance * exogenous plasmid isolation Subject RIV: EE - Microbiology, Virology Impact factor: 4.165, year: 2015

  11. Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup

    Directory of Open Access Journals (Sweden)

    Birgit eWolters

    2015-01-01

    Full Text Available Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs. These genes are often located on mobile genetic elements. In biogas plants (BGPs, organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10-5 to 10-7. Transconjugants (n = 101 were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tetA, sul1, qacE∆1, intI1 and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical for this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer.

  12. Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

    Directory of Open Access Journals (Sweden)

    Jing-Jing Chen

    2017-12-01

    Full Text Available The glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation and LXYL-P1–2-EP2 (EP2, T368E mutation, were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

  13. Characterization of a broad host-spectrum virulent Salmonella bacteriophage fmb-p1 and its application on duck meat.

    Science.gov (United States)

    Wang, Changbao; Chen, Qiming; Zhang, Chong; Yang, Jie; Lu, Zhaoxin; Lu, Fengxia; Bie, Xiaomei

    2017-05-15

    The aim of this study was to find a virulent bacteriophage for the biocontrol of Salmonella in duck meat. A broad host-spectrum virulent phage, fmb-p1, was isolated and purified from an duck farm, and its host range was determined to include S. Typhimurium, S. Enteritidis, S. Saintpaul, S. Agona, S. Miami, S. Anatum, S. Heidelberg and S. Paratyphi-C. Electron microscopy and genome sequencing showed that fmb-p1 belongs to the family Siphoviridae. The genome of fmb-p1 is composed of a 43,327-bp double-stranded DNA molecule with 60 open reading frames and a total G+C content of 46.09%. There are no deleterious sequences or genes encoding known harmful products in the phage fmb-p1 genome. Phage fmb-p1 was stable under different temperature (40-75°C), pH (4-10) and NaCl solutions (1-11%). The phage treatment (9.9×10 9 PFU/cm 2 ) caused a peak reduction in S. Typhimurium of 4.52 log CFU/cm 2 in ready-to-eat (RTE) duck meat, whereas potassium sorbate treatment (PS, 2mg/cm 2 ) resulted in a 0.05-0.12 log reduction. Compared to PS treatment, there was significant difference in the S. Typhimurium reduction (P˂0.05) by phage treatment at both 4°C and 25°C. The results suggested that phage could be applied to reduce Salmonella, on commercial poultry products. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Photoluminescence of heterostructures with GaP1−xNx and GaP1−x−yNxAsy layers grown on GaP and Si substrates by molecular-beam epitaxy

    International Nuclear Information System (INIS)

    Lazarenko, A. A.; Nikitina, E. V.; Sobolev, M. S.; Pirogov, E. V.; Denisov, D. V.; Egorov, A. Yu.

    2015-01-01

    The structural and optical properties of heterostructures containing GaP 1−x N x ternary and GaP 1−x−y N x As y quaternary alloy layers are discussed. The heterostructures are grown by molecular-beam epitaxy on GaP and Si substrates. The structures are studied by the high-resolution X-ray diffraction technique and photoluminescence measurements in a wide temperature range from 10 to 300 K. In the low-temperature photoluminescence spectra of the alloys with a low nitrogen fraction (x < 0.007), two clearly resolved narrow lines attributed to the localized states of nitrogen pairs and the phonon replicas of these lines are observed

  15. Effect of orbital alignment on the forward and reverse electronic energy transfer Ca(4s5p 1P1)+Marrow-right-leftCa(4s5p 3P/sub J/)+M with rare gases

    International Nuclear Information System (INIS)

    Bussert, W.; Neuschaefer, D.; Leone, S.R.; Departments of Physics and Chemistry, University of Colorado, Boulder, Colorado 80309-0440)

    1987-01-01

    Effects of orbital alignment on the relative cross sections for electronic energy transfer are determined for the near resonant transfer between Ca(4s5p 1 P 1 ) and Ca(4s5p 3 P/sub J/) states with rare gas collision partners. The experiments are carried out by pulsed laser excitation in a crossed beam. The results for the forward direction, 1 P to 3 P, formulated in terms of the ratio of the maximum to minimum transfer probability are: 3 He 1.61 +- 0.05; He 1.60 +- 0.03; Ne 1.55 +- 0.10; Ar 1.52 +- 0.21; for Kr, transfer occurs, but no preference is distinguishable within 1 +- 0.2; Xe 1.44 +- 0.06. The results for He, Ne, and Ar indicate a clear preference in the transfer for the initially prepared molecular Pi state. For Xe the molecular Σ state is dominant. The energy transfer is also carried out in the reverse direction, 3 P 1 to 1 P, for He and Xe, obtaining 1.65 +- 0.10 and 1.94 +- 0.22, respectively. Analysis of the state preparation suggests that the reverse direction favors the asymptotic molecular Σ state for He and the molecular Pi state for Xe. These alignment results provide a first experimental determination of the dominant electronic states involved in a collisional energy transfer process

  16. Material properties and modeling characteristics for MnFeP1-xAsx materials for application in magnetic refrigeration

    DEFF Research Database (Denmark)

    Engelbrecht, Kurt; Nielsen, Kaspar Kirstein; Bahl, Christian R.H.

    2013-01-01

    Compounds of MnFeP1-xAsx have received attention recently for their use in active magnetic regenerators (AMR) because of their relatively high isothermal entropy change and adiabatic temperature change with magnetization. However, the materials also generally exhibit a significant magnetic...... impact of hysteresis is a key element to guide successful material development and synthesis. The properties of a magnetocaloric MnFeP1-xAsx compound are characterized as a function of temperature and applied magnetic field, and the results are used to assess the effects of hysteresis on magnetocaloric...... and thermal hysteresis, and it is not well understood how the hysteresis will affect performance in a practical AMR device. The amount of hysteresis shown by a material can be controlled to an extent by tuning the processing conditions used during material synthesis; therefore, knowledge of the practical...

  17. Small organic compounds enhance antigen loading of class II major histocompatibility complex proteins by targeting the polymorphic P1 pocket

    DEFF Research Database (Denmark)

    Höpner, Sabine; Dickhaut, Katharina; Hofstätter, Maria

    2006-01-01

    immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated...... the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently......-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce...

  18. The Level of Mite Dermatophagoides’ Allergens (Der-p 1 and Der-f 1 in Birjand

    Directory of Open Access Journals (Sweden)

    Mohammad Fereidouni

    2014-06-01

    Full Text Available Background: House dust mite allergens especially pyroglyphid species are among the most important indoor allergens and have an important role in development of asthma and allergies. Materials and Methods: In current study, the level of two main allergens from mites including Der-p1 and Der-f 1 in dust of 28 homes in Birjand city was measured by ELISA method. Results: All samples were negative for Der-p1. Low leverl of Der-f 1 was detected in one sample. Prevalence of asthma, rhinitis and rhinoconjunctivitis was 2%, 28% and 15% respectively. Conclusion: The results of this study suggest that House dust mites could not grow in Birjand climate.

  19. Filled and empty states of Zn-TPP films deposited on Fe(001)-p(1×1)O.

    Science.gov (United States)

    Bussetti, Gianlorenzo; Calloni, Alberto; Yivlialin, Rossella; Picone, Andrea; Bottegoni, Federico; Finazzi, Marco

    2016-01-01

    Zn-tetraphenylporphyrin (Zn-TPP) was deposited on a single layer of metal oxide, namely an Fe(001)- p (1×1)O surface. The filled and empty electronic states were measured by means of UV photoemission and inverse photoemission spectroscopy on a single monolayer and a 20 monolayer thick film. The ionization energy and the electron affinity of the organic film were deduced and the interface dipole was determined and compared with data available in the literature.

  20. Filled and empty states of Zn-TPP films deposited on Fe(001-p(1×1O

    Directory of Open Access Journals (Sweden)

    Gianlorenzo Bussetti

    2016-10-01

    Full Text Available Zn-tetraphenylporphyrin (Zn-TPP was deposited on a single layer of metal oxide, namely an Fe(001-p(1×1O surface. The filled and empty electronic states were measured by means of UV photoemission and inverse photoemission spectroscopy on a single monolayer and a 20 monolayer thick film. The ionization energy and the electron affinity of the organic film were deduced and the interface dipole was determined and compared with data available in the literature.

  1. Characterization of the human T cell response to rye grass pollen allergens Lol p 1 and Lol p 5.

    Science.gov (United States)

    Burton, M D; Papalia, L; Eusebius, N P; O'Hehir, R E; Rolland, J M

    2002-12-01

    Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals. Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3H-thymidine uptake and IL-5 and IFN-gamma in culture supernatants analysed by ELISA. Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found. For Lol p 1 these spanned residues 19-38, 109-128, 154-173, 190-209, and for Lol p 5 37-56, 100-119, 145-164, 154-173, 190-209, 217-236 and 226-245. IL-5 and IFN-gamma were produced by T cells cultured with proliferation-inducing peptides. T cell responses to RGP major allergens have been extensively characterized, providing fundamental information for developing T cell-targeted immunotherapy for RGP allergy.

  2. Detection of S1-P1 and S3-P3 interactions between papain and four synthetic substrates

    Directory of Open Access Journals (Sweden)

    Emmanuel M Papamichael

    1999-01-01

    Full Text Available In this study, the S1 - P1 and S3 - P3 interactions between papain and four synthetic peptide substrates were found as to be important. The values of Km were estimated as to be practically identical between these substrates; this latter is supporting the conclusions obtained by considering the estimated values of other kinetic parameters. Nevertheless, based on the estimated kcat and/or k cat/Km parameters of the used substrates, we concluded that an aromatic ring at the P3 position, and a positively charged side chain of the residue at the P1 position of the synthetic substrates were favored considerably their interaction with papain.Neste estudo, o S1 - P1 e S3 - P3, interações entre papaina e quatro substratos sintéticos de pepetídios foram considerados importantes. Os valores de Km foram estimados e são praticamente idênticos entre esses substratos; Isso dá suporte as conclusões obtidas, considerando os valores parâmetros cinéticos estimados. No obstante, baseou na estimação parâmetros kcat e/ou k cat /Km dos substratos utilizados. Se pode concluir que um anel aromático na posição P3, e uma corrente carregada positivamente da cadeia do resíduo na posição P1 dos substratos sintéticos favoreceram interação com a papaina.

  3. A lifetime measurement of the 1s2p 3P1 level in helium-like Mg and Al

    International Nuclear Information System (INIS)

    Armour, I.A.; Silver, J.D.; Traebert, E.; Oxford Univ.

    1981-01-01

    The lifetimes of the 1s2p 3 P 1 levels of helium-like magnesium and aluminium have been measured by the beam-foil decay curve technique. The 1s 2 -1s2p transitions were observed with a curved-crystal x-ray spectrometer. Decay curves taken under systematically varied conditions have been evaluated using several techniques including a cascade model. The results are in agreement with most of the theoretical predictions. (author)

  4. Ukpebor p1

    African Journals Online (AJOL)

    BIG TIMMY

    motorcycles, cassava milling machine and the re- suspension of dust particles while sweeping and fugitive harmattan dust from the trans sahara desert. Table 2: Concentration of Outdoor Heavy Metals and Particulate Matter at Iyowa (µgm )-3. Table 3: Kitchen Concentration of Heavy Metals and Particulate Matter at Iyowa ...

  5. A through calculation of 1,100 MWe PWR large break LOCA by THYDE-P1 EM model

    International Nuclear Information System (INIS)

    Kanazawa, Masayuki; Asahi, Yoshiro; Hirano, Masashi

    1984-07-01

    THYDE-P1 is a code to analyze both the blowdown and refill-reflood phases of loss-of-coolant accidents (LOCAs) of pressurized water reactors (PWRs). Up to now, THYDE-P1 has been applied to various experiment analyses, which show its high capability to analyze LOCAs as a best estimate (BE) calculation code. In this report, evaluation model (EM) calculation method, especialy in the blowdown and refill phases, is established equivalently to WREM/J2 which is regarded as appropriate for an EM calculation code, and the results of them are compared and discussed. The present calculation was the first executed by THYDE-P1-EM, and was performed as Sample Calculation Run 80 which was a part of a series of THYDE-P sample calculations. The calculation was carried out from the LOCA initiation till 400 seconds for a guillotine break at the cold leg of a commercial 1,100 MWe PWR plant. The calculated results agreed well to that of the WREM/J2 code. (author)

  6. Discovery of a potential lead compound for treating leprosy with dapsone resistance mutation in M. leprae folP1.

    Science.gov (United States)

    Nisha, J; Ramanathan, K; Nawaz Khan, F; Dhanasekaran, D; Shanthi, V

    2016-06-21

    Dapsone resistance is a serious impediment to the implementation of the present leprosy control strategies. In the recent past, many studies have been undertaken to address the antibiotic activity and binding pattern of dapsone against both native and mutant (Pro55Leu) folP1. Yet, there is no well-developed structural basis for understanding drug action and there is dire need for new antibacterial therapies. In the present study, molecular simulation techniques were employed alongside experimental strategies to address and overcome the mechanism of dapsone resistance. In essence, we report the identification of small molecule compounds to effectively and specifically inhibit the growth of M. leprae through targeting dihydropteroate synthase, encoded by folP1 which is involved in folic acid synthesis. Initially, ADME and toxicity studies were employed to screen the lead compounds, using dapsone as standard drug. Subsequently, molecular docking was employed to understand the binding efficiency of dapsone and its lead compounds against folP1. Further, the activity of the screened lead molecule was studied by means of molecular dynamics simulation techniques. Furthermore, we synthesized 4-(2-fluorophenylsulfonyl)benzenamine, using (2-fluorophenyl)boronic acid and 4-aminobenzenesulfonyl chloride, and the compound structure was confirmed by (1)H NMR and (13)C NMR spectroscopic techniques. Most importantly, the antibacterial activity of the compound was also examined and compared against dapsone. Overall, the result from our analysis suggested that CID21480113 (4-(2-fluorophenylsulfonyl)benzenamine) could be developed into a promising lead compound and could be effective in treating dapsone resistant leprosy cases.

  7. Comparison of two automated assays of BTM (CTX and P1NP) and reference intervals in a Danish population.

    Science.gov (United States)

    Jørgensen, N R; Møllehave, L T; Hansen, Y B L; Quardon, N; Lylloff, L; Linneberg, A

    2017-07-01

    Bone turnover markers are used for monitoring osteoporosis treatment. Therefore, we evaluated the agreement between different assays for CTX and PINP and established reference intervals in a cohort of 2300 individuals. We found poor agreement between assays and different reference intervals. This highlights the importance of harmonization of the assays. Two reference markers for bone turnover have been proposed: CTX bone resorption and P1NP for bone formation. The purpose of the current study was to establish reference intervals for the two markers in a Danish cohort and to determine the agreement on the two platforms. Fasting sera from 2308 individuals (1250 males and 1058 females, age range 24-76 years) participating in the Health2006 study were analyzed for CTX and P1NP using the automated IDS-iSYS analyzer and the automated Cobas e411 analyzer. Participants in anti-osteoporotic treatment were excluded, while subjects on hormonal contraceptives were included. There was significant disagreement between both the two P1NP assays with a mean difference of -3 μg/L (LoA -19 to 14) (p reference intervals for each sex: 25-29, 30-39, and 40-80 years for men, and 25-29, >30 (pre-menopausal), and >30 years (post-menopausal) for women. There is significant disagreement between the IDS-iSYS and Roche Cobas assays for both reference markers. Consequently, the reference intervals for an adult, healthy population are different depending on the analysis method used. Therefore, repeated measurements of patient samples used for monitoring of treatment should be done on the same assay. Moreover, assay-specific reference intervals should be used. Harmonization of assays for BTM is highly warranted.

  8. Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

    Science.gov (United States)

    Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

    2010-12-01

    Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

  9. Effect of rapid thermal annealing on InP1−xBix grown by molecular beam epitaxy

    International Nuclear Information System (INIS)

    Wu, X Y; Wang, K; Pan, W W; Wang, P; Li, Y Y; Song, Y X; Gu, Y; Yue, L; Xu, H; Zhang, Z P; Cui, J; Gong, Q; Wang, S M

    2015-01-01

    The effect of post-growth rapid thermal annealing on structural and optical properties of InP 1−x Bi x thin films was investigated. InPBi shows good thermal stability up to 500 °C and a modest improvement in photoluminescence (PL) intensity with an unchanged PL spectral feature. Bismuth outdiffusion from InPBi and strain relaxation are observed at about 600 °C. The InPBi sample annealed at 800 °C shows an unexpected PL spectrum with different energy transitions. (paper)

  10. The subcellular distribution of the human ribosomal "stalk" components: P1, P2 and P0 proteins

    DEFF Research Database (Denmark)

    Tchórzewski, Marek; Krokowski, Dawid; Rzeski, Wojciech

    2003-01-01

    The ribosomal "stalk" structure is a distinct lateral protuberance located on the large ribosomal subunit in prokaryotic, as well as in eukaryotic cells. In eukaryotes, this ribosomal structure is composed of the acidic ribosomal P proteins, forming two hetero-dimers (P1/P2) attached......-proteins that are not actively transported into the nucleus; moreover, this might imply that the "stalk" constituents are assembled onto the ribosomal particle at the very last step of ribosomal maturation, which takes part in the cell cytoplasm....

  11. Comparison of two automated assays of BTM (CTX and P1NP) and reference intervals in a Danish population

    DEFF Research Database (Denmark)

    Jørgensen, N R; Møllehave, L T; Hansen, Y B L

    2017-01-01

    -osteoporotic treatment were excluded, while subjects on hormonal contraceptives were included. RESULTS: There was significant disagreement between both the two P1NP assays with a mean difference of -3 μg/L (LoA -19 to 14) (p ... (pre-menopausal), and >30 years (post-menopausal) for women. CONCLUSIONS: There is significant disagreement between the IDS-iSYS and Roche Cobas assays for both reference markers. Consequently, the reference intervals for an adult, healthy population are different depending on the analysis method used...

  12. Response surface optimization of carbon and nitrogen sources for nuclease P1 production by Penicillium citrinum F-5-5

    International Nuclear Information System (INIS)

    Liang Xinle; Huang Yingying; Zhang Hong; Chen Min; Liu Xuan

    2011-01-01

    Penicillium citrinum F-5-5, a nuclease P1 high-producing strain with 978.6 U/ml in potato glucose medium, was derived from the original Penicillium citrinum CICC 4011 with 60 Co γ-rays irradiation mutation and then protoplasts fusion treatment. Culture components were optimized for the nuclease P1 production, and response surface methodology was applied for the critical medium components(carbon and nitrogen sources) which were preselected by Plackett-Burman design approach. Glucose, soluble starch and corn steep powder showed significant effects on production of nuclease. Central composite design was used for the optimization levels by software Minitab 15, and it showed that, the optimal values for the concentration of glucose, soluble starch and corn steep powder were 30.89, 42.46 and 11.60 g/L, respectively. With this medium,an enzyme activity of 1687.16 U/ml could be obtained theoretically. Using this optimized medium, an experimental enzyme activity of 1672.6 U/ml was reached. (authors)

  13. Band gap calculations of the semiconductor BNxP1−x using modified Becke–Johnson approximation

    International Nuclear Information System (INIS)

    Benkraouda, M.; Amrane, N.

    2013-01-01

    Highlights: ► The Modified Becke–Johnson scheme gives a very accurate band gap. ► We have shown the invalidity of Vegard’s linear rule for BN x P 1−x . ► The band gap changes with alloy concentration are important in band gap engineering. - Abstract: In this work, the electronic properties of BN, BP and BN x P 1−x compounds have been investigated by means of first-principles density-functional total-energy calculation using the all-electron full potential linear augmented plane-wave method (FP-LAPW). The (FP-LAPW) method was used within the density functional theory (DFT) along with the Engel–Vosko and Becke–Johnson exchange correlation potential. The energy bands along high symmetry directions, the density of states and bowing distributions are calculated. The results have been discussed in terms of previously existing experimental and theoretical data, and comparisons with similar compounds have been made. Analysis of band structure suggests direct and pseudo-direct band gaps for both compounds.

  14. New insights into alkylammonium-functionalized clinoptilolite and Na-P1 zeolite: Structural and textural features

    Science.gov (United States)

    Muir, Barbara; Matusik, Jakub; Bajda, Tomasz

    2016-01-01

    The area of zeolites' application could be expanded by utilizing their surfaces. Zeolites are frequently modified to increase their hydrophobicity and to generate the negative charge of the surface. The main objective of the study was to investigate and compare the features of natural clinoptilolite and synthetic zeolite Na-P1 modified by selected surfactants involving quaternary ammonium salts. The FTIR study indicates that with increasing carbon chain length in the surfactant attached to the zeolites surface the molecules adopt a more disordered structure. FTIR was also used to determine the efficiency of surface modification. Thermal analysis revealed that the presence of surfactant results in additional exothermic effects associated with the breaking of electrostatic bonds between zeolites and surfactants. The mass losses are in line with ECEC and CHN data. The textural study indicates that the synthetic zeolite Na-P1 has better sorption properties than natural clinoptilolite. The modification process always reduces the SBET and porosity of the material. With an increasing carbon chain length of surfactants all the texture parameters decrease.

  15. Univariate Lp and ɭ p Averaging, 0 < p < 1, in Polynomial Time by Utilization of Statistical Structure

    Directory of Open Access Journals (Sweden)

    John E. Lavery

    2012-10-01

    Full Text Available We present evidence that one can calculate generically combinatorially expensive Lp and lp averages, 0 < p < 1, in polynomial time by restricting the data to come from a wide class of statistical distributions. Our approach differs from the approaches in the previous literature, which are based on a priori sparsity requirements or on accepting a local minimum as a replacement for a global minimum. The functionals by which Lp averages are calculated are not convex but are radially monotonic and the functionals by which lp averages are calculated are nearly so, which are the keys to solvability in polynomial time. Analytical results for symmetric, radially monotonic univariate distributions are presented. An algorithm for univariate lp averaging is presented. Computational results for a Gaussian distribution, a class of symmetric heavy-tailed distributions and a class of asymmetric heavy-tailed distributions are presented. Many phenomena in human-based areas are increasingly known to be represented by data that have large numbers of outliers and belong to very heavy-tailed distributions. When tails of distributions are so heavy that even medians (L1 and l1 averages do not exist, one needs to consider using lp minimization principles with 0 < p < 1.

  16. The Complete Sequence of the Acacia ligulata Chloroplast Genome Reveals a Highly Divergent clpP1 Gene.

    Directory of Open Access Journals (Sweden)

    Anna V Williams

    Full Text Available Legumes are a highly diverse angiosperm family that include many agriculturally important species. To date, 21 complete chloroplast genomes have been sequenced from legume crops confined to the Papilionoideae subfamily. Here we report the first chloroplast genome from the Mimosoideae, Acacia ligulata, and compare it to the previously sequenced legume genomes. The A. ligulata chloroplast genome is 174,233 bp in size, comprising inverted repeats of 38,225 bp and single-copy regions of 92,798 bp and 4,985 bp [corrected]. Acacia ligulata lacks the inversion present in many of the Papilionoideae, but is not otherwise significantly different in terms of gene and repeat content. The key feature is its highly divergent clpP1 gene, normally considered essential in chloroplast genomes. In A. ligulata, although transcribed and spliced, it probably encodes a catalytically inactive protein. This study provides a significant resource for further genetic research into Acacia and the Mimosoideae. The divergent clpP1 gene suggests that Acacia will provide an interesting source of information on the evolution and functional diversity of the chloroplast Clp protease complex.

  17. Development and characterization of murine monoclonal antibody specific for the P1.4 PorA proteins from strain B:4:P1.(7b.4. of Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    María Elena Pérez

    2011-08-01

    Full Text Available Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained.

  18. Immunogenicity and In Vitro and In Vivo Protective Effects of Antibodies Targeting a Recombinant Form of the Streptococcus mutans P1 Surface Protein

    OpenAIRE

    Batista, Milene Tavares; Souza, Renata D.; Ferreira, Ewerton L.; Robinette, Rebekah; Crowley, Paula J.; Rodrigues, Juliana F.; Brady, L. Jeannine; Ferreira, Luís C. S.; Ferreira, Rita C. C.

    2014-01-01

    Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P139–512), produced in Bacillus subtilis and encompassing a functional dom...

  19. Variability of Outer Membrane Protein P1 and Its Evaluation as a Vaccine Candidate against Experimental Otitis Media due to Nontypeable Haemophilus influenzae: an Unambiguous, Multifaceted Approach

    Science.gov (United States)

    Bolduc, Gilles R.; Bouchet, Valérie; Jiang, Ru-Zhang; Geisselsoder, Janet; Truong-Bolduc, Que Chi; Rice, Peter A.; Pelton, Stephen I.; Goldstein, Richard

    2000-01-01

    Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (≤95% similarity) were identified among 42 strains. When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with an ompP1 mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine and (ii) use of a phylogenically characterized collection of representative isolates in concert with gene sequencing, cloning, gene inactivation, and animal testing offers an efficient, rational, and rigorous strategy for evaluating the potential problems associated with variability of vaccine targets and specificity of related immune responses. PMID:10899849

  20. 35S Promoter Methylation in Kanamycin-Resistant Kalanchoe (Kalanchoe pinnata L.) Plants Expressing the Antimicrobial Peptide Cecropin P1 Transgene.

    Science.gov (United States)

    Shevchuk, T V; Zakharchenko, N S; Tarlachkov, S V; Furs, O V; Dyachenko, O V; Buryanov, Y I

    2016-09-01

    Transgenic kalanchoe plants (Kalanchoe pinnata L.) expressing the antimicrobial peptide cecropin P1 gene (cecP1) under the control of the 35S cauliflower mosaic virus 35S RNA promoter and the selective neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase gene promoter were studied. The 35S promoter methylation and the cecropin P1 biosynthesis levels were compared in plants growing on media with and without kanamycin. The low level of active 35S promoter methylation further decreases upon cultivation on kanamycin-containing medium, while cecropin P1 synthesis increases.

  1. Nuclear magnetic resonance study of reaction of p-(1,1', 3,3'-tetramethylbutyl)phenol with phosphoric pentoxide

    International Nuclear Information System (INIS)

    Didi, Mohamed Amine; Elias Abdelhamid

    1998-12-01

    Some aspects dealing with the mechanisms and the kinetics of the reaction between phosphorus pentoxide and p-(1,1',3,3'-tetramethylbutyl)phenol were investigated, by means of the 31P nmr technique . The kinetic model considered showed that only the time and, to a lesser extent, the temperature of the reaction seem to the yield. The reactant mode ratio does not exhibit any effect upon the MOPPA DOPPA ratio. A series of adequate experiments based on the 2 factorial 3 plane method allowed to confirm these results. The tripyroesters were identified as being the longest intermediates detected by nmr. In the reaction mixture no traces of phosphoric triester (t- TOPPA) were detected

  2. Precision Measurement Method for Branching Fractions of Excited P1/2 States Applied to Ca+40

    Science.gov (United States)

    Ramm, Michael; Pruttivarasin, Thaned; Kokish, Mark; Talukdar, Ishan; Häffner, Hartmut

    2013-07-01

    We present a method for measuring branching fractions for the decay of J=1/2 atomic energy levels to lower-lying states based on time-resolved recording of the atom’s fluorescence during a series of population transfers. We apply this method to measure the branching fractions for the decay of the 4P1/22 state of Ca+40 to the 4S1/22 and 3D3/22 states to be 0.935 65(7) and 0.064 35(7), respectively. The measurement scheme requires that at least one of the lower-lying states be long lived. The method is insensitive to fluctuations in laser light intensity and magnetic field and is readily applicable to various atomic species due to its simplicity. Our result distinguishes well among existing state-of-the-art theoretical models of Ca+.

  3. Blood Group Antigens C, Lub and P1 May Have a Role in HIV Infection in Africans.

    Science.gov (United States)

    Motswaledi, Modisa Sekhamo; Kasvosve, Ishmael; Oguntibeju, Oluwafemi Omoniyi

    2016-01-01

    Botswana is among the world's countries with the highest rates of HIV infection. It is not known whether or not this susceptibility to infection is due to genetic factors in the population. Accumulating evidence, however, points to the role of erythrocytes as potential mediators of infection. We therefore sought to establish the role, if any, of some erythrocyte antigens in HIV infection in a cross-section of the population. 348 (346 HIV-negative and 2 HIV-positive) samples were obtained from the National Blood Transfusion Service as residual samples, while 194 HIV-positive samples were obtained from the Botswana-Harvard HIV Reference Laboratory. Samples were grouped for twenty three antigens. Chi-square or Fischer Exact analyses were used to compare the frequencies of the antigens in the two groups. A stepwise, binary logistic regression was used to study the interaction of the various antigens in the light of HIV-status. The Rh antigens C and E were associated with HIV-negative status, while blood group Jka, P1 and Lub were associated with HIV-positive status. A stepwise binary logistic regression analysis yielded group C as the most significant protective blood group while Lub and P1 were associated with significantly higher odds ratio in favor of HIV-infection. The lower-risk-associated group C was significantly lower in Africans compared to published data for Caucasians and might partially explain the difference in susceptibility to HIV-1. The most influential antigen C, which also appears to be protective, is significantly lower in Africans than published data for Caucasians or Asians. On the other hand, there appear to be multiple antigens associated with increased risk that may override the protective role of C. A study of the distribution of these antigens in other populations may shed light on their roles in the HIV pandemic.

  4. Structure determination of Ga As (110) p (1 x 1) - Sb using scanned-energy photoelectron diffraction

    International Nuclear Information System (INIS)

    Ascolani, H.; Asensio, M.C.; Fritzsche, W.

    1996-01-01

    Photoelectron diffraction (PD) in the scanned-energy mode has proven to be a powerfull tool for structural determination of the first few surface layers. The scanned-energy mode involves the measurement of the intensity of photoelectrons emitted from a core level as a function of the incident photon energy for different emission directions. The atom specificity of PD allows the investigation of the local structure of adsorbed atoms without interference of the substrate. In addition, if a measurable chemical shift exists, this technique is also able to discriminate between atoms of the same species adsorbed in inequivalent sites. The Ga As (110) p (1x1)-Sb (1 ML) surface represents a prototype system to study atom adsorption on III-V semiconductors. The epitaxial continued layer structure (ECLS) is generally accepted as the geometry corresponding to this surface, although some authors have claimed that the p 3 model forms a stable geometry equivalent to the ECLS, and that it provides an equally good description of their experimental results. So far, the conclusions about the atomic structure of this surface had been derived on the basis of indirect methods. This work exploits to the utmost the possibilities of analysis offered by the scanned-energy PD technique, namely, chemical shift analysis and direct inversion. The energy spectrum of Sb-4d photoelectrons emitted from the Ga As (110)-p (1x1) Sb surface has two chemically-shifted components. We have inverted the scanned-energy photoelectron diffraction data corresponding to these two components to obtain the positions of the Ga and As atoms which are nearest neighbors of two inequivalent Sb atoms. Our results contradict various models proposed for this surface and are consistent only with the ECLS. For a more detailed atomic structure determination, the best fit between experiment and multiple-scattering calculations was determined by a trial-and-error procedure. (author)

  5. CIGS P1, P2, P3 Scribing Processes using a Pulse Programmable Industrial Fiber Laser: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Rekow, M.; Murison, R.; Panarello, T.; Dunsky, C.; Dinkel, C.; Nikumb, S.; Pern, F. J.; Mansfield, L.

    2010-10-01

    We describe a novel set of laser processes for the CIGS P1, P2 and P3 scribing steps, the development of which has been enabled by a unique pulse-programmable fiber laser. We find that the unique pulse control properties of this 1064 nm wavelength laser have significant effects on the material removal dynamics of the various film layers in the CIGS material system. In the case of the P2 and P3 processes, the shaped pulses create new laser/material interaction effects that permit the material to be cleanly and precisely removed with zero Heat Affected Zone (HAZ) at the edges of the scribe. The new P2 and P3 processes we describe demonstrate the first use of infrared nanosecond laser pulses that eliminate the HAZ and the consequent localized compositional changes in the CIGS absorber material that result in poor shunt resistance. SEM micrographs and EDX compositional scans are presented. For the P1 scribe, we process the bi-layer molybdenum from the film side as well as through the glass substrate. Microscopic inspection and compositional analysis of the scribe lines are not sufficient to determine electrical and optical performance in working PV modules. Therefore, to demonstrate the applicability of the infrared pulse-programmable laser to all three scribing processes for thin-film CIGS, we fabricate small-size multiple-cell monolithically interconnected mini-modules in partnership with the National Renewable Energy Laboratory (Golden, Colorado). A total of four mini-modules are produced, two utilizing all laser scribing, and two with the P2 and P3 steps mechanically scribed (by a third party) for reference. Mini-module performance data measured at NREL is presented, and we also discuss the commercialization potential of the new single-laser CIGS scribing process. Finally we present a phenomenological model to describe this physics underlying this novel ablation process.

  6. Measurement of IgE antibodies against purified grass pollen allergens (Lol p 1, 2, 3 and 5) during immunotherapy.

    Science.gov (United States)

    Van Ree, R; Van Leeuwen, W A; Dieges, P H; Van Wijk, R G; De Jong, N; Brewczyski, P Z; Kroon, A M; Schilte, P P; Tan, K Y; Simon-Licht, I F; Roberts, A M; Stapel, S O; Aalberse, R C

    1997-01-01

    IgE titres tend to rise early after the start of immunotherapy, followed by a decline to pre-immunotherapy levels or lower. We were interested to know whether the early increase in IgE antibodies includes new specificities of IgE, and whether these responses persist. Sera of 64 patients undergoing grass pollen immunotherapy were tested for IgE against four purified grass pollen allergens: Lol p 1, 2, 3, and 5. At least two serum samples were taken, one before the start of therapy and one between 5 and 18 months after the first immunization (mean: 10 months). The mean IgE responses to Lol p 1, 2 and 3 showed a moderate but not significant increase. In contrast, the mean IgE response to Lol p 5 showed a significant decrease of > 30%. IgE against total Lohum perenne pollen extract moderately increased (> 20%), showing that a RAST for total pollen is not always indicative for the development of IgE against its major allergens. For > 40% of the patients it was found that IgE against one or more of the four allergens increased, while IgE against the remaining allergen(s) decreased. For 10 sera the ratio of IgE titres against at least two allergens changed by at least a factor of 5. The changes in specific IgE also included conversions from negative (< 0.1 RU) to positive (0.6 to 5.0 RU) for five patients. For two patients, the induction of these 'new' IgE antibodies against major allergens was shown to result in a response that was persistent over several years. Although active induction of new IgE specificities by immunotherapy was not really proven, the observations in this study indicate that monitoring of IgE against purified (major) allergens is necessary to evaluate changes in specific IgE in a reliable way.

  7. Intermittent PTHrP(1–34) Exposure Augments Chondrogenesis and Reduces Hypertrophy of Mesenchymal Stromal Cells

    Science.gov (United States)

    Fischer, Jennifer; Aulmann, Antje; Dexheimer, Verena; Grossner, Tobias

    2014-01-01

    Phenotype instability and premature hypertrophy prevent the use of human mesenchymal stromal cells (MSCs) for cartilage regeneration. Aim of this study was to investigate whether intermittent supplementation of parathyroid hormone-related protein (PTHrP), as opposed to constant treatment, can beneficially influence MSC chondrogenesis and to explore molecular mechanisms below catabolic and anabolic responses. Human MSCs subjected to chondrogenic induction in high-density culture received PTHrP(1–34), forskolin, dbcAMP, or PTHrP(7–34) either constantly or via 6-h pulses (three times weekly), before proteoglycan, collagen type II, and X deposition; gene expression; and alkaline phosphatase (ALP) activity were assessed. While constant application of PTHrP(1–34) suppressed chondrogenesis of MSCs, pulsed application significantly increased collagen type 2 (COL2A1) gene expression and the collagen type II, proteoglycan, and DNA content of pellets after 6 weeks. Collagen type 10 (COL10A1) gene expression was little affected but Indian hedgehog (IHH) expression and ALP activity were significantly downregulated by pulsed PTHrP. A faster response to PTHrP exposure was recorded for ALP activity over COL2A1 regulation, suggesting that signal duration is critical for catabolic versus anabolic reactions. Stimulation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling by forskolin reproduced major effects of both treatment modes, whereas application of PTHrP(7–34) capable of protein kinase C (PKC) signaling was ineffective. Pulsed PTHrP exposure of MSCs stimulated chondrogenesis and reduced endochondral differentiation apparently uncoupling chondrogenic matrix deposition from hypertrophic marker expression. cAMP/PKA was the major signaling pathway triggering the opposing effects of both treatment modes. Intermittent application of PTHrP represents an important novel means to improve chondrogenesis of MSCs and may be considered as a supporting clinical

  8. THE PLASMA ENVIRONMENT IN COMETS OVER A WIDE RANGE OF HELIOCENTRIC DISTANCES: APPLICATION TO COMET C/2006 P1 (MCNAUGHT)

    International Nuclear Information System (INIS)

    Shou, Y.; Combi, M.; Gombosi, T.; Toth, G.; Jia, Y.-D.; Rubin, M.

    2015-01-01

    On 2007 January 12, comet C/2006 P1 (McNaught) passed its perihelion at 0.17 AU. Abundant remote observations offer plenty of information on the neutral composition and neutral velocities within 1 million kilometers of the comet nucleus. In early February, the Ulysses spacecraft made an in situ measurement of the ion composition, plasma velocity, and magnetic field when passing through the distant ion tail and the ambient solar wind. The measurement by Ulysses was made when the comet was at around 0.8 AU. With the constraints provided by remote and in situ observations, we simulated the plasma environment of Comet C/2006 P1 (McNaught) using a multi-species comet MHD model over a wide range of heliocentric distances from 0.17 to 1.75 AU. The solar wind interaction of the comet at various locations is characterized and typical subsolar standoff distances of the bow shock and contact surface are presented and compared to analytic solutions. We find the variation in the bow shock standoff distances at different heliocentric distances is smaller than the contact surface. In addition, we modified the multi-species model for the case when the comet was at 0.7 AU and achieved comparable water group ion abundances, proton densities, plasma velocities, and plasma temperatures to the Ulysses/SWICS and SWOOPS observations. We discuss the dominating chemical reactions throughout the comet-solar wind interaction region and demonstrate the link between the ion composition near the comet and in the distant tail as measured by Ulysses

  9. THE PLASMA ENVIRONMENT IN COMETS OVER A WIDE RANGE OF HELIOCENTRIC DISTANCES: APPLICATION TO COMET C/2006 P1 (MCNAUGHT)

    Energy Technology Data Exchange (ETDEWEB)

    Shou, Y.; Combi, M.; Gombosi, T.; Toth, G. [Department of Atmospheric, Oceanic and Space Sciences, University of Michigan, Ann Arbor, MI (United States); Jia, Y.-D. [IGPP, and EPSS, University of California, Los Angeles, CA 90095 (United States); Rubin, M. [Physikalisches Institut, University of Bern, Sidlerstrasse. 5, CH-3012 Bern (Switzerland)

    2015-08-20

    On 2007 January 12, comet C/2006 P1 (McNaught) passed its perihelion at 0.17 AU. Abundant remote observations offer plenty of information on the neutral composition and neutral velocities within 1 million kilometers of the comet nucleus. In early February, the Ulysses spacecraft made an in situ measurement of the ion composition, plasma velocity, and magnetic field when passing through the distant ion tail and the ambient solar wind. The measurement by Ulysses was made when the comet was at around 0.8 AU. With the constraints provided by remote and in situ observations, we simulated the plasma environment of Comet C/2006 P1 (McNaught) using a multi-species comet MHD model over a wide range of heliocentric distances from 0.17 to 1.75 AU. The solar wind interaction of the comet at various locations is characterized and typical subsolar standoff distances of the bow shock and contact surface are presented and compared to analytic solutions. We find the variation in the bow shock standoff distances at different heliocentric distances is smaller than the contact surface. In addition, we modified the multi-species model for the case when the comet was at 0.7 AU and achieved comparable water group ion abundances, proton densities, plasma velocities, and plasma temperatures to the Ulysses/SWICS and SWOOPS observations. We discuss the dominating chemical reactions throughout the comet-solar wind interaction region and demonstrate the link between the ion composition near the comet and in the distant tail as measured by Ulysses.

  10. Intermittent PTHrP(1-34) exposure augments chondrogenesis and reduces hypertrophy of mesenchymal stromal cells.

    Science.gov (United States)

    Fischer, Jennifer; Aulmann, Antje; Dexheimer, Verena; Grossner, Tobias; Richter, Wiltrud

    2014-10-15

    Phenotype instability and premature hypertrophy prevent the use of human mesenchymal stromal cells (MSCs) for cartilage regeneration. Aim of this study was to investigate whether intermittent supplementation of parathyroid hormone-related protein (PTHrP), as opposed to constant treatment, can beneficially influence MSC chondrogenesis and to explore molecular mechanisms below catabolic and anabolic responses. Human MSCs subjected to chondrogenic induction in high-density culture received PTHrP(1-34), forskolin, dbcAMP, or PTHrP(7-34) either constantly or via 6-h pulses (three times weekly), before proteoglycan, collagen type II, and X deposition; gene expression; and alkaline phosphatase (ALP) activity were assessed. While constant application of PTHrP(1-34) suppressed chondrogenesis of MSCs, pulsed application significantly increased collagen type 2 (COL2A1) gene expression and the collagen type II, proteoglycan, and DNA content of pellets after 6 weeks. Collagen type 10 (COL10A1) gene expression was little affected but Indian hedgehog (IHH) expression and ALP activity were significantly downregulated by pulsed PTHrP. A faster response to PTHrP exposure was recorded for ALP activity over COL2A1 regulation, suggesting that signal duration is critical for catabolic versus anabolic reactions. Stimulation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling by forskolin reproduced major effects of both treatment modes, whereas application of PTHrP(7-34) capable of protein kinase C (PKC) signaling was ineffective. Pulsed PTHrP exposure of MSCs stimulated chondrogenesis and reduced endochondral differentiation apparently uncoupling chondrogenic matrix deposition from hypertrophic marker expression. cAMP/PKA was the major signaling pathway triggering the opposing effects of both treatment modes. Intermittent application of PTHrP represents an important novel means to improve chondrogenesis of MSCs and may be considered as a supporting clinical

  11. Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptococcus mutans P1 surface protein.

    Science.gov (United States)

    Batista, Milene Tavares; Souza, Renata D; Ferreira, Ewerton L; Robinette, Rebekah; Crowley, Paula J; Rodrigues, Juliana F; Brady, L Jeannine; Ferreira, Luís C S; Ferreira, Rita C C

    2014-12-01

    Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  12. HDL activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) promotes regeneration and suppresses fibrosis in the liver

    DEFF Research Database (Denmark)

    Ding, Bi-Sen; Liu, Catherine H; Sun, Yue

    2016-01-01

    repair" phenotype was recapitulated in mice that lack S1P1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic...

  13. IgE Abs to Der p 1 and Der p 2 as diagnostic markers of house dust mite allergy as defined by a bronchoprovocation test

    Directory of Open Access Journals (Sweden)

    Takafumi Minami

    2015-01-01

    Conclusions: IgE to nDer p 1 and/or rDer p 2 was highly predictive of allergen-induced IAR. These findings validate the clinical usefulness of measuring the levels of IgE to nDer p 1 and rDer p 2 as a diagnostic tool for genuine HDM allergy.

  14. Regulation and function of transaldolase isoenzymes involved in sugar and one-carbon metabolism in the ribulose monophosphate cycle methylotroph Arthrobacter P1

    NARCIS (Netherlands)

    Levering, P.R.; Dijkhuizen, Lubbert

    In the facultative methylotroph Arthrobacter P1 the enzyme transaldolase plays an important role in both the pentose phosphate pathway and in the ribulose monophosphate cycle of formaldehyde fixation. Among gluconate-negative mutants of Arthrobacter P1 strains occurred which also were unable to grow

  15. Medium optimization for nuclease P1 production by Penicillium citrinum in solid-state fermentation using polyurethane foam as inert carrier

    NARCIS (Netherlands)

    Zhu, Y.; Knol, W.; Smits, J.P.; Bol, J.

    1996-01-01

    A solid-state fermentation system, using polyurethane foam as an inert carrier, was used for the production of nuclease P1 by Penicillium citrinum. Optimization of nuclease P1 production was carried out using a synthetic liquid medium. After a two-step medium optimization using a fractional

  16. A neutralizing recombinant single chain antibody, scFv, against BaP1, A P-I hemorrhagic metalloproteinase from Bothrops asper snake venom.

    Science.gov (United States)

    Castro, J M A; Oliveira, T S; Silveira, C R F; Caporrino, M C; Rodriguez, D; Moura-da-Silva, A M; Ramos, O H P; Rucavado, A; Gutiérrez, J M; Magalhães, G S; Faquim-Mauro, E L; Fernandes, I

    2014-09-01

    BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of

  17. Bothrops asper metalloproteinase BaP1 is inhibited by alpha(2)-macroglobulin and mouse serum and does not induce systemic hemorrhage or coagulopathy.

    Science.gov (United States)

    Escalante, Teresa; Rucavado, Alexandra; Kamiguti, Aura S; Theakston, R David G; Gutiérrez, José María

    2004-02-01

    The ability of the P-I metalloproteinase BaP1, isolated from the venom of the snake Bothrops asper, to induce systemic bleeding, thrombocytopenia and defibrinogenation was assessed in an experimental mouse model. Intravenous administration of BaP1 caused neither systemic bleeding nor any evidence of pathology in lungs, kidneys, liver, heart and brain. Moreover, there were no alterations in the whole blood clotting time or in platelet numbers. In addition, BaP1 did not inhibit collagen-induced platelet aggregation in vitro. Proteolytic and hemorrhagic activities of BaP1 were readily inhibited by the plasma proteinase inhibitor, alpha(2)-macroglobulin, and normal mouse serum also inhibited hemorrhage. Such inhibition may explain why BaP1 induces multiple local tissue-damaging effects, but is largely devoid of systemic toxicity.

  18. All IncP-1 plasmid subgroups, including the novel e subgroup, are prevalent in the influent of a Danish wastewater treatment plant

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Burmølle, Mette; Meisner, Annelein

    2009-01-01

    The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281 bp fragment of trfA from all currently sequenced...... IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, a, ß, ¿ and d, were present in the wastewater treatment plant...... influent. Also sequences representing the recently described fifth subgroup, the e subgroup, were detected in the wastewater. Thus, these results confirm the presence of at least five phylogenetically distinct subgroups of IncP-1 plasmids and represent the first time that sequences associated with plasmids...

  19. Pre- and Post-perihelion Observations of C/2009 P1 (Garradd): Evidence for an Oxygen-rich Heritage?

    Science.gov (United States)

    Disanti, Michael Antonio; Villanueva, Geronimo Luis; Paganini, Lucas; Bonev, Boncho P.; Keane, Jacqueline V.; Meech, Karen J.; Mumma, Michael Jon

    2013-01-01

    We conducted pre- and post-perihelion observations of Comet C/2009 P1 (Garradd) on UT 2011 October 13 and 2012 January 8, at heliocentric distances of 1.83 and 1.57 AU, respectively, using the high-resolution infrared spectrometer (NIRSPEC) at the Keck II 10-m telescope on Mauna Kea, HI. Pre-perihelion, we obtained production rates for nine primary volatiles (native ices): H2O, CO, CH3OH, CH4, C2H6, HCN, C2H2, H2CO, and NH3. Post-perihelion, we obtained production rates for three of these (H2O, CH4, and HCN) and sensitive upper limits for three others (C2H2, H2CO, and NH3). CO was enriched and C2H2 was depleted, yet C2H6 and CH3OH were close to their currentmean values asmeasured in a dominant group of Oort cloud comets. This may indicate processing of its pre-cometary ices in a relatively oxygen-rich environment. Our measurements indicate consistent pre- and post-perihelion abundance ratios relative to H2O, suggesting we were measuring compositional homogeneity among measured species to the depths in the nucleus sampled. However, the overall gas production was lower post-perihelion despite its smaller heliocentric distance on January 8. This is qualitatively consistent with other studies of C/2009 P1, perhaps due to seasonal differences in the heating of one or more active regions on the nucleus. On October 13, the water profile showed a pronounced excess towards the Sun-facing hemisphere that was not seen in other molecules, including H2O on January 8, nor in the dust continuum. Inter-comparison of profiles from October 13 permitted us to quantify contributions due to release of H2O from the nucleus, and fromits release in the coma. This resulted in the latter source contributing 25-30% of the total observed water within our slit, which covered roughly +/-300 km by +/-4500 km from the nucleus. We attribute this excess H2O, which peaked at a mean projected distance of 1300-1500 km from the nucleus, to release from water-rich, relatively pure icy grains

  20. Sphingosine-1-Phosphate Induces Dose-Dependent Chemotaxis or Fugetaxis of T-ALL Blasts through S1P1 Activation.

    Directory of Open Access Journals (Sweden)

    Carolina V Messias

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive sphingolipid involved in several physiological processes including cell migration and differentiation. S1P signaling is mediated through five G protein-coupled receptors (S1P1-S1P5. S1P1 is crucial to the exit of T-lymphocytes from the thymus and peripheral lymphoid organs through a gradient of S1P. We have previously observed that T-ALL and T-LBL blasts express S1P1. Herein we analyzed the role of S1P receptors in the migratory pattern of human T-cell neoplastic blasts. S1P-triggered cell migration was directly related to S1P1 expression. T-ALL blasts expressing low levels of S1P1 mRNA (HPB-ALL did not migrate toward S1P, whereas those expressing higher levels of S1P1 (MOLT-4, JURKAT and CEM did migrate. The S1P ligand induced T-ALL cells chemotaxis in concentrations up to 500 nM and induced fugetaxis in higher concentrations (1000-10000 nM through interactions with S1P1. When S1P1 was specifically blocked by the W146 compound, S1P-induced migration at lower concentrations was reduced, whereas higher concentrations induced cell migration. Furthermore, we observed that S1P/S1P1 interactions induced ERK and AKT phosphorylation, and modulation of Rac1 activity. Responding T-ALL blasts also expressed S1P3 mRNA but blockage of this receptor did not modify migratory responses. Our results indicate that S1P is involved in the migration of T-ALL/LBL blasts, which is dependent on S1P1 expression. Moreover, S1P concentrations in the given microenvironment might induce dose-dependent chemotaxis or fugetaxis of T-ALL blasts.

  1. Structural Analysis of Der p 1–Antibody Complexes and Comparison with Complexes of Proteins or Peptides with Monoclonal Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Osinski, Tomasz; Pomés, Anna; Majorek, Karolina A.; Glesner, Jill; Offermann, Lesa R.; Vailes, Lisa D.; Chapman, Martin D.; Minor, Wladek; Chruszcz, Maksymilian (INDOOR); (UV); (SC)

    2015-05-29

    Der p 1 is a major allergen from the house dust mite, Dermatophagoides pteronyssinus, that belongs to the papain-like cysteine protease family. To investigate the antigenic determinants of Der p 1, we determined two crystal structures of Der p 1 in complex with the Fab fragments of mAbs 5H8 or 10B9. Epitopes for these two Der p 1–specific Abs are located in different, nonoverlapping parts of the Der p 1 molecule. Nevertheless, surface area and identity of the amino acid residues involved in hydrogen bonds between allergen and Ab are similar. The epitope for mAb 10B9 only showed a partial overlap with the previously reported epitope for mAb 4C1, a cross-reactive mAb that binds Der p 1 and its homolog Der f 1 from Dermatophagoides farinae. Upon binding to Der p 1, the Fab fragment of mAb 10B9 was found to form a very rare α helix in its third CDR of the H chain. To provide an overview of the surface properties of the interfaces formed by the complexes of Der p 1–10B9 and Der p 1–5H8, along with the complexes of 4C1 with Der p 1 and Der f 1, a broad analysis of the surfaces and hydrogen bonds of all complexes of Fab–protein or Fab–peptide was performed. This work provides detailed insight into the cross-reactive and specific allergen–Ab interactions in group 1 mite allergens. The surface data of Fab–protein and Fab–peptide interfaces can be used in the design of conformational epitopes with reduced Ab binding for immunotherapy.

  2. Factor VIII S373L: mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A.

    Science.gov (United States)

    Johnson, D J; Pemberton, S; Acquila, M; Mori, P G; Tuddenham, E G; O'Brien, D P

    1994-04-01

    A novel CRM+ mutation, factor VIII position 373 serine to leucine substitution (FVIII 373-Leu) was identified during a survey of Factor VIII (FVIII) mutations. We have purified the variant protein from the patient's plasma in order to allow further characterisation of the molecule. The CRM+ plasma contained 120% Factor VIII antigen (FVIII:Ag) and 6% Factor VIII coagulant activity (FVIII:C). After purification the mutant FVIII was subjected to thrombin proteolysis, and was thereby activated 5.6-fold compared with 7-fold for wild type molecule. Subsequently, spontaneous inactivation of the mutant was much slower than noted for wild type FVIII. Western blot analysis using monoclonal antibodies demonstrated that thrombin cleavage of FVIII 373-Leu at positions 740 and 1689 were normal but that cleavage at position 372 was completely absent. Crystallographic coordinates of the active site of thrombin complexed to fibrinopeptide A were used to explore possible mechanistic reasons for the failure of thrombin to cleave the mutant FVIII at position 372. Steric hindrance between the mutant side chain and the side chain of the P1 residue was apparent. We conclude that the functional defect of FVIII 373-Leu results from the inability of thrombin to cleave the mutant at position 372-373, and propose that this is due to steric hindrance by the side chain of leucine 373, preventing correct formation of the enzyme substrate complex.

  3. Elimination of contaminant Escherichia coli chromosomal DNA from preparations of P1 artificial chromosome recombinants facilitates directed subcloning.

    Science.gov (United States)

    Davidson, H; Doherty, A; Stevenson, B; Boyd, A C; Porteous, D J

    1999-06-01

    The subcloning of large inserts (>50 kbp) from P1-derived artificial chromosomes (PACs) was found to be hindered by the presence of contaminating Escherichia coli chromosomal fragments which, because of their smaller median size, are recovered preferentially as unwanted subclones. A significant fraction of contaminating DNA was seen to persist after conventional plasmid purification methods. We describe a rigorous protocol for eliminating the bulk of contamination that involves plasmid isolation on commercially available silica-based columns followed by three pulsed field gel electrophoresis steps. Using this, we were able to subclone 55, 85 and 90 kbp PAC inserts but failed to subclone a 195 kbp PAC insert. After surveying a range of DNA purification methods, we devised an optimised protocol that allowed us to subclone the 195 kbp insert. The optimised protocol, which reliably yields DNA with essentially no contaminating material, consists of plasmid isolation on silica-based columns followed by treatment with highly purified DNaseI and retrieval by electroelution of restriction-digested DNA electrophoresed on a single pulsed field gel. By inference it is applicable to the purification of large inserts from other single-copy plasmid vectors such as bacterial artificial chromosomes (BACs).

  4. P1-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors with Potent Antiviral Activity against Drug-Resistant Viruses

    Energy Technology Data Exchange (ETDEWEB)

    DeGoey, David A.; Grampovnik, David J.; Chen, Hui-Ju; Flosi, William J.; Klein, Larry L.; Dekhtyar, Tatyana; Stoll, Vincent; Mamo, Mulugeta; Molla, Akhteruzzaman; Kempf, Dale J. (Abbott)

    2013-03-07

    Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

  5. Bragg gravity-gradiometer using the 1S0–3P1 intercombination transition of 88Sr

    Science.gov (United States)

    del Aguila, R. P.; Mazzoni, T.; Hu, L.; Salvi, L.; Tino, G. M.; Poli, N.

    2018-04-01

    We present a gradiometer based on matter-wave interference of alkaline-earth-metal atoms, namely 88Sr. The coherent manipulation of the atomic external degrees of freedom is obtained by large-momentum-transfer Bragg diffraction, driven by laser fields detuned away from the narrow 1S0–3P1 intercombination transition. We use a well-controlled artificial gradient, realized by changing the relative frequencies of the Bragg pulses during the interferometer sequence, in order to characterize the sensitivity of the gradiometer. The sensitivity reaches 1.5 × 10‑5 s‑2 for an interferometer time of 20 ms, limited only by geometrical constraints. We observed extremely low sensitivity of the gradiometric phase to magnetic field gradients, approaching a value 104 times lower than the sensitivity of alkali-atom based gradiometers, limited by the interferometer sensitivity. An efficient double-launch technique employing accelerated red vertical lattices from a single magneto-optical trap cloud is also demonstrated. These results highlight strontium as an ideal candidate for precision measurements of gravity gradients, with potential application in future precision tests of fundamental physics.

  6. Effective inhibition of lytic development of bacteriophages lambda, P1 and T4 by starvation of their host, Escherichia coli.

    Science.gov (United States)

    Łoś, Marcin; Golec, Piotr; Łoś, Joanna M; Weglewska-Jurkiewicz, Anna; Czyz, Agata; Wegrzyn, Alicja; Wegrzyn, Grzegorz; Neubauer, Peter

    2007-02-26

    Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, lambda, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

  7. Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1

    Directory of Open Access Journals (Sweden)

    Sheetal Bhasin

    2012-01-01

    Full Text Available The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI, an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS. HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production.

  8. Optimization of Fermentation Medium for the Production of Glucose Isomerase Using Streptomyces sp. SB-P1.

    Science.gov (United States)

    Bhasin, Sheetal; Modi, H A

    2012-01-01

    The combination of medium ingredients has a profound influence on the metabolic pathways running in the microorganism which regulates the production of numerous metabolites. Glucose isomerase (GI), an enzyme with huge potential in the market, can isomerise glucose into fructose. GI is used widely for the production of High-Fructose Corn Syrup (HFCS). HFCS is used as a sweetener in food and pharmaceutical industries. Streptomyces are well-known producers of numerous enzymes including glucose isomerase. An array of 75 isolates was screened for the production of glucose isomerase. The isolate Streptomyces sp. SB-P1 was found to produce maximum amount of extracellular GI. Sucrose and raffinose among pure carbon sources and corn cob and wheat husk among crude agro residues were found to yield high enzyme titers. Potassium nitrate among pure nitrogen sources and soy residues among crude sources gave maximum production. Quantitative effect of carbon, nitrogen, and inducer on GI was also determined. Plackett-Burman design was used to study the effect of different medium ingredients. Sucrose and xylose as carbon sources and peptone and soy residues as nitrogen sources proved to be beneficial for GI production.

  9. The role of a P1-type ATPase from Pseudomonas fluorescens SBW25 in copper homeostasis and plant colonization.

    Science.gov (United States)

    Zhang, Xue-Xian; Rainey, Paul B

    2007-05-01

    The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 possesses a putative copper-transporting P1-type ATPase (CueA) that is induced on the plant surfaces. Using a chromosomally-integrated cueA-lacZ fusion, we show that transcription of cueA can be induced (in vitro) by ions of copper, silver, gold, and mercury. To investigate the biological significance of cueA, a nonpolar cueA deletion mutant (SBW25 delta cueA) was constructed. This mutant strain displayed a twofold reduction in its tolerance to copper compared with the wild-type strain; however, no change was observed in the sensitivity of the mutant strain to silver, gold, or mercury ions. To obtain insight into the ecological significance of cueA, the competitive ability of SBW25 delta cueA was determined relative to wild-type SBW25 in three environments (none contained added copper): minimal M9 medium, the root of sugar beet (Beta vulgaris), and the root of pea (Pisum sativum). Results showed that the fitness of SBW25 delta cueA was not different from the wild type in laboratory medium but was compromised in the two plant environments. Taken together, these data demonstrate a functional role for CueA in copper homeostasis and reveal an ecologically significant contribution to bacterial fitness in the plant rhizosphere. They also suggest that copper ions accumulate on plant surfaces.

  10. Modeling Tolerance Development for the Effect on Heart Rate of the Selective S1P1 Receptor Modulator Ponesimod.

    Science.gov (United States)

    Lott, Dominik; Lehr, Thorsten; Dingemanse, Jasper; Krause, Andreas

    2017-09-15

    Ponesimod is a selective sphingosine-1-phosphate-1 (S1P 1 ) receptor modulator currently under investigation for the treatment of multiple sclerosis. S1P receptor modulators reduce heart rate following treatment initiation. This effect disappears with repeated dosing, enabling development of innovative uptitration regimens to optimize patient safety. There are currently no published pharmacokinetic/pharmacodynamic models describing the heart rate reduction of S1P receptor modulators in humans. The model developed here provides quantification of this effect for ponesimod. A direct-effect I max model with estimated maximum reduction of 45%, tolerance development, and circadian variation best described this effect. The pooled data from nine clinical studies enabled characterization of interindividual variability. The model was used to simulate different treatment regimens to compare the effect of high initial doses vs. gradual uptitration with respect to the occurrence of bradycardia. The results indicate a better safety profile when using gradual uptitration. The model allows studying dosing regimens not clinically tested in silico. © 2017 American Society for Clinical Pharmacology and Therapeutics.

  11. Effective inhibition of lytic development of bacteriophages λ, P1 and T4 by starvation of their host, Escherichia coli

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2007-02-01

    Full Text Available Abstract Background Bacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful. Results Here, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats. Conclusion Bacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

  12. Inflammatory effects of BaP1 a metalloproteinase isolated from Bothrops asper snake venom: leukocyte recruitment and release of cytokines.

    Science.gov (United States)

    Fernandes, Cristina Maria; Zamuner, Stella Regina; Zuliani, Juliana Pavan; Rucavado, Alexandra; Gutiérrez, José Maria; Teixeira, Catarina de Fátima Pereira

    2006-04-01

    The inflammatory events induced by BaP1, a 22.7 kDa metalloproteinase isolated from Bothrops asper snake venom, were studied. BaP1 i.p. injection in mice induced a marked inflammatory cell infiltrate into peritoneal cavity of animals with predominance of neutrophils in the early phase followed by mononuclear cells in the late period. Inhibition of enzymatic activity of BaP1 by chelation with EDTA resulted in a drastic reduction of this effect. In addition, BaP1 induced a significant increase of blood neutrophil numbers before its accumulation in peritoneal cavity, thus suggesting a stimulatory action of BaP1 on mechanisms of cell mobilization from bone marrow reserve compartments. A reduction in the number of neutrophils was observed in the exudate when antibodies against LECAM-1, CD18 and LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of BaP1. In contrast, there was no effect with antibodies against ICAM-1 and PECAM-1. Moreover, a conspicuous increment in the levels of IL-1 and TNF-alpha, but not of LTB4, was observed in peritoneal washes collected from mice injected with BaP1. It is concluded that BaP1 induces in vivo a marked leukocyte influx, which parallels an increased number of these cells in the blood, and is associated to the expression of specific leukocyte adhesion molecules and release of chemotactic inflammatory cytokines. Since BaP1 is a P-I class metalloproteinase, these results indicate that the proteolytic domain of metalloproteinases per se can trigger specific inflammatory events.

  13. Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

    Science.gov (United States)

    Farsky, S H; Gonçalves, L R; Gutiérrez, J M; Correa, A P; Rucavado, A; Gasque, P; Tambourgi, D V

    2000-01-01

    The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a. PMID:11200361

  14. Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiyuan [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); An, Byoung Ha [Department of Food and Nutrition, College of Life Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Kim, Min Jung; Park, Jong Hoon [Department of Biological Science, Sookmyung Women’s University, Seoul (Korea, Republic of); Kang, Young Sook [Department of Pharmacy, College of Pharmacy, Sookmyung Women’s University, Seoul (Korea, Republic of); Chang, Minsun, E-mail: minsunchang@sm.ac.kr [Department of Medical and Pharmaceutical Science, College of Science, Sookmyung Women’s University, Seoul (Korea, Republic of)

    2014-09-26

    Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1 (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.

  15. Glutathione-S-transferase (GST) P1, GSTM1, exercise, ozone and asthma incidence in school children.

    Science.gov (United States)

    Islam, T; Berhane, K; McConnell, R; Gauderman, W J; Avol, E; Peters, J M; Gilliland, F D

    2009-03-01

    Because asthma has been associated with exercise and ozone exposure, an association likely mediated by oxidative stress, we hypothesised that glutathione-S-transferase (GST)P1, GSTM1, exercise and ozone exposure have interrelated effects on the pathogenesis of asthma. Associations of the well characterised null variant of GSTM1 and four single nucleotide polymorphisms (SNPs) that characterised common variation in the GSTP1 locus with new onset asthma in a cohort of 1610 school children were examined. Children's exercise and ozone exposure were classified using participation in team sports and community annual average ozone levels, respectively. A two SNP model involving putatively functional variants (rs6591255, rs1695 (Ile105Va)) best captured the association between GSTP1 and asthma. The risk of asthma was lower for those with the Val allele of Ile105Val (hazard ratio (HR) 0.60, 95% CI 0.4 to 0.8) and higher for the variant allele of rs6591255 (HR 1.40, 95% CI 1.1 to 1.9). The risk of asthma increased with level of exercise among ile(105) homozygotes but not among those with at least one val(105) allele (interaction p value = 0.02). The risk was highest among ile(105) homozygotes who participated in >or=3 sports in the high ozone communities (HR 6.15, 95% CI 2.2 to 7.4). GSTM1 null was independently associated with an increased risk of asthma and showed little variation with air pollution or GSTP1 genotype. These results were consistent in two independent fourth grade cohorts recruited in 1993 and 1996. Children who inherit a val(105) variant allele may be protected from the increased risk of asthma associated with exercise, especially in high ozone communities. GSTM1 null genotype was associated with an increased risk of asthma.

  16. Three different up-titration regimens of ponesimod, an S1P1 receptor modulator, in healthy subjects.

    Science.gov (United States)

    Scherz, Michael W; Brossard, Patrick; D'Ambrosio, Daniele; Ipek, Murat; Dingemanse, Jasper

    2015-06-01

    Ponesimod is a selective S1P1 receptor modulator, and induces dose-dependent reduction of circulating lymphocytes upon oral dosing. Previous studies showed that single doses up to 75 mg or multiple doses up to 40 mg once daily are well tolerated, and heart rate (HR) reduction and atrio-ventricular conduction delays upon treatment initiation are reduced by gradual up-titration to the maintenance dose. This single-center, open-label, randomized, multiple-dose, 3-treatment, 3-way crossover study compared the tolerability, safety, pharmacokinetics, cardiodynamics, and effects on lymphocytes of 3 different up-titration regimens of ponesimod in healthy male and female subjects. Up-titration regimens comprised escalating periods of b.i.d. dosing (2.5 or 5 mg) and q.d. dosing (10 or 20 mg or both). After the third up-titration period a variable-duration washout period of 1-3 days was followed by re-challenge with a single 20-mg dose of ponesimod. Adverse events were transient and mild to moderate in intensity, not different between regimens. HR decrease after the first dose was greater than after all subsequent doses, including up-titration doses. Little or no HR change was observed with morning doses of b.i.d. regimens, suggesting that 2.5 and 5 mg b.i.d. are sufficient to sustain cardiac desensitization for the 12-hours dosing interval. © 2015, The American College of Clinical Pharmacology.

  17. Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotroph Arthrobacter P1

    NARCIS (Netherlands)

    Attwood, Margaret M.; Arfman, Nico; Weusthuis, Ruud A.; Dijkhuizen, Lubbert

    1992-01-01

    When Arthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD+-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular

  18. Role of PTHrP(1-34) Pulse Frequency Versus Pulse Duration to Enhance Mesenchymal Stromal Cell Chondrogenesis.

    Science.gov (United States)

    Fischer, Jennifer; Ortel, Marlen; Hagmann, Sebastien; Hoeflich, Andreas; Richter, Wiltrud

    2016-12-01

    Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix, and avoidance of hypertrophy being prime challenges. We recently demonstrated that parathyroid hormone-related protein (PTHrP) is a promising factor to direct chondrogenesis of MSCs towards an articular phenotype, since intermittent PTHrP application stimulated cartilage matrix production and reduced undesired hypertrophy. We here investigated the role of frequency, pulse duration, total exposure time, and underlying mechanisms in order to unlock the full potential of PTHrP actions. Human MSC subjected to in vitro chondrogenesis for six weeks were exposed to 2.5 nM PTHrP(1-34) pulses from days 7 to 42. Application frequency was increased from three times weekly (3 × 6 h/week) to daily maintaining either the duration of individual pulses (6 h/day) or total exposure time (18 h/week; 2.6 h/day). Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline-phosphatase activity by 87%. High total exposure time significantly reduced cell proliferation at day 14. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP-induced suppression of the hypertrophic markers MEF2C and IBSP. COL10A1, RUNX2, and MMP13 expression remained unaltered. Decreased IGFBP-2, -3, and -6 expression suggested modulated IGF-I availability in PTHrP groups, while drop of SOX9 protein levels during the PTHrP-pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP-pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF- and SOX9-related mechanisms. J. Cell. Physiol. 231: 2673-2681, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Backup expression of the PhaP2 phasin compensates for phaP1 deletion in Herbaspirillum seropedicae, maintaining fitness and PHB accumulation

    Directory of Open Access Journals (Sweden)

    Luis Paulo Silveira Alves

    2016-05-01

    Full Text Available Phasins are important proteins controlling PHB granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈ 50 % in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was 6-fold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50% of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria.

  20. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  1. GIS-NaP1 zeolite microspheres as potential water adsorption material: Influence of initial silica concentration on adsorptive and physical/topological properties.

    Science.gov (United States)

    Sharma, Pankaj; Song, Ju-Sub; Han, Moon Hee; Cho, Churl-Hee

    2016-03-11

    GIS-NaP1 zeolite samples were synthesized using seven different Si/Al ratios (5-11) of the hydrothermal reaction mixtures having chemical composition Al2O3:xSiO2:14Na2O:840H2O to study the impact of Si/Al molar ratio on the water vapour adsorption potential, phase purity, morphology and crystal size of as-synthesized GIS-NaP1 zeolite crystals. The X-ray diffraction (XRD) observations reveal that Si/Al ratio does not affect the phase purity of GIS-NaP1 zeolite samples as high purity GIS-NaP1 zeolite crystals were obtained from all Si/Al ratios. Contrary, Si/Al ratios have remarkable effect on the morphology, crystal size and porosity of GIS-NaP1 zeolite microspheres. Transmission electron microscopy (TEM) evaluations of individual GIS-NaP1 zeolite microsphere demonstrate the characteristic changes in the packaging/arrangement, shape and size of primary nano crystallites. Textural characterisation using water vapour adsorption/desorption, and nitrogen adsorption/desorption data of as-synthesized GIS-NaP1 zeolite predicts the existence of mix-pores i.e., microporous as well as mesoporous character. High water storage capacity 1727.5 cm(3) g(-1) (138.9 wt.%) has been found for as-synthesized GIS-NaP1 zeolite microsphere samples during water vapour adsorption studies. Further, the total water adsorption capacity values for P6 (1299.4 mg g(-1)) and P7 (1388.8 mg g(-1)) samples reveal that these two particular samples can absorb even more water than their own weights.

  2. On functional bases of the first-order differential invariants for non-conjugate subgroups of the Poincaré group $P(1,4$

    Directory of Open Access Journals (Sweden)

    V.M. Fedorchuk

    2008-11-01

    Full Text Available It is established which functional bases of the first-order differential invariants of the splitting and non-splitting subgroups of the Poincaré group $P(1,4$ are invariant under the subgroups of the extended Galilei group $widetilde G(1,3 subset P(1,4$. The obtained sets of functional bases are classified according to dimensions.

  3. The potential of P1 site alterations in peptidomimetic protease inhibitors as suggested by virtual screening and explored by the use of C-C-coupling reagents.

    Science.gov (United States)

    Weik, Steffen; Luksch, Torsten; Evers, Andreas; Böttcher, Jark; Sotriffer, Christoph A; Hasilik, Andrej; Löffler, Hans-Gerhard; Klebe, Gerhard; Rademann, Jörg

    2006-04-01

    A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.

  4. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    International Nuclear Information System (INIS)

    Tejedor, F.; Amils, R.; Ballesta, J.P.G.

    1987-01-01

    The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

  5. Extremophiles Survival to Simulated Space Conditions: An Astrobiology Model Study

    Science.gov (United States)

    Mastascusa, V.; Romano, I.; Di Donato, P.; Poli, A.; Della Corte, V.; Rotundi, A.; Bussoletti, E.; Quarto, M.; Pugliese, M.; Nicolaus, B.

    2014-09-01

    In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

  6. Structural and Functional Characterization of an Archaeal Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated Complex for Antiviral Defense (CASCADE)

    DEFF Research Database (Denmark)

    Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K

    2011-01-01

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA....... The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5......a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2...

  7. Blister formation and skin damage induced by BaP1, a haemorrhagic metalloproteinase from the venom of the snake Bothrops asper.

    Science.gov (United States)

    Rucavado, A; Núñez, J; Gutiérrez, J M

    1998-01-01

    Blister formation and skin damage can be induced by BaP1, a haemorrhagic metalloproteinase from the venom of the snake Bothrops asper. Pathological changes in the skin were investigated after intramuscular injections of Bothrops asper haemorrhagic metalloproteinase BaP1. Blisters developed within the first hour, with separation of epidermis from the dermal-epidermal junction, whereas acantholysis of epithelial cells was not observed. After the third hour there was ulceration with formation of a proteinaceous scab and inflammatory infiltrate. By 7 to 14 days there was evidence of a regenerative process in dermis and epidermis. Haemorrhage occurred in both dermis and hypodermis as a consequence of BaP1 injection, together with damage of sebaceous glands and an inflammatory reaction in which enlarged macrophages were the predominant cell type. Zymography assays showed the presence of several endogenous metalloproteinases in the exudate, skin homogenates and plasma. In addition, BaP1 was detected in exudates and plasma by immunoblotting. This technique also demonstrated the presence of components immunologically related to laminin and collagen type IV in exudates. It is suggested that BaP1, and probably endogenous matrix metalloproteinases, degrade some protein components at the dermal-epidermal junction, inducing the formation of blisters. PMID:9797720

  8. Evaluation of the level of house dust mite allergens, Der p 1 and Der f 1 in Iranian homes, a nationwide study.

    Science.gov (United States)

    Fereidouni, M; Fereidouni, F; Hadian, M; Nourani Hasankiadeh, Sh; Mazandarani, M; Ziaee, M

    2013-01-01

    Exposure to house dust mites (HDMs) is a major risk factor for the development of allergic symptoms. HDMs are worldwide in distribution. Assessing these allergens in each area is a critical step in evaluating the risk of sensitisation and controlling allergic symptoms. The aim of the present study was to evaluate the level of major HDMs, Der p 1 and Der f 1, in various parts of Iran. In 2009, 257 dust samples were obtained from living rooms' carpets in seven cities throughout Iran with different geoclimatic conditions. The level of Der p 1 and Der f 1 was measured by commercial ELISA. Detectable level of Der p 1 and Der f 1 levels were only found in Gorgan and Sari, two cities near the Caspian Sea with moderate temperatures and high relative humidity. In both of these cities, Der f 1 was more frequent than Der p 1 (100% vs. 85%) and was found to be at a higher level than Der p 1 (geometric mean 3128 vs. 439ng/g dust, Pdust mites in Iran are restricted to the Caspian Sea coastal areas, and in other parts, due to seasonal variations of temperature and humidity mites are not able to grow well and therefore are not an important risk factor for sensitisation and respiratory allergies. Copyright © 2012 SEICAP. Published by Elsevier Espana. All rights reserved.

  9. The Effects of Temperature and Growth Phase on the Lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii

    DEFF Research Database (Denmark)

    Jensen, Sara Munk; Neesgaard, Vinnie Lund; Skjoldbjerg, Sandra Landbo Nedergaard

    2015-01-01

    The functionality of the plasma membrane is essential for all organisms. Adaption to high growth temperatures imposes challenges and Bacteria, Eukarya, and Archaea have developed several mechanisms to cope with these. Hyperthermophilic archaea have earlier been shown to synthesize tetraether...... at three different temperatures, with samples withdrawn during lag, exponential, and stationary phases. Three abundant tetraether lipid classes and one diether lipid class were monitored. Beside the expected increase in the number of cyclopentane moieties with higher temperature in both archaea, we...... observed previously unreported changes in the average cyclization of the membrane lipids throughout growth. The average number of cyclopentane moieties showed a significant dip in exponential phase, an observation that might help to resolve the currently debated biosynthesis pathway of tetraether lipids....

  10. Infrared spectroscopic study of the structural and functional properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii.

    Science.gov (United States)

    Dzafić, E; Klein, O; Goswami, P; Kühlbrandt, W; Mäntele, W

    2009-06-01

    In this study, structural, functional, and mechanistic properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na(+)/H(+) antiporters are generally responsible for the regulation of cytoplasmic pH and Na(+) concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive. The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to alpha-helical segments. In contrast, the temperature-induced structural changes for beta-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.

  11. PET imaging of inflammation and adenocarcinoma xenografts using vascular adhesion protein 1 targeting peptide {sup 68}Ga-DOTAVAP-P1: comparison with {sup 18}F-FDG

    Energy Technology Data Exchange (ETDEWEB)

    Autio, Anu; Ujula, Tiina; Luoto, Pauliina [University of Turku and Turku University Hospital, Turku PET Centre, Turku (Finland); Salomaeki, Satu [University of Turku and Turku University Hospital, Turku PET Centre, Turku (Finland); University of Turku, Department of Chemistry, Turku (Finland); Jalkanen, Sirpa [University of Turku, MediCity Research Laboratory, Turku (Finland); National Public Health Institute, Turku (Finland); Roivainen, Anne [University of Turku and Turku University Hospital, Turku PET Centre, Turku (Finland); University of Turku, Turku Centre for Disease Modeling, Turku (Finland)

    2010-10-15

    The aim of this study was to evaluate inflammation and tumour imaging with a vascular adhesion protein 1 (VAP-1) targeting peptide {sup 68}Ga-DOTAVAP-P1 in comparison with {sup 18}F-FDG. Rats with both subcutaneous human pancreatic adenocarcinoma xenografts and turpentine oil-induced acute sterile inflammation were evaluated by dynamic positron emission tomography (PET) and by digital autoradiography of tissue cryosections. Subsequently, the autoradiographs were combined with histological and immunohistological analysis of the sections. {sup 68}Ga-DOTAVAP-P1 delineated acute, sterile inflammation comparable with {sup 18}F-FDG. However, the tumour uptake of {sup 68}Ga-DOTAVAP-P1 was low in contrast to prominent {sup 18}F-FDG uptake. The standardised uptake values of inflammation and tumours by PET were 1.1 {+-} 0.4 (mean {+-} SEM) and 0.4 {+-} 0.1 for {sup 68}Ga-DOTAVAP-P1 and 2.0 {+-} 0.5 and 1.6 {+-} 0.8 for {sup 18}F-FDG, respectively. In addition, PET studies showed inflammation to muscle and tumour to muscle ratios of 5.1 {+-} 3.1 and 1.7 {+-} 0.3 for {sup 68}Ga-DOTAVAP-P1 and 6.2 {+-} 0.7 and 4.6 {+-} 2.2 for {sup 18}F-FDG, respectively. Immunohistochemistry revealed increased expression of luminal VAP-1 on the endothelium at the site of inflammation and low expression in the tumour The {sup 68}Ga-DOTAVAP-P1 PET was able to visualise inflammation better than tumour, which was in accordance with the luminal expression of VAP-1 on vasculature in these experimental models. (orig.)

  12. Pin p 1 is a major allergen in pine nut and the first food allergen described in the plant group of gymnosperms.

    Science.gov (United States)

    Cabanillas, Beatriz; Crespo, Jesus F; Maleki, Soheila J; Rodriguez, Julia; Novak, Natalija

    2016-11-01

    This study aimed to report the complete sequence of a 2S albumin purified from pine nut and to analyze its allergenic properties. Individual recognition of this protein by serum IgE from pine nut-allergic patients was assessed. IgE cross-linking capacity was analyzed in a basophil activation test. Inhibition of IgE-binding and stability to heating was also assessed. The complete nucleotide sequence was obtained and a phylogenetic study was carried out. 2S albumin from pine nut (registered as Pin p 1.0101) was recognized by IgE of 75% of sera. The allergen was heat-stable and had a robust capacity to inhibit IgE-binding to whole pine nut extract. The IgE cross-linking capacity of Pin p 1 on basophils was also demonstrated. Despite the low homology of Pin p 1 sequence with other allergenic 2S albumins from angiosperms, Pin p 1 contains the typical skeleton of 8 cysteine residues, important for its α-helixes enriched structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery.

    Science.gov (United States)

    Schmitz, Karl R; Carney, Daniel W; Sello, Jason K; Sauer, Robert T

    2014-10-28

    Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.

  14. K(+) currents produce P(1) in the RW CAP: evidence from DC current bias, K(+) channel blockade and recordings from cochlea and brainstem.

    Science.gov (United States)

    Brown, D J; McMahon, C M; Patuzzi, R B

    2004-04-01

    Tone-burst-evoked compound action potentials (CAP) from the guinea pig round window (RW) are altered by DC current injection through the RW. The CAP waveform consists of a series of interleaved negative and positive peaks (N(1), P(1), N(2), P(2) etc.) of decreasing amplitude. During positive DC current injection (around +50 microA) the positive peaks are depressed substantially and there is an overall negative baseline shift of the waveform following the N(1). Negative current injection (around -50 microA) increased the positive peaks, in particular P(1), and produced an overall positive baseline shift following the N(1) peak. Results support our hypothesis that the first and dominant N(1) peak in the RW CAP is due to depolarising Na(+) currents into the primary afferent dendrites and axons within the cochlea, and that the P(1) potential is largely due to the exit of the hyperpolarising K(+) currents in the same cells. We have reached this conclusion on the basis of the sign and latency of the N(1) and P(1) components at the RW, beneath the myelin layers around the spiral ganglion cells, at the internal auditory meatus (IAM) within the brain case, and on the basis of the differential susceptibility of the various peaks to perfusion of lidocaine in the cochlear nucleus, sectioning of the cochlear nerve at the IAM, application of the K(+) channel blockers 4-amino-pyridine and tetraethylammonium within the cochlea, and DC current biasing at the RW.

  15. Group 5 allergens of timothy grass (Phl p 5) bear cross-reacting T cell epitopes with group 1 allergens of rye grass (Lol p 1).

    Science.gov (United States)

    Müller, W D; Karamfilov, T; Bufe, A; Fahlbush, B; Wolf, I; Jäger, L

    1996-04-01

    Selected human T cell clones reactive with group 5 allergens of timothy grass (Phl p 5) were cross-stimulated in specific proliferation assays with group 1 allergens of rye grass (Lol p 1). Such interspecies cross-reactivities result obviously from structural motifs presented on defined Phl p 5 fragments as shown with recombinant Phl p 5 products.

  16. Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues

    DEFF Research Database (Denmark)

    Jiang, Longguang; Zhao, Baoyu; Xu, Peng

    2015-01-01

    Abstract Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (∼70 fold) to the target...

  17. Determination of the effective radiative lifetimes of the 6 3P1 atomic mercury level in low-pressure mercury discharges

    International Nuclear Information System (INIS)

    van de Weijer, P.; Cremers, R.M.M.

    1984-01-01

    Experiments are described in which low-pressure mercury, mercury-argon and mercury-krypton discharges were irradiated with a dye laser pulse at 365.5 nm, thus exciting mercury atoms from the metastable 6 3 P 2 level to the 6 3 D 2 level. The 6 3 D 2 level decays radiatively to the 6 P levels. By recording the time dependence of the overpopulation in the 6 3 P 1 and the 6 1 P 1 level at the fluorescence signals at 254 nm and 185 nm, respectively, the effective radiative lifetime of these levels were determined. The effective radiative lifetime of the 6 3 P 1 level was measured in the k 0 R regime 0.1-500. The 6 1 P 1 lifetime was determined for the following discharge conditions: tube diameter 10-36 mm, mercury density 7.10 18 -2.10 21 m -3 , and noble gas pressure 0, 130, 400 Pa

  18. Integrated synthesis of zeolites 4A and Na-P1 using coal fly ash for application in the formulation of detergents and swine wastewater treatment.

    Science.gov (United States)

    Cardoso, Ariela M; Horn, Martha B; Ferret, Lizete S; Azevedo, Carla M N; Pires, Marçal

    2015-04-28

    Several researchers have reported zeolite synthesis using coal ash for a wide range of applications. However, little attention has been given to green processes, including moderate synthesis conditions, using waste as raw material and effluent reuse or reduction. In this study, Brazilian coal fly ashes were used for integrated synthesis of zeolites 4A and Na-P1 by two different routes and under moderate operating conditions (temperature and pressure). Both procedures produced zeolites with similar conversions (zeolite 4A at 82% purity and zeolite Na-P1 at 57-61%) and high CEC values (zeolites 4A: 4.5meqCa(2+)g(-1) and zeolites Na-P1: 2.6-2.8meqNH4(+)g(-1)). However, process 1 generated less effluent for the zeolite mass produced (7mLg(-1)), with low residual Si and Al levels and 74% of the Si available in the coal fly ash incorporated into the zeolite, while only 55% is used in process 2. For use as a builder in detergents, synthetic zeolite 4A exhibited conformity parameters equal to or greater than those of the commercial zeolite adopted as reference. Treatment of swine wastewater with zeolite Na-P1 resulted in a high removal capacity for total ammoniacal nitrogen (31mgg(-1)). Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1. Formaldehyde is the inducing signal for the synthesis of the RuMP cycle enzyme hexulose phosphate synthase

    NARCIS (Netherlands)

    Croes, L.M.; Dijkhuizen, L.

    The inducing potential of formaldehyde on the synthesis of hexulose phosphate synthase, a key enzyme of the RuMP cycle in Arthrobacter P1, was investigated in resting cell suspensions. Induction of this enzyme only occurred at formaldehyde concentrations of 0.5 mM and below. No evidence was obtained

  20. Regulation of methylamine and formaldehyde metabolism in Arthrobacter P1. Effect of pulse-wise addition of "heterotrophic" substrates to C1 substrate-limited continuous cultures

    NARCIS (Netherlands)

    Croes, L.M.; Tiesma, L.; Dijkhuizen, L.

    1986-01-01

    The regulation of methylamine and formaldehyde metabolism in Arthrobacter P1 was investigated in carbon-limited continuous cultures. To avoid toxic effects of higher formaldehyde concentrations, formaldehyde-limited cultures were established in smooth substrate transitions from choline-limitation.

  1. Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii.

    Science.gov (United States)

    Kedrov, Alexej; Wegmann, Susanne; Smits, Sander H J; Goswami, Panchali; Baumann, Hella; Muller, Daniel J

    2007-08-01

    Integral membrane proteins are involved in virtually every cellular process. Precisely regulating these machineries would allow controlling many human and vertebrate diseases. Embedded into cellular membranes, membrane proteins establish molecular interactions that sensitively react to environmental changes and to molecular compounds, such as ligands or inhibitors. We applied atomic force microscopy (AFM) to image the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii, and single-molecule force spectroscopy (SMFS) to probe molecular interactions that drive the protein structure-function relationship. High-resolution AFM topographs showed the dimeric assembly of MjNhaP1 being reconstituted into a lipid bilayer. SMFS of MjNhaP1 unraveled molecular interactions stabilizing individual structural domains. Transmembrane domains exhibited certain probabilities to unfold individually or cooperatively with other domains resulting in different unfolding pathways. Helices VIII and X established pH sensitive interactions altering significantly upon MjNhaP1 activation, while removal of the ligand (Na(+)) destabilized the entire antiporter except helix VIII. It is assumed that Asp234/235 of helix VIII are involved in the ligand-binding site and that helix X plays a functional role in the activation of the transporter.

  2. Dormancy in Deinococcus sp. UDEC-P1 as a survival strategy to escape from deleterious effects of carbon starvation and temperature.

    Science.gov (United States)

    Guerra, Matías; González, Karina; González, Carlos; Parra, Boris; Martínez, Miguel

    2015-09-01

    Dormancy is characterized by low metabolism and absence of protein synthesis and cellular division enabling bacterial cells to survive under stress. The aim was to determine if carbon starvation and low temperature are factors that modify the proportion of dormant/active cells in Deinococcus sp. UDEC-P1. By flow cytometry, RedoxSensor Green (RSG) was used to quantify metabolic activity and Propidium Iodide (PI) to evaluate membrane integrity in order to determine the percentage of dormant cells. Cell size and morphology were determined using scanning electronic microscopy. Under carbon starvation at 30°C, Deinococcus sp. UDEC-P1 increased its proportion of dormant cells from 0.1% to 20%, decreased the count of culturable cells and average cell volume decreased 7.1 times. At 4°C, however, the proportion of dormant cells increased only to 6%, without a change in the count of culturable cells and an average cellular volume decrease of 4.1 times and 3% of the dormant cells were able to be awakened. Results indicate a greater proportion of dormant Deinococcus sp. UDEC-P1 cells at 30ºC and it suggests that carbon starvation is more deleterious condition at 30ºC than 4ºC. For this reason Deinococcus sp. UDEC-P1 cells are more likely to enter into dormancy at higher temperature as a strategy to survive. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  3. A specific and efficient photoreaction between E. coli RNA polymerase and T+1 in the lacUV5 or deoP1 promoter

    DEFF Research Database (Denmark)

    Jeppesen, Claus; Jensen, Kaj Frank; Nielsen, Peter E.

    1988-01-01

    Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when...

  4. Re-Evaluation of Binding Properties of Recombinant Lymphocyte Receptors NKR-P1A and CD69 to Chemically Synthesized Glycans and Peptides

    Czech Academy of Sciences Publication Activity Database

    Rozbeský, Daniel; Krejzová, Jana; Křenek, Karel; Prchal, J.; Hrabal, R.; Kožíšek, Milan; Weignerová, Lenka; Fiore, M.; Dumy, P.; Renaudet, O.; Křen, Vladimír

    2014-01-01

    Roč. 15, č. 1 (2014), s. 1271-1283 E-ISSN 1422-0067 R&D Projects: GA MŠk(CZ) LD13042 Institutional support: RVO:61388971 ; RVO:61388963 Keywords : CD69 * NKR-P1A * NMR titration Subject RIV: CE - Biochemistry Impact factor: 2.862, year: 2014

  5. IncP-1 and PromA group plasmids are major providers of horizontal gene transfer capacities across bacteria in the mycosphere of different soil fungi

    NARCIS (Netherlands)

    Zhang, Miaozhi; Visser, Sander; Pereira e Silva, Michele C.; van Elsas, Jan Dirk

    Plasmids of the IncP-1 beta group have been found to be important carriers of accessory genes that enhance the ecological fitness of bacteria, whereas plasmids of the PromA group are key agents of horizontal gene transfer in particular soil settings. However, there is still a paucity of knowledge

  6. CENTRAL AUDTIORY DEVELOPMENT IN CHILDREN WITH HEARING LOSS: CLINICAL RELEVANCE OF THE P1 CAEP BIOMARKER IN HEARING-IMPAIRED CHILDREN WITH MULTIPLE DISABILITIES.

    Science.gov (United States)

    Sharma, Anu; Glick, Hannah; Campbell, Julia; Biever, Allison

    2013-09-01

    First, we review the development and plasticity of the central auditory pathways in infants and children with hearing loss who are fitted with cochlear implants (CIs). Second, we describe case studies demonstrating the clinical utility of the P1 central auditory evoked potential (CAEP) for evaluating cortical auditory maturation in the rapidly increasing number of cochlear-implanted children who have multiple disabilities. Children who receive CIs provide a platform to examine the trajectories of deprivation-induced and experience-dependent plasticity in the central auditory system. We review the evidence for, and time limits of sensitive periods for cortical auditory maturation framing an optimal period for cochlear implantation. Finally, we evaluate the use of the P1 biomarker as an objective assessment tool in the special case of children with multiple disabilities. The P1 response was useful in assessing central auditory maturation in patients with CHARGE association, ANSD, and Pallister-Killian Syndrome concomitant with hearing loss. The presence of co-existing disabilities in addition to hearing loss poses unique challenges regarding both pre-intervention evaluation and post-intervention rehabilitation for children with multiple disabilities. When combined with a standard audiological test battery, the P1 CAEP biomarker has a useful role in objectively evaluating the maturation of central auditory pathways to determine the effectiveness of various intervention strategies in hearing-impaired children with multiple disabilities.

  7. Immuno-modulatory activity of Ganoderma lucidum-derived polysacharide on human monocytoid dendritic cells pulsed with Der p 1 allergen

    Directory of Open Access Journals (Sweden)

    Lo Shih-Yen

    2011-05-01

    Full Text Available Abstract Background Ganoderma lucidum-derived polysaccharide (PS-G can rapidly and effectively promote the activation and maturation of immature dendritic cells (DCs, suggesting that PS-G possesses the capacity to regulate immune responses. This study aimed to clarify the immunologic effect of PS-G on monocyte-derived dendritic cells (MD-DCs from asthmatic children allergic to house dust mites. The MD-DCs were stimulated for 24 h with the related allergen, Der p 1, in the presence or absence of PS-G. Cell surface markers and phagocytic capacity were assessed by FACS analysis, and key polarizing cytokines (IL-12 p40, IL-12 p70, IL-6, IL-23, and IL-10 were quantified. The subsequent regulatory effect of pulsed MD-DCs on naïve T cells was evaluated by determining the T-cell cytokine profile. Results PS-G induced the maturation of MD-DCs and decreased phagocytic capacity, even if pulsed with Der p 1. After incubation with PS-G and Der p 1, MD-DCs produced higher amounts of IL-12 p70, IL-12 p40, IL-6, IL-23, and IL10 than Der p 1-pulsed DCs. Furthermore, type 1 helper T (Th1 cell cytokine (INF-γ production was highly increased when naïve autologous T cells were co-cultured with Der p 1-pulsed MD-DCs. Naïve T cells stimulated by MD-DCs pulsed with Der p 1 failed to produce proliferation of T-cells, whereas the addition of PS-G to Der p 1 induced a significant proliferation of T-cells similar to that observed with PS-G alone. Conclusion The presence of PS-G in an allergen pulse promoted allergic MD-DCs to produce IL-12 p70, IL-12 p40, IL-6, IL-23, and IL-10, and exerted an effect on shifting the immune balance towards Th1 in children with allergic asthma.

  8. Mechanisms of vasodilation to PTH 1-84, PTH 1-34, and PTHrP 1-34 in rat bone resistance arteries.

    Science.gov (United States)

    Benson, T; Menezes, T; Campbell, J; Bice, A; Hood, B; Prisby, R

    2016-05-01

    Parathyroid hormone (PTH) augments bone metabolism and bone mass when given intermittently. Enhanced blood flow is requisite to support high tissue metabolism. The bone arteries are responsive to all three PTH analogs, which may serve to augment skeletal blood flow during intermittent PTH administration. PTH augments bone metabolism. Yet, mechanisms by which PTH regulates bone blood vessels are unknown. We deciphered (1) endothelium-dependent and endothelium-independent vasodilation to PTH 1-84, PTH 1-34, and PTHrP 1-34, (2) the signaling pathways (i.e., endothelial nitric oxide synthase [eNOS], cyclooxygenase [COX], protein kinase C [PKC], and protein kinase A [PKA]), and (3) receptor activation. Femoral principal nutrient arteries (PNAs) were given cumulative doses (10(-13)-10(-8) M) of PTH 1-84, PTH 1-34, and PTHrP 1-34 with and without signaling pathway blockade. Vasodilation was also determined following endothelial cell removal (i.e., denudation), PTH 1 receptor (PTH1R) inhibition and to sodium nitroprusside (SNP; a nitric oxide [NO] donor). Vasodilation was lowest to PTH 1-34, and maximal dilation was highest to PTHrP 1-34. Inhibition of eNOS reduced vasodilation to PTH 1-84 (-80 %), PTH 1-34 (-66 %), and PTHrP 1-34 (-48 %), evidencing the contribution of NO. Vasodilation following denudation was eliminated (PTH 1-84 and PTHrP 1-34) and impaired (PTH 1-34, 17 % of maximum), highlighting the importance of endothelial cells for PTH signaling. Denuded and intact PNAs responded similarly to SNP. Both PKA and PKC inhibition diminished vasodilation in all three analogs to varying degrees. PTH1R blockade reduced vasodilation to 1, 12, and 12 % to PTH 1-84, PTH 1-34, and PTHrP 1-34, respectively. Vasodilation of femoral PNAs to the PTH analogs occurred via activation of the endothelial cell PTH1R for NO-mediated events. PTH 1-84 and PTHrP 1-34 primarily stimulated PKA signaling, and PTH 1-34 equally stimulated PKA and PKC signaling.

  9. Ponesimod, a selective S1P1 receptor modulator: a potential treatment for multiple sclerosis and other immune-mediated diseases

    Science.gov (United States)

    D’Ambrosio, Daniele; Freedman, Mark S.; Prinz, Joerg

    2016-01-01

    The first oral treatment for relapsing multiple sclerosis, the nonselective sphingosine-1-phosphate receptor (S1PR) modulator fingolimod, led to identification of a pivotal role of sphingosine-1-phosphate and one of its five known receptors, S1P1R, in regulation of lymphocyte trafficking in multiple sclerosis. Modulation of S1P3R, initially thought to cause some of fingolimod’s side effects, prompted the search for novel compounds with high selectivity for S1P1R. Ponesimod is an orally active, selective S1P1R modulator that causes dose-dependent sequestration of lymphocytes in lymphoid organs. In contrast to the long half-life/slow elimination of fingolimod, ponesimod is eliminated within 1 week of discontinuation and its pharmacological effects are rapidly reversible. Clinical data in multiple sclerosis have shown a dose-dependent therapeutic effect of ponesimod and defined 20 mg as a daily dose with desired efficacy, and acceptable safety and tolerability. Phase II clinical data have also shown therapeutic efficacy of ponesimod in psoriasis. These findings have increased our understanding of psoriasis pathogenesis and suggest clinical utility of S1P1R modulation for treatment of various immune-mediated disorders. A gradual dose titration regimen was found to minimize the cardiac effects associated with initiation of ponesimod treatment. Selectivity for S1P1R, rapid onset and reversibility of pharmacological effects, and an optimized titration regimen differentiate ponesimod from fingolimod, and may lead to better safety and tolerability. Ponesimod is currently in phase III clinical development to assess efficacy and safety in relapsing multiple sclerosis. A phase II study is also ongoing to investigate the potential utility of ponesimod in chronic graft versus host disease. PMID:26770667

  10. Zea mays (L. P1 locus for cob glume color identified as a post-domestication selection target with an effect on temperate maize genomes

    Directory of Open Access Journals (Sweden)

    Chuanxiao Xie

    2013-10-01

    Full Text Available Artificial selection during domestication and post-domestication improvement results in loss of genetic diversity near target loci. However, the genetic locus associated with cob glume color and the nature of the genomic pattern surrounding it was elusive and the selection effect in that region was not clear. An association mapping panel consisting of 283 diverse modern temperate maize elite lines was genotyped by a chip containing over 55,000 evenly distributed SNPs. Ten-fold resequencing at the target region on 40 of the panel lines and 47 tropical lines was also undertaken. A genome-wide association study (GWAS for cob glume color confirmed the P1 locus, which is located on the short arm of chromosome 1, with a − log10P value for surrounding SNPs higher than the Bonferroni threshold (α/n, α < 0.001 when a mixed linear model (MLM was implemented. A total of 26 markers were identified in a 0.78 Mb region surrounding the P1 locus, including 0.73 Mb and 0.05 Mb upstream and downstream of the P1 gene, respectively. A clear linkage disequilibrium (LD block was found and LD decayed very rapidly with increasing physical distance surrounding the P1 locus. The estimates of π and Tajima's D were significantly (P < 0.001 lower at both ends compared to the locus. Upon comparison of temperate and tropical lines at much finer resolution by resequencing (180-fold finer than chip SNPs, a more structured LD block pattern was found among the 40 resequenced temperate lines. All evidence indicates that the P1 locus in temperate maize has not undergone neutral evolution but has been subjected to artificial selection during post-domestication selection or improvement. The information and analytical results generated in this study provide insights as to how breeding efforts have affected genome evolution in crop plants.

  11. Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, TvDyP1 and TvVP2, Identified from the Secretome of Trametes versicolor.

    Science.gov (United States)

    Amara, Sawsan; Perrot, Thomas; Navarro, David; Deroy, Aurélie; Benkhelfallah, Amine; Chalak, Amani; Daou, Marianne; Chevret, Didier; Faulds, Craig B; Berrin, Jean-Guy; Morel-Rouhier, Mélanie; Gelhaye, Eric; Record, Eric

    2018-04-15

    Trametes versicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase Tv DyP1 and the versatile peroxidase Tv VP2. Both peroxidases were heterologously produced in Escherichia coli , biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and Tv DyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while Tv VP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and Tv DyP1 was globally found to be more stable than Tv VP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of Tv VP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, Tv DyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and Tv VP2 was shown to accept a wider range of reducing substrates than Tv DyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with Tv VP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and Tv VP2 presented a greater ability to oxidize and decolorize the dye substrates than Tv DyP1. IMPORTANCE Trametes versicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the

  12. Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures.

    Science.gov (United States)

    Milewska, Klaudia; Węgrzyn, Grzegorz; Szalewska-Pałasz, Agnieszka

    2015-09-01

    The presence of natural plasmids has been reported for many Shewanella isolates. However, knowledge about plasmid replication origin and segregation mechanisms is not extensive for this genus. Shewanella baltica is an important species in the marine environment due to its denitrification ability in oxygen-deficient zones and the potential role in bioremediation processes. However, no information about possible use of plasmid vectors in this species has been reported to date. Here we report that plasmids with ColE1-type and plasmid P1 origin can transform S. baltica and replicate in this bacterium. Without the antibiotic selection pressure plasmid maintenance is less efficient than in Escherichia coli. Nevertheless, cultivation of S. baltica in the presence of appropriate antibiotics caused relatively stable maintenance of ColE1-like and P1-derived plasmids. This indicates that plasmid-based genetic manipulations and gene transfer in S. baltica are possible. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Branching ratio and angular distribution of ejected electrons from Eu 4f76p1/2 n d auto-ionizing states

    International Nuclear Information System (INIS)

    Wu Xiao-Rui; Shen Li; Zhang Kai; Dai Chang-Jian; Yang Yu-Na

    2016-01-01

    The branching ratios of ions and the angular distributions of electrons ejected from the Eu 4f 7 6p 1/2 n d auto-ionizing states are investigated with the velocity-map-imaging technique. To populate the above auto-ionizing states, the relevant bound Rydberg states have to be detected first. Two new bound Rydberg states are identified in the region between 41150 cm −1 and 44580 cm −1 , from which auto-ionization spectra of the Eu 4f 7 6p 1/2 n d states are observed with isolated core excitation method. With all preparations above, the branching ratios from the above auto-ionizing states to different final ionic states and the angular distributions of electrons ejected from these processes are measured systematically. Energy dependence of branching ratios and anisotropy parameters within the auto-ionization spectra are carefully analyzed, followed by a qualitative interpretation. (paper)

  14. Thermal magnetized D-branes on R{sup 1,p} x T{sup d-p-1} in the generalized thermo-field dynamics approach

    Energy Technology Data Exchange (ETDEWEB)

    Nardi, R [Centro Brasileiro de Pesquisas Fisicas (CBPF), R. Dr. Xavier Sigaud 150, 22290-180 Rio de Janeiro, RJ (Brazil); Santos, M A [Departamento de Fisica e QImica, Universidade Federal do EspIrito Santo (UFES), Av. Fernando Ferarri S/N-Goiabeiras, 29060-900 Vitoria-ES (Brazil); Vancea, I V, E-mail: rnardi@cbpf.br, E-mail: masantos@cce.ufes.br, E-mail: ionvancea@ufrrj.br [Grupo de Fisica Teorica e Matematica Fisica, Departamento de Fisica, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Cx. Postal 23851, BR 465 Km 7, 23890-000 Seropedica, RJ (Brazil)

    2011-06-10

    We construct the D-brane states at finite temperature in thermal equilibrium in the R{sup 1,p} x T{sup d-p-1} spacetime in the presence of cold (unthermalized) Kalb-Ramond (KR) and U(1) gauge potential background. To this end, we first generalize the thermo-field dynamics to wrapped closed strings. This generalization is consistent with the spatial translation invariance on the string world-sheet. Next, we determine the thermal string vacuum and define the entropy operator. From these data we calculate the entropy of the closed string and the free energy. Finally, we define the thermal D-brane states in R{sup 1,p} x T{sup d-p-1} in the presence of a cold constant KR field and U(1) gauge potential as the boundary states of the thermal closed string and compute their entropy.

  15. Electronic band structure calculations for GaxIn1−xASyP1−y alloys lattice matched to InP

    International Nuclear Information System (INIS)

    Bechiri, A; Benmakhlouf, F; Allouache, H; Bacha, S; Bouarissa, N

    2012-01-01

    A pseudopotential formalism coupled with the virtual crystal approximation are applied to study the effect of compositional disorder upon electronic band structure of cubic Ga x In 1−x As y P 1−y quarternary alloys lattice matched to InP. The effects of compositional variations are properly included in the calculations. Very good agreement is obtained between the calculated values and the available experimental data for the lattice–matched alloy to InP. The absorption at the fundamental optical gaps is found to be direct within a whole range of the y composition whatever the lattice-matching to the substrate of interest. The alloy system Ga x In 1−x As y P 1−y lattice matched to InP is suggested to be suitable for an efficient light emitting device (ELED) material.

  16. High-resolution absorption spectrum of the 61S0 → 63P1 transition in mercury with a Cw dye laser

    International Nuclear Information System (INIS)

    Crane, J.K.; Erbert, G.V.; Mostek, S.D.; Kerlin, R.C.; Paisner, J.A.

    1985-01-01

    Using a stabilized single-frequency commercial dye laser and an external cavity doubling crystal, we have measured the isotope shifts of mercury for the 6 1 S 0 → 6 3 P 1 transition with an accuracy of 4 MHz. We describe the method for generating single-frequency light at 2537 A and compare the results of our measurements of the isotope shifts with previous work. 7 refs., 5 figs., 1 tab

  17. Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Sepulveda, Hugo; Aguilar, Rodrigo; Prieto, Catalina P; Bustos, Francisco; Aedo, Sócrates; Lattus, José; van Zundert, Brigitte; Palma, Veronica; Montecino, Martin

    2017-09-01

    Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519-2527, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. PENERAPAN METODE RETAD UNTUK MENGURANGI WAKTU SET UP PADA MESIN MILLING P1 DAN P2 DEPARTEMEN MACHINING PT. KUBOTA INDONESIA

    Directory of Open Access Journals (Sweden)

    Sriyanto Sriyanto

    2012-02-01

    Full Text Available Pengurangan waktu produksi dalam suatu proses produksi dapat dilakukan dengan meminimalkan waktu setup pada proses produksi tersebut. Untuk mengurangi waktu setup diperlukan suatu cara untuk membantu operator dalam melaksanakan proses milling sehingga dapat meminimalkan waktu setup serta dapat menghilangkan elemen kerja yang tidak produktif tersebut. Metode RETAD (Rapid Exchange of Tooling and Dies merupakan pengembangan dari metode SMED (Single Minuite Exchange of Dies yang bertujuan mengurangi waktu setup, menghapus scrap dan rework. Dari hasil pengolahan data dan analisis untuk proses milling pada mesin Milling Vertikal P1, waktu elemen kerja membersihkan jig dari geram dapat dikurangi dari 5.53 detik sebelum perbaikan menjadi 1.54 detik setelah perbaikan dan untuk mesin Milling Vertikal P2, waktu elemen kerja membersihkan jig dari geram dapat  dikurangi dari 5.15 detik menjadi 1.54 detik. Dari hasil di atas maka dapat disimpulkan bahwa terjadi perbaikan waktu setup pada proses milling pada Mesin Milling Vertikal P1 dan P2 di Cylinder Head Line, perbandingan waktu standar proses milling dari 258.505 detik sebelum perbaikan menjadi 254.518 detik setelah perbaikan pada mesin Milling Vertikal P1 dan 256.002 detik sebelum perbaikan menjadi 252.392 detik setelah perbaikan pada mesin Milling Vertikal P2 Untuk penelitian ini hasil tersebut merupakan hasil maksimal yang dapat dicapai, namun dapat dikembangkan lebih lanjut pada mesin-mesin produksi lainnya. Kata kunci           : RETAD, waktu setup, elemen kerja

  19. Construction of a Streptomyces lydicus A01 transformant with a chit42 gene from Trichoderma harzianum P1 and evaluation of its biocontrol activity against Botrytis cinerea.

    Science.gov (United States)

    Wu, Qiong; Bai, Linquan; Liu, Weicheng; Li, Yingying; Lu, Caige; Li, Yaqian; Fu, Kehe; Yu, Chuanjin; Chen, Jie

    2013-04-01

    Streptomyces lydicus A01 and Trichoderma harzianum P1 are potential biocontrol agents of fungal diseases in plants. S. lydicus A01 produces natamycin to bind the ergosterol of the fungal cell membrane and inhibits the growth of Botrytis cinerea. T. harzianum P1, on the other hand, features high chitinase activity and decomposes the chitin in the cell wall of B. cinerea. To obtain the synergistic biocontrol effects of chitinase and natamycin on Botrytis cinerea, this study transformed the chit42 gene from T. harzianum P1 to S. lydicus A01. The conjugal transformant (CT) of S. lydicus A01 with the chit42 gene was detected using polymerase chain reaction (PCR). Associated chitinase activity and natamycin production were examined using the 3, 5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry, respectively. The S. lydicus A01-chit42 CT showed substantially higher chitinase activity and natamycin production than its wild type strain (WT). Consequently, the biocontrol effects of S. lydicus A01-chit42 CT on B. cinerea, including inhibition to spore germination and mycelial growth, were highly improved compared with those of the WT. Our research indicates that the biocontrol effect of Streptomyces can be highly improved by transforming the exogenous resistance gene, i.e. chit42 from Trichoderma, which not only enhances the production of antibiotics, but also provides a supplementary function by degrading the cell walls of the pathogens.

  20. Identification of paramagnetic nitrogen centers (P1) in diamond crystallites synthesized via the sintering of detonation nanodiamonds at high pressure and temperature

    Science.gov (United States)

    Osipov, V. Yu.; Shakhov, F. M.; Efimov, N. N.; Minin, V. V.; Kidalov, S. V.; Vul', A. Ya.

    2017-06-01

    Diamond single crystals synthesized from powder detonation nanodiamonds (DNDs) by means of treatment at high pressures ( P 7 GPa) and temperatures ( T > 1300°C) have been studied by electron paramagnetic resonance (EPR). A key feature of treatment (high-pressure high-temperature (HPHT) sintering) is the use of low molecular weight alcohols in the process. The appearance of a hyperfine EPR signal structure due to "paramagnetic nitrogen" (P1 centers) is explained by the growth of submicron and micron diamond single crystals from DND nanocrystals by the oriented attachment and coalescence mechanism. Such growth and coarsening of crystals appreciably decreases the concentration of paramagnetic centers, the presence of which hinders the detection of a hyperfine structure in the EPR signal from P1 centers, in the near-surface areas of coalesced and grown together DND particles. It has been shown that the concentration of paramagnetic defects of all types decreases to 3.1 × 1018 g-1 ( 60 ppm) during HPHT treatment at T = 1650°C. This causes the successful identification of P1 centers, whose fraction is no less than 40% of the total amount of paramagnetic centers in microcrystals synthesized by HPHT sintering.

  1. Exceptional disfavor for proline at the P + 1 position among AGC and CAMK kinases establishes reciprocal specificity between them and the proline-directed kinases.

    Science.gov (United States)

    Zhu, Guozhi; Fujii, Koichi; Belkina, Natalya; Liu, Yin; James, Michael; Herrero, Juan; Shaw, Stephen

    2005-03-18

    To precisely regulate critical signaling pathways, two kinases that phosphorylate distinct sites on the same protein substrate must have mutually exclusive specificity. Evolution could assure this by designing families of kinase such as basophilic kinases and proline-directed kinase with distinct peptide specificity; their reciprocal peptide specificity would have to be very complete, since recruitment of substrate allows phosphorylation of even rather poor phosphorylation sites in a protein. Here we report a powerful evolutionary strategy that assures distinct substrates for basophilic kinases (PKA, PKG and PKC (AGC) and calmodulin-dependent protein kinase (CAMK)) and proline-directed kinase, namely by the presence or absence of proline at the P + 1 position in substrates. Analysis of degenerate and non-degenerate peptides by in vitro kinase assays reveals that proline at the P + 1 position in substrates functions as a "veto" residue in substrate recognition by AGC and CAMK kinases. Furthermore, analysis of reported substrates of two typical basophilic kinases, protein kinase C and protein kinase A, shows the lowest occurrence of proline at the P + 1 position. Analysis of crystal structures and sequence conservation provides a molecular basis for this disfavor and illustrate its generality.

  2. Cytomegalovirus evasion of innate immunity by subversion of the NKR-P1B:Clr-b missing-self axis.

    Science.gov (United States)

    Voigt, Sebastian; Mesci, Aruz; Ettinger, Jakob; Fine, Jason H; Chen, Peter; Chou, Wayne; Carlyle, James R

    2007-05-01

    Cytomegaloviruses are known to encode several gene products that function to subvert MHC-dependent immune recognition. Here we characterize a rat cytomegalovirus (RCMV) C-type lectin-like (RCTL) gene product with homology to the Clr ligands for the NKR-P1 receptors. RCMV infection rapidly extinguished host Clr-b expression, thereby sensitizing infected cells to killing by natural killer (NK) cells. However, the RCTL protein functioned as a decoy ligand to protect infected cells from NK killing via direct interaction with the NKR-P1B inhibitory receptor. In vivo, an RCTL mutant virus displayed diminished virulence in an NK-dependent and strain-specific manner, suggesting that host NKR-P1 polymorphisms have evolved to avert the viral decoy mechanism while maintaining Clr-b recognition to preserve self tolerance. These findings reveal a unique strategy adopted by cytomegaloviruses to evade MHC-independent self-nonself discrimination. The existence of lectin-like genes in several poxviruses suggests that this may represent a common theme for viral evasion of innate immunity.

  3. Association of glutathione S-transferase (GSTM1, T1 and P1 gene polymorphisms with type 2 diabetes mellitus in north Indian population

    Directory of Open Access Journals (Sweden)

    Bid H

    2010-01-01

    Full Text Available Background: Diabetes mellitus is associated with an increased production of reactive oxygen species (ROS and a reduction in antioxidant defense. The oxidative stress becomes evident as a result of accumulation of ROS in conditions of inflammation and Type 2 diabetes mellitus (T2DM. The genes involved in redox balance, which determines the susceptibility to T2DM remain unclear. In humans, the glutathione S-transferase (GST family comprises several classes of GST isozymes, the polymorphic variants of GSTM1, T1 and P1 genes result in decreased or loss of enzyme activity. Aims: The present study evaluated the effect of genetic polymorphisms of the GST gene family on the risk of developing T2DM in the North Indian population. Settings and Design: GSTM1, T1 and P1 polymorphisms were genotyped in 100 T2DM patients and 200 healthy controls from North India to analyze their association with T2DM susceptibility. Materials and Methods: Analysis of GSTM1 and GSTT1 gene polymorphisms was performed by multiplex polymerase chain reaction (PCR and GSTP1 by PCR-Restriction Fragment Length Polymorphism (RFLP. Statistical Analysis: Fisher′s exact test and χ2 statistics using SPSS software (Version-15.0. Results: We observed significant association of GSTM1 null (P=0.004, OR= 2.042, 95%CI= 1.254-3.325 and GSTP1 (I/V (P=0.001, OR= 0.397, 95%CI=0.225-0.701 with T2DM and no significant association with GSTT1 (P=0.493. The combined analysis of the three genotypes GSTM1 null, T1 present and P1 (I/I demonstrated an increase in T2DM risk (P= 0.005, OR= 2.431 95% CI=1.315-4.496. Conclusions: This is the first study showing the association of a combined effect of GSTM1, T1 and P1 genotypes in a representative cohort of Indian patients with T2DM. Since significant association was seen in GSTM1 null and GSTP1 (I/V and multiple association in GSTM1 null, T1 present and P1 (I/I, these polymorphisms can be screened in the population to determine the diabetic risk.

  4. Desensitization by progressive up-titration prevents first-dose effects on the heart: guinea pig study with ponesimod, a selective S1P1 receptor modulator.

    Directory of Open Access Journals (Sweden)

    Markus Rey

    Full Text Available Ponesimod, a selective S1P1 receptor modulator, reduces the blood lymphocyte count in all tested species by preventing egress of T and B cells from thymus and peripheral lymphoid organs. In addition, ponesimod transiently affects heart rate and atrioventricular (AV conduction in humans, effects not observed in mice, rats, and dogs with selective S1P1 receptor modulators, suggesting that the regulation of heart rate and rhythm is species dependent. In the present study, we used conscious guinea pigs implanted with a telemetry device to investigate the effects of single and multiple oral doses of ponesimod on ECG variables, heart rate, and blood pressure. Oral administration of ponesimod did not affect the sinus rate (P rate but dose-dependently induced AV block type I to III. A single oral dose of 0.1 mg/kg had no effect on ECG variables, while a dose of 3 mg/kg induced AV block type III in all treated guinea pigs. Repeated oral dosing of 1 or 3 mg/kg ponesimod resulted in rapid desensitization, so that the second dose had no or a clearly reduced effect on ECG variables as compared with the first dose. Resensitization of the S1P1 receptor in the heart was concentration dependent. After desensitization had been induced by the first dose of ponesimod, the cardiac system remained desensitized as long as the plasma concentration was ≥75 ng/ml. By using a progressive up-titration regimen, the first-dose effect of ponesimod on heart rate and AV conduction was significantly reduced due to desensitization of the S1P1 receptor. In summary, conscious guinea pigs implanted with a telemetry device represent a useful model to study first-dose effects of S1P1 receptor modulators on heart rate and rhythm. This knowledge was translated to a dosing regimen of ponesimod to be tested in humans to avoid or significantly reduce the first-dose effects.

  5. Whole-gene positive selection, elevated synonymous substitution rates, duplication, and indel evolution of the chloroplast clpP1 gene.

    Directory of Open Access Journals (Sweden)

    Per Erixon

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

  6. Growth of GaP and AlGaP on GaP(1 1 1)B using gas-source molecular-beam-epitaxy

    Science.gov (United States)

    Barakat, J.-B.; Dadgostar, S.; Hestroffer, K.; Bierwagen, O.; Trampert, A.; Hatami, F.

    2017-11-01

    We present an initial study of the influence of the growth parameters on the surface morphology and on the interface quality of homoepitaxial GaP(1 1 1) and heteroepitaxial GaP/AlGaP(1 1 1) grown on GaP(1 1 1)B substrates using Gas-Source Molecular Beam Epitaxy (GSMBE). Three different surface reconstructions are identified in the RHEED patterns during the growth runs. The Root Mean Square (RMS) surface roughness measured post-growth by AFM ranges from 3 to 10 nm over 10 × 10 μm2 areas, for a film thickness of 100-600 nm. The results of 2θ-ω XRD scans on (1 1 1) and (3 1 1) planes reveal a stacking disorder in the AlGaP layer and further XRD phi-scan measurements on GaP (3 1 1) show strong peaks with 3-fold rotational symmetry and additional of 3-fold weak peaks indicating only a negligible fraction of the twinned crystal orientation in the substrate. TEM images of these samples show a smooth interface between the AlGaP layer and GaP substrate, and reveal the presence of a high density of extended defects such as stacking faults, twinning and dislocations lines in AlGaP layer whereas the GaP layer appears as pure Zinc-Blende. Further TEM analysis reveals composition and local strain variations for GaP/AlGaP samples associated with an undulated surface.

  7. Simultaneous pyrosequencing of the 16S rRNA, IncP-1 trfA, and merA genes

    DEFF Research Database (Denmark)

    Holmsgaard, Peter Nikolai; Sørensen, Søren Johannes; Hansen, Lars H.

    2013-01-01

    The use of amplicon pyrosequencing makes it possible to produce thousands of sequences of the same gene at relatively low costs. Here we show that it is possible to simultaneously sequence the 16S rRNA gene, IncP-1 trfA gene and mercury reductase gene (merA) as a way for screening the diversity...... of several genes in the same samples. As a proof-of-concept two different soil samples and a wastewater sample were screened. Multiplexing identifiers (MIDs) and sequencing adapters were added to amplicons using a tailed PCR ...

  8. 25-Hydroxy vitamin-D, obesity, and associated variables as predictors of breast cancer risk and tamoxifen benefit in NSABP-P1

    OpenAIRE

    Amir, Eitan; Cecchini, Reena S.; Ganz, Patricia A.; Costantino, Joseph P.; Beddows, Samantha; Hood, Nicola; Goodwin, Pamela J.

    2012-01-01

    Observational studies suggest that host factors are associated with breast cancer risk. The influence of obesity, vitamin-D status, insulin resistance, inflammation, and elevated adipocytokines in women at high risk of breast cancer is unknown. The NSABP-P1 trial population was used for a nested case–control study. Cases were drawn from those who developed invasive breast cancer and controls selected from unaffected participants (≤4 per case) matched for age, race, 5 year Gail score, and geog...

  9. High-level expression of soluble form of mouse natural killer cell receptor NKR-P1C(B6) in Escherichia coli

    Czech Academy of Sciences Publication Activity Database

    Rozbeský, Daniel; Kavan, Daniel; Chmelík, Josef; Novák, Petr; Vaněk, Ondřej; Bezouška, Karel

    2011-01-01

    Roč. 77, č. 2 (2011), s. 178-184 ISSN 1046-5928 R&D Projects: GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR GAP207/10/1040; GA AV ČR IAA500200620; GA MŠk LC07017; GA MŠk LC545; GA MŠk 1M0505 Institutional research plan: CEZ:AV0Z50200510 Keywords : Natural killer cell * NKR-P1C receptor * NK1.1 antigen Subject RIV: EC - Immunology Impact factor: 1.587, year: 2011

  10. The use of radiation trapping in the measurement of the electron excitation cross section for the production of the 1s4 (3P1) level of Ne

    International Nuclear Information System (INIS)

    Miers, R.E.; Gastineau, J.E.; Phillps, M.H.; Anderson, L.W.; Lin, C.C.

    1981-01-01

    The authors report the use of laser induced fluorescence for the first measurement of the electron excitation cross section for the production of the 1s 4 ( 3 P 1 ) level of Ne. Radiation trapping is used to lengthen the effective lifetime of the 1s 4 level allowing for the electron excitation cross section of the 1s 4 level to be measured in a manner similar to the measurement of electron excitation cross sections of the metastable 1s 3 and 1s 5 levels. (Auth.)

  11. A genetic study of SSV1, the prototypical fusellovirus.

    Directory of Open Access Journals (Sweden)

    Eric eIverson

    2012-06-01

    Full Text Available Viruses of thermophilic Archaea are unique in both their structures and genomic sequences. The most widespread and arguably best studied are the lemon-shaped fuselloviruses. The spindle-shaped virus morphology is unique to Archaea but widespread therein. The best studied fusellovirus is SSV1 from Beppu Japan, which infects Sulfolobus solfataricus. Very little is known about the function of the genes in the SSV1 genome. Recently we have developed genetic tools to analyze these genes. In this study, we have deleted three SSV1 open reading frames ranging from completely conserved to poorly conserved: VP2, d244, and b129. Deletion of the universally conserved ORF b129, which encodes a predicted transcriptional regulator, results in loss of infectivity. Deletion of the poorly-conserved predicted DNA binding protein gene VP2 yields viable virus that is indistinguishable from wild-type Deletion of the well-conserved ORF d244 that encodes a predicted nuclease yields viable virus. However infection of Sulfolobus solfataricus with virus lacking ORF d244 dramatically retards host growth, compared to the wild-type virus.

  12. Four crystal structures of human LLT1, a ligand of human NKR-P1, in varied glycosylation and oligomerization states

    Energy Technology Data Exchange (ETDEWEB)

    Skálová, Tereza, E-mail: t.skalova@gmail.com [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Bláha, Jan [Charles University Prague, Hlavova 8, 128 40 Praha (Czech Republic); Harlos, Karl [University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Dušková, Jarmila [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Koval’, Tomáš [Academy of Sciences of the Czech Republic, v.v.i., Heyrovského nám. 2, 162 06 Praha 6 (Czech Republic); Stránský, Jan; Hašek, Jindřich [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Vaněk, Ondřej [Charles University Prague, Hlavova 8, 128 40 Praha (Czech Republic); Dohnálek, Jan, E-mail: t.skalova@gmail.com [Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Praha 4 (Czech Republic); Academy of Sciences of the Czech Republic, v.v.i., Heyrovského nám. 2, 162 06 Praha 6 (Czech Republic)

    2015-03-01

    Four crystal structures of human LLT1, a ligand of human NKR-P1, are reported. Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc{sub 2}Man{sub 5} glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

  13. First-Principle Study of the Structural, Electronic, and Optical Properties of Cubic InNxP1-x Ternary Alloys under Hydrostatic Pressure

    Science.gov (United States)

    Hattabi, I.; Abdiche, A.; Moussa, R.; Riane, R.; Hadji, K.; Soyalp, F.; Varshney, Dinesh; Syrotyuk, S. V.; Khenata, R.

    2016-09-01

    In this article, we present results of the first-principle study of the structural, electronic, and optical properties of the InN, InP binary compounds and their related ternary alloy InNxP1-x in the zinc-blend (ZB) phase within a nonrelativistic full potential linearised augmented plan wave (FP-LAPW) method using Wien2k code based on the density functional theory (DFT). Different approximations of exchange-correlation energy were used for the calculation of the lattice constant, bulk modulus, and first-order pressure derivative of the bulk modulus. Whereas the lattice constant decreases with increasing nitride composition x. Our results present a good agreement with theoretical and experimental data. The electronic band structures calculated using Tran-Blaha-modified Becke-Johnson (TB-mBJ) approach present a direct band gap semiconductor character for InNxP1-x compounds at different x values. The electronic properties were also calculated under hydrostatic pressure for (P=0.00, 5.00, 10.0, 15.0, 20.0, 25.0 GPa) where it is found that the InP compound change from direct to indirect band gap at the pressure P≥7.80 GPa. Furthermore, the pressure effect on the dielectric function and the refractive index was carried out. Results obtained in our calculations present a good agreement with available theoretical reports and experimental data.

  14. Identification of two conserved cis-acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species.

    Science.gov (United States)

    Chen, Aiqun; Gu, Mian; Sun, Shubin; Zhu, Lingling; Hong, Shuai; Xu, Guohua

    2011-03-01

    • In this study, six putative promoter regions of phosphate transporter Pht1;3, Pht1;4 and Pht1;5 genes were isolated from eggplant and tobacco using the inverse polymerase chain reaction (iPCR). The isolated sequences show evolutionary conservation and divergence within/between the two groups of Pht1;3 and Pht1;4/Pht1;5. • Histochemical analyses showed that all six promoter fragments were sufficient to drive β-glucuronidase (GUS) expression specifically in arbuscular mycorrhizal (AM) tobacco roots and were confined to distinct cells containing AM fungal structures (arbuscules or intracellular hyphae). • A series of promoter truncation and mutation analyses combined with phylogenetic footprinting of these promoters revealed that at least two cis-regulatory elements--the mycorrhiza transcription factor binding sequence (MYCS) first identified in this study and P1BS--mediated the transcriptional activation of the AM-mediated inorganic phosphate (Pi) transporter genes. Deletion or partial mutation of either of the two motifs in the promoters could cause a remarkable decrease, or even complete absence, of the promoter activity. • Our results propose that uptake of inorganic phosphate (Pi) by AM fungi is regulated, at least partially, in an MYCS- and P1BS-dependent manner in eudicot species. Our finding offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi signalling pathways. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  15. Studies on the O-specific polysaccharide of the lipopolysaccharide from the Pseudomonas mediterranea strain C5P1rad1, a bacterium pathogenic of tomato and chrysanthemum.

    Science.gov (United States)

    Zdorovenko, Evelina L; Cimmino, Alessio; Marchi, Guido; Shashkov, Alexander S; Fiori, Mario; Knirel, Yuriy A; Evidente, Antonio

    2017-08-07

    An O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Pseudomonas mediterranea strain C5P1rad1, the causal agents of tomato pith necrosis and Chrysanthemum stem rot, and studied by one- and two-dimensional 1 H and 13 C NMR spectroscopy. The following structure of the trisaccharide repeating unit of the OPS was established, which, to our knowledge, is unique among the known bacterial polysaccharide structures: →4)-β-d-ManpNAc3NAcA-(1 → 4)-β-d-ManpNAc3NAcA-(1 → 3)-α-d-QuipNAc4NAc-(1→ where QuiNAc4NAc and ManNAc3NAcA indicate 2,4-diacetamido-2,4,6-trideoxyglucose and 2,3-diacetamido-2,3-dideoxymannuronic acid, respectively. Pre-treatment of leaves with LPS or OPS preparations at 250 and 50 μg mL -1 did not inhibit development of a hypersensitivity reaction induced by P. mediterranea C5P1rad1 on tobacco, tomato and chrysanthemum plants. The same preparations at 250 μg mL -1 partially prevented elicitation of the hypersensitivity reaction by Pseudomonas syringae KVPT7RC on chrysanthemum but not tobacco and tomato. Copyright © 2017. Published by Elsevier Ltd.

  16. Structure-based design of potent HIV-1 protease inhibitors with modified P1-biphenyl ligands: synthesis, biological evaluation, and enzyme-inhibitor X-ray structural studies.

    Science.gov (United States)

    Ghosh, Arun K; Yu, Xufen; Osswald, Heather L; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T; Mitsuya, Hiroaki

    2015-07-09

    We report the design, synthesis, X-ray structural studies, and biological evaluation of a novel series of HIV-1 protease inhibitors. We designed a variety of functionalized biphenyl derivatives to make enhanced van der Waals interactions in the S1 subsite of HIV-1 protease. These biphenyl derivatives were conveniently synthesized using a Suzuki-Miyaura cross-coupling reaction as the key step. We examined the potential of these functionalized biphenyl-derived P1 ligands in combination with 3-(S)-tetrahydrofuranyl urethane and bis-tetrahydrofuranyl urethane as the P2 ligands. Inhibitor 21e, with a 2-methoxy-1,1'-biphenyl derivative as P1 ligand and bis-THF as the P2 ligand, displayed the most potent enzyme inhibitory and antiviral activity. This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray crystal structure of related Boc-derivative 17a-bound HIV-1 protease provided important molecular insight into the ligand-binding site interactions of the biphenyl core in the S1 subsite of HIV-1 protease.

  17. Molecular and biochemical properties of two P1B2-ATPases, CsHMA3 and CsHMA4, from cucumber.

    Science.gov (United States)

    Migocka, Magdalena; Papierniak, Anna; Maciaszczyk-Dziubinska, Ewa; Posyniak, Ewelina; Kosieradzka, Anna

    2015-06-01

    P1B-ATPases (heavy metal ATPases, HMAs) constitute a multigenic subfamily of P-ATPases involved in the transport of monovalent and divalent heavy metals in plant cells. Here, we present the organization of genes encoding the HMA family in the cucumber genome and report the function and biochemical properties of two cucumber proteins homologous to the HMA2-4-like plant HMAs. Eight genes encoding putative P1B -ATPases were identified in the cucumber genome. Among them, CsHMA3 was predominantly expressed in roots and up-regulated by Pb, Zn and Cd excess, whereas the CsHMA4 transcript was most abundant in roots and flowers of cucumber plants, and elevated under Pb and Zn excess. Expression of CsHMA3 in Saccharomyces cerevisiae enhanced yeast tolerance to Cd and Pb, whereas CsHMA4 conferred increased resistance of yeast cells to Cd and Zn. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether, the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells. © 2014 John Wiley & Sons Ltd.

  18. IgE profiles of Bermuda grass pollen sensitised patients evaluated by Phleum pratense allergens Phl P 1, 2, 4, 5, 6 , 7, 11, 12.

    Science.gov (United States)

    Rossi, Renato E; Monasterolo, Giorgio; Prina, Paolo; Coco, Giuseppe; Operti, Daniela; Rossi, Lucilla

    2008-06-01

    Despite the difference in geographical dominance of certain grasses, a high degree of allergenic similarity or cross-reactivity between Bermuda grass pollen (BGP) and timothy grass pollen (TGP) has been previously demonstrated. The aim of the present study was to ascertain the sensitisation to TGP in 411 patients known for their reactivity to BGP extracts by analysing their reactivity to crude timothy pollen extract and timothy pollen purified allergens, establishing their specific IgE-profiles. Using the immunoenzymatic CAP method we evaluated IgE-specific antibodies for BGP- and TGP- extracts and the timothy recombinant (r) and natural (n) allergens rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12. BGP-IgE positive patients (median = 8.0 kUA/l, 2.8-22.2 kUA/l 25th-75th percentile) simultaneously had IgE positive results for TGP (100% of subjects)(median = 48.9 kUA/l, 19.8- > 100 kUA/l 25th-75th percentile) and high prevalence of sensitization to 6/8 Phleum pratense allergens (Phl p 1, 2, 4, 5, 6, 11, markers of genuine sensitisation to TGP) other than profilin and calcium binding protein. More than 72% of BGP allergic patients were co-sensitised to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6. A decrease of total and specific IgE with patients' age was observed. Our data show that all BGP-allergic patients simultaneously exhibit higher IgE antibody levels to recombinant and natural P. pratense allergens as well as to crude TGP extract. This suggests that when choosing an immunotherapeutic regimen for BGP-sensitised patients (after establishing their IgE profile via purified TGP-allergens), subcutaneous or sublingual TGP-extract vaccines in appropriate doses, in order to influence T epitope specificity, might be beneficial. Though extremely uncommon, in cases where a patient is exclusively BGP allergen-sensitised, BGP-extract therapy is the appropriate therapeutic response.

  19. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  20. [Role and related mechanism of S1P/S1P1 signal pathway during post conditioning of hypertrophic cardiomyocytes].

    Science.gov (United States)

    Bao, X H; Li, H X; Tao, J; Li, X M; Yang, Y N; Ma, Y T; Chen, B D

    2016-05-24

    To study the role and mechanism of sphingosine-1-phosphate (S1P)/ sphingosine-1-phosphate receptor 1(S1P1) signal pathway during post conditioning of hypertrophic cardiomyocytes. Neonatal rat cardiomyocytes were isolated and cultured, then stimulated by norepinephrine (NE) to induce cardiomyocytes hypertrophy. Using tri-gas incubator to create hypoxia and reoxygenation enviroment to mimic ischemia-reperfusion and postconditioning. Hypertrophic cardiomyoctyes were divided into five groups according to the presence or absence of various drugs and postconditiong and relevant signal pathways changes were detected: (1) IPost group (hypoxia+ postconditioning); (2) IPost+ S1P group (cells were pretreated with S1P (1 μmol/L) for 2 h before IPost); (3) IPost+ W-146+ S1P group (cells in IPost+ W-146+ S1P group were pretreated with S1P1 inhibitor W-146 (0.4 μmol/L) for 20 min); (4) IPost+ PD98059+ S1P group (cells in IPost+ S1P group were pretreated with MAPK antagonist PD98059 (125 μmol/L) for 20 min); (5) IPost+ LY-294002+ S1P group (cells in IPost+ S1P group were pretreated with PI3K antagonist LY294002 (0.1 μmol/L) for 20 min). Apoptosis was detected by flow cytometry and protein expression of relevant signal pathways were detected by Western blot. (1)Apoptosis rate was significantly increased in hypoxia/reoxygenation (27.90±4.49)% group compared with normal control group (7.97±2.18)%, which could be significantly reduced in IPost group (15.90±1.77)% (all P<0.05). (2)Apoptosis rate and caspase-3 expression were both significantly lower in IPost+ S1P and IPost+ S1P+ LY-294002 groups than in IPost and IPost+ S1P+ W-146 and IPost+ S1P+ PD98059 group (all P<0.05). (3)p-ERK1/2 expression was significantly higher in IPost+ S1P and IPost+ S1P+ LY-294002 group than in IPost and IPost+ S1P+ W-146 group and IPost+ S1P+ PD98059 group (all P<0.05) while p-Akt expression was similar among IPost, IPost+ S1P+ W-146 and IPost+ S1P+ PD98059 groups. p-ERK1/2 and p-Akt levels in

  1. Laboratory observation of the 3P1-3P0 transition of the neutral carbon atom by submillimeter-wave absorption spectroscopy

    International Nuclear Information System (INIS)

    Yamamoto, Satoshi; Saito, Shuji

    1991-01-01

    The 3P1-3P0 transitions of the neutral C-12 and C-13 atoms are observed by laboratory microwave spectroscopy. The carbon atom is produced by discharging a gaseous mixture of CO and He at the liquid nitrogen temperature. The transition frequency is precisely determined for (C-12)T to be 492160.651 + or - 0.055 MHz, where the error is three standard deviations in the frequency measurements. Two hyperfine components, F = 3/2-1/2 and F = 1/2-1/2, are observed for (C-13)I at 492164.276 + or - 0.072 MHz and 492160.147 + or - 0.168 MHz, respectively. The precise laboratory values are recommended as rest frequencies in astronomical observations. 24 refs

  2. Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2017-11-01

    Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

  3. Physical mapping of the Bloom syndrome region by the identification of YAC and P1 clones from human chromosome 15 band q26.1

    Energy Technology Data Exchange (ETDEWEB)

    Straughen, J.; Groden, J. [Univ. of Cincinnati College of Medicine, OH (United States); Ciocci, S. [New York Blood Center, NY (United States)] [and others

    1996-07-01

    The gene for Bloom syndrome (BLM) has been mapped to human chromosome 15 band q26.1 by homozygosity mapping. Further refinement of the location of BLM has relied upon linkage-disequilibrium mapping and somatic intragenic recombination. In combination with these mapping approaches and to identify novel DNA markers and probes for the BLM candidate region, a contiguous representation of the 2-Mb region that contains the BLM gene was generated and is presented here. YAC and P1 clones from the region have been identified and ordered by using previously available genetic markers in the region along with newly developed sequence-tagged sites from radiation-restriction map of the 2-Mb region that allowed estimation of the distance between polymorphic microsatellite loci is also reported. This map and the DNA markers derived from it were instrumental in the recent identification of the BLM gene. 25 refs., 3 figs., 3 tabs.

  4. The collectins CL-L1, CL-K1 and CL-P1, and their roles in complement and innate immunity

    DEFF Research Database (Denmark)

    Hansen, Soren W K; Ohtani, Katsuki; Roy, Nitai

    2016-01-01

    Both the complement system and collectins play important roles in our innate immune system. The collectins, which are characterized by their inclusion of a collagen-like region and a calcium-dependent carbohydrate recognition domain, are pattern recognition molecules and include the well...... system, in innate immunity and their possible association with disease development and pathogenesis....... that CL-L1, CL-K1 and CL-P1 play important roles in host defense by recognizing a variety of microorganisms and interacting with effector proteins, including complement components. The recent establishment of the existence of CL-K1 in the circulation in form of heteromeric complexes with CL-L1 (known...

  5. Effects of 100 years wastewater irrigation on resistance genes, class 1 integrons and IncP-1 plasmids in Mexican soil

    Directory of Open Access Journals (Sweden)

    Sven eJechalke

    2015-03-01

    Full Text Available Long-term irrigation with untreated wastewater can lead to an accumulation of antibiotic substances and antibiotic resistance genes in soil. However, little is known so far about effects of wastewater, applied for decades, on the abundance of IncP-1 plasmids and class 1 integrons which may contribute to the accumulation and spread of resistance genes in the environment, and their correlation with heavy metal concentrations.Therefore, a chronosequence of soils that were irrigated with wastewater from zero to 100 years was sampled in the Mezquital Valley in Mexico in the dry season. The total community DNA was extracted and the absolute and relative abundance (relative to 16S rRNA genes of antibiotic resistance genes (tet(W, tet(Q, aadA, class 1 integrons (intI1, quaternary ammonium compound resistance genes (qacE+qacEΔ1 and IncP-1 plasmids (korB were quantified by real-time PCR. Except for intI1 and qacE+qacEΔ1 the abundances of selected genes were below the detection limit in non-irrigated soil. Confirming the results of a previous study, the absolute abundance of 16S rRNA genes in the samples increased significantly over time (linear regression model, p < 0.05 suggesting an increase in bacterial biomass due to repeated irrigation with wastewater. Correspondingly, all tested antibiotic resistance genes as well as intI1 and korB significantly increased in abundance over the period of 100 years of irrigation. In parallel, concentrations of the heavy metals Zn, Cu, Pb, Ni, and Cr significantly increased. However, no significant positive correlations were observed between the relative abundance of selected genes and years of irrigation, indicating no enrichment in the soil bacterial community due to repeated wastewater irrigation or due to a potential co-selection by increasing concentrations of heavy metals.

  6. Expression of FlHMA3, a P1B2-ATPase from Festulolium loliaceum, correlates with response to cadmium stress.

    Science.gov (United States)

    Guo, Qiang; Meng, Lin; Humphreys, Mike W; Scullion, John; Mur, Luis A J

    2017-03-01

    Heavy metal ATPase 3 (HMA3), a P 1B2 -ATPase, is a key tonoplast transporter involved in mediating the vacuolar sequestration of cadmium (Cd) to detoxify the intake of this element by plants. HMA3 expression in response to Cd stress has not been previously examined in the grass hybrid species Festulolium loliaceum (Huds.) P. Fourn. In this study, FlHMA3 isolated from F. loliaceum was found to comprise 833 amino acid residues with 77% homology to the rice OsHMA3. Transient expression of FlHMA3 fused to enhanced green fluorescent protein in Arabidopsis protoplasts suggested its localization to vacuolar membranes. Quantitative real-time RT-PCR analysis of F. loliaceum revealed that FlHMA3 is expressed predominantly within roots and up-regulated by excess Cd. Over the 168 h treatment, Cd content of F. loliaceum roots was significantly higher than that of shoots, regardless of external CdCl 2 concentrations. A significant positive correlation was found between FlHMA3 expression and Cd accumulation in roots of F. loliaceum seedlings subjected to 10-100 mg L -1 CdCl 2 for 168 h or, in a separate experiment, to 25 or 100 mg L -1 CdCl 2 for the same duration. These findings provide evidence that FlHMA3 encodes a vacuolar P 1B2 -ATPase that may play an important role in Cd 2+ sequestration into root cell vacuoles, thereby limiting the entry of Cd 2+ into the cytoplasm and reducing Cd 2+ toxicity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

    Science.gov (United States)

    Watanabe, Leandra; Shannon, John D.; Valente, Richard H.; Rucavado, Alexandra; Alape-Girón, Alberto; Kamiguti, Aura S.; Theakston, R. David G.; Fox, Jay W.; Gutiérrez, José María; Arni, Raghuvir K.

    2003-01-01

    BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152, as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1–152), comprising four α-helices and a five-stranded β-sheet, from the minor subdomain, which is formed by a single α-helix and several loops. The catalytic zinc ion is coordinated by the Nɛ2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix. PMID:14500885

  8. Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities.

    Science.gov (United States)

    Watanabe, Leandra; Shannon, John D; Valente, Richard H; Rucavado, Alexandra; Alape-Girón, Alberto; Kamiguti, Aura S; Theakston, R David G; Fox, Jay W; Gutiérrez, José María; Arni, Raghuvir K

    2003-10-01

    BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

  9. Functional Consequences of Human Immunodeficiency Virus Escape from an HLA-B*13-Restricted CD8+ T-Cell Epitope in p1 Gag Protein▿

    Science.gov (United States)

    Prado, Julia G.; Honeyborne, Isobella; Brierley, Ian; Puertas, Maria Carmen; Martinez-Picado, Javier; Goulder, Philip J. R.

    2009-01-01

    The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI429-437 (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness. PMID:18945768

  10. Functional consequences of human immunodeficiency virus escape from an HLA-B*13-restricted CD8+ T-cell epitope in p1 Gag protein.

    Science.gov (United States)

    Prado, Julia G; Honeyborne, Isobella; Brierley, Ian; Puertas, Maria Carmen; Martinez-Picado, Javier; Goulder, Philip J R

    2009-01-01

    The observed association between HLA-B*13 and control of human immunodeficiency virus type 1 (HIV-1) infection has been linked to the number of Gag-specific HLA-B*13-restricted cytotoxic T-cell (CTL) responses identified. To date, the Gag escape mutations described that result in an in vitro fitness cost to the virus have been located within structural protein p24 only. Here we investigated the hypothesis that CTL escape mutations within other regions of HIV Gag may also reduce viral fitness and contribute to immune control. We analyzed an HLA-B*13-restricted CTL response toward an epitope in p1 Gag, RQANFLGKI(429-437) (RI9), where amino acid variation at Gag residues 436 and 437 is associated with HLA-B*13 expression. In this work, we assessed the impact of amino acid substitutions at these positions on CTL recognition and on HIV-1 fitness. We demonstrated that substitutions I437L and I437M largely abrogate CTL recognition and reduce viral fitness while variants K436R and I437V have only a marginal effect on recognition and fitness. Examination of the patterns of protein synthesis indicated that the loss of fitness in the I437L and I437M mutants is associated with the accumulation of unprocessed Gag precursors. A significant reduction in ribosomal frameshifting efficiency was observed with I437M, suggesting that this mechanism contributes to the observed reduced fitness of this virus. These studies illustrate the apparent trade-off available to the virus between evasion of CTL recognition in p1 Gag and the functional consequences for viral fitness.

  11. A uniform method for the simultaneous blood group phenotyping of Fya, Fyb, Jka, Jkb, S, s̅, P1, k applying lateral-flow technique.

    Science.gov (United States)

    Caesar, A; Meyer, S; Trost, N; Neuenschwander, K; Geisen, C; Frey, B M; Gassner, C; Schwind, P

    2018-02-01

    A lateral flow assay for simultaneous blood group typing of ABO, RhD, C, E, c, e, Cw and K with stable end-point and without centrifugation is in routine use since several years (MDmulticard ® ). The typing of extended phenotype parameters belonging to the Duffy, Kidd, MNSs blood group systems and others, however, has not yet been demonstrated for this technique. Reliable detection of Fy x , a weak Fy b phenotype with a pronounced quantitative reduction of the number of Fy b antigens on the erythrocyte surface, remains a weakness of current serological blood grouping techniques. The performance characteristics of the following reagents were evaluated in donor and patient samples in lateral flow technology (MDmulticard ® ): Anti-Fy a , -Fy b , -Jk a , -Jk b , -S, -s̅, -P1 and -k. The sensitivity to detect Fy x was in addition evaluated with Fy x positive samples, which had been preselected by MALDI-TOF MS-based genotyping. All results obtained with the MDmulticard ® were in full accordance with those of the CE-certified reference products for all the eight reagent formulations used: Anti-Fy a , -Fy b , -Jk a , -Jk b , -S, -s̅, -P1 and -k. Also, all Fy x phenotypes of the selected population of 93 positive samples, originally identified by MALDI-TOF MS-based genotyping, were reliably detected by the lateral flow assay. Extended phenotype blood group parameters, including the serologically challenging Fy x phenotype, can be determined simultaneously, rapidly and accurately using the lateral flow (MDmulticard ® ) technology, even in cases when IgG class antibodies are the only source of diagnostic antibodies. © 2017 International Society of Blood Transfusion.

  12. The detection and sequencing of a broad-host-range conjugative IncP-1β plasmid in an epidemic strain of Mycobacterium abscessus subsp. bolletii.

    Directory of Open Access Journals (Sweden)

    Sylvia Cardoso Leão

    Full Text Available BACKGROUND: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,267-bp [corrected] circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2155 could not be demonstrated. CONCLUSIONS/SIGNIFICANCE: The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.

  13. Quasimolecular emission near the Xe(5p 56s 1,3 P 1 - 5p 6 1 S 0) and Kr (4p 55s 1,3 P 1 - 4p 6 1 S 0) resonance lines induced by collisions with He atoms

    Science.gov (United States)

    Alekseeva, O. S.; Devdariani, A. Z.; Grigorian, G. M.; Lednev, M. G.; Zagrebin, A. L.

    2017-02-01

    This study is devoted to the theoretical investigation of the quasimolecular emission of Xe*-He and Kr*-He collision pairs near the Xe (5p 56s 1,3 P 1 - 5p 6 1 S 0) and Kr (4p 55s 1,3 P 1 - 4p 6 1 S 0) resonance atomic lines. The potential curves of the quasimolecules Xe(5p 56s) + He and Kr(4p 55s) + He have been obtained with the use of the effective Hamiltonian and pseudopotential methods. Based on these potential curves the processes of quasimolecular emission of Xe*+He and Kr*+He mixtures have been considered and the spectral distributions I(ħΔω) of photons emitted have been obtained in the framework of quasistatic approximation.

  14. Vapour liquid equilibria of monocaprylin plus palmitic acid or methyl stearate at P = (1.20 and 2.50) kPa by using DSC technique

    International Nuclear Information System (INIS)

    Cunico, Larissa P.; Damaceno, Daniela S.; Matricarde Falleiro, Rafael M.; Sarup, Bent; Abildskov, Jens; Ceriani, Roberta; Gani, Rafiqul

    2015-01-01

    Highlights: • Novel VLE data for binary mixtures involving a partial acylglycerol (monocaprylin). • Use of a promising technique for measuring VLE/vapour pressure data (DSC technique). • The consistency of experimental data is analysed by a proposed methodology. • Regressed parameters are given for excess Gibbs thermodynamic models. • New regressed parameters are presented for UNIFAC to account for nonidealities. - Abstract: The Differential Scanning Calorimetry (DSC) technique is used for measuring isobaric (vapour + liquid) equilibria for two binary mixtures: {monocaprylin + palmitic acid (system 1) or methyl stearate (system 2)} at two different pressures P = (1.20 and 2.50) kPa. The obtained PTx data are correlated by Wilson, NRTL and UNIQUAC models. The original UNIFAC group contribution method is also considered and new binary interaction parameters for the main groups CH 2 , CCOO, OH and COOH are regressed, to account for the non-idealities found in these lipid systems. Established thermodynamic consistency tests are applied and attest the quality of the measured data. In terms of relevance of the selected components, system 1 can be found in the purification and deodorization steps during the production of edible oils, while, system 2 can be found in the purification steps of biodiesel. It should be noted that no such data could be found in the open literature, not only for the specific components selected but also for the combination of the classes of components considered; that is, acylglycerol plus fatty acid or fatty ester.

  15. Results on $B_{s,d}\\to\\mu\\mu$ decays and measurement of $P'_5$ and $P_1$ parameters in $B_0\\to K^* \\mu \\mu$ decay

    CERN Document Server

    Lacaprara, Stefano

    2017-01-01

    Phenomena beyond the standard model (SM) can manifest themselves indirectly, by affecting the production and decay of SM particles. \\\\The features of $B_{d,s}\\to\\mu\\mu$ decays are sensitive probes of physics beyond the Standard Model. This talk will review the results on these decays from the data collected by the CMS experiment. \\\\The decay $B_0\\to K^*\\mu\\mu$ is a flavor-changing neutral current (FCNC) process particularly sensitive to new physics, since it is heavily suppressed in the SM. Recent results from the LHCb collaboration show a tension with respect to SM prediction of more than 3 sigmas, and the Belle collaboration reported a discrepancy almost as large. We will present results of an angular analysis done by the CMS experiment at the LHC, using p-p data collected at $\\sqrt{s} = 8~\\text{TeV}$, corresponding to an integrated luminosity of L = $20~\\text{fb}^{-1}$. The analysis is focused to measure the angular parameter $P'_5$, as well as $P_1$, as a function of the di-muon invariant mass.

  16. Unexpectedly large difference of the electron density at the nucleus in the 4p ^2{P}_{{1}/{2},{3}/{2}} fine-structure doublet of Ca^+

    Science.gov (United States)

    Shi, C.; Gebert, F.; Gorges, C.; Kaufmann, S.; Nörtershäuser, W.; Sahoo, B. K.; Surzhykov, A.; Yerokhin, V. A.; Berengut, J. C.; Wolf, F.; Heip, J. C.; Schmidt, P. O.

    2017-01-01

    We measured the isotope shift in the ^2{S}_{{1}/{2}} → ^2{P}_{{3}/{2}} (D2) transition in singly ionized calcium ions using photon recoil spectroscopy. The high accuracy of the technique enables us to compare the difference between the isotope shifts of this transition to the previously measured isotopic shifts of the ^2{S}_{{1}/{2}} → ^2{P}_{{1}/{2}} (D1) line. This so-called splitting isotope shift is extracted and exhibits a clear signature of field shift contributions. From the data, we were able to extract the small difference of the field shift coefficient and mass shifts between the two transitions with high accuracy. This J-dependence is of relativistic origin and can be used to benchmark atomic structure calculations. As a first step, we use several ab initio atomic structure calculation methods to provide more accurate values for the field shift constants and their ratio. Remarkably, the high-accuracy value for the ratio of the field shift constants extracted from the experimental data is larger than all available theoretical predictions.

  17. Electron-photon angular correlation measurements of He (1 1S0-2 1P1) excitation by electron impact at 80 eV

    International Nuclear Information System (INIS)

    Steph, N.C.; Golden, D.E.

    1980-01-01

    The electron-photon angular correlation function was measured between 80-eV electrons which excited the 2 1 P 1 state of helium and 58.4-nm photons from the decay of that state for electron scattering angles ranging from 5 0 to 100 0 . The data have been analyzed to yield values of the ratio lambda of the differential cross section for exciting the M/sub j/=0 sublevel to the total differential cross section and the magnitude vertical-barchivertical-bar of the phase difference between the M/sub j/=0 and M/sub j/=1 excitation amplitudes. The data agree with all previous measurements within one standard deviation, with the exception of the large-angle values of lambda obtained by Hollywood, Crowe, and Williams. Possible causes of these discrepancies are discussed. The values of lambda and vertical-barchivertical-bar obtained in this work agree quite well with those given by the distorted-wave calculations of Madison over the entire angular range

  18. Elastic Properties and the Band Gap of AlNxP1-x Semiconductor Alloy: A Comparative Study of Various Ab Initio Approaches

    Directory of Open Access Journals (Sweden)

    M. P. Polak

    2016-01-01

    Full Text Available Structural and elastic properties of AlNxP1-x, a novel semiconductor alloy, are studied from the first principles in both zinc-blende and wurtzite structures. Performances of the finite difference (FD method and the density functional perturbation theory (DFPT are tested and compared. Both of these methods are applied to two different approaches of alloy simulation, a supercell of 16 and 32 atoms (for zinc-blende and wurtzite structures, resp. and the alchemical mixing (AM method, where the pseudopotentials are mixed in an appropriate way to form an alloy. All elastic properties, including the elastic tensors, elastic moduli, Poisson’s ratio, B/G, and relaxation coefficient, as well as lattice parameters are calculated using all said methods. Conclusions about the use of the approaches investigated in this paper and about their performance are drawn. In addition, in both crystal structures, the band gap is studied in the whole composition range using the MBJLDA functional. The band gap bowings are unusually high, which confirms earlier reports.

  19. Resolution of the diadenosine 5',5'''-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase α

    International Nuclear Information System (INIS)

    Baril, E.; Bonin, P.; Burstein, D.; Mara, K.; Zamecnik, P.

    1983-01-01

    A diadenosine 5',5'''-P 1 ,P 4 -tetraphosphate (Ap 4 A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase α [deoxy-nucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells. The Ap 4 A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatograpy on butylagarose resolves the Ap 4 A binding activity from the DNA polymerase. The Ap 4 A binding activity is protein in nature since the binding of Ap 4 A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap 4 A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO 4 /polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [ 32 P]Ap 4 A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap 4 A

  20. Analysis of mechanism of PM2.5 and house dust mite antigen Der p1 in attack stage of child asthma.

    Science.gov (United States)

    Cheng, Q; Yang, C-Y; Guo, B-Y; Wei, X; Liu, M

    2017-05-01

    We analyzed the influence of PM2.5 and house dust mite antigen Der p1 on the treatment of child asthma attack. A total of 96 children with asthma attack were included into the study. The patients were randomly divided into the PM2.5 group, the house dust mite antigen group, the synergistic group and the control group (n= 24 in each group). The PM2.5 concentration in the PM2.5 group was twice higher than standard level (≤ the average value of PM2.5 in local air). All cases were given with same treatment, and the treatment effects were compared and analyzed. It was found that the asthma control rate in the control group was significantly higher than that in the PM2.5 group and the house dust mite antigen group, and the synergistic group was the lowest. The control time in the synergistic group was significantly longest, followed by the PM2.5 group and the house dust mite antigen group, and the control group was significantly short (pasthma attack by an inflammatory reaction and oxidative stress.

  1. The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

    Science.gov (United States)

    Turner, Andrew G; Ong, Cheryl-Lynn Y; Djoko, Karrera Y; West, Nicholas P; Davies, Mark R; McEwan, Alastair G; Walker, Mark J

    2017-06-01

    Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA ( P erR-regulated m etal t ransporter A ), a P 1B-4 -type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448Δ pmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448Δ pmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen. Copyright © 2017 American Society for Microbiology.

  2. Identification and validation of FaP1D7, a putative marker associated with the biosynthesis of methyl butanoate in cultivated strawberry (Fragaria x ananassa).

    Science.gov (United States)

    Gor, Mian Chee; Candappa, Chrishani; de Silva, Thishakya; Mantri, Nitin; Pang, Edwin

    2017-12-12

    Breeding strawberry (Fragaria x ananassa) with enhanced fruit flavour is one of the top breeding goals of many strawberry-producing countries. Although several genes involved in the biosynthetic pathways of key aroma compounds have been identified, the development and application of molecular markers associated with fruit flavour remain limited. This study aims to identify molecular markers closely linked to genes controlling strawberry aroma. A purpose-built Subtracted Diversity Array (SDA) known as Fragaria Discovery Panel (FDP) was used for marker screening. Polymorphic sequences associated with key aroma compounds were identified from two DNA bulks with extreme phenotypes, established using 50 F 1 progeny plants derived from Juliette X 07-102-41 cross, two strawberry genotypes differing in aroma profile. A total of 49 polymorphic markers for eight key aroma compounds were detected using genotypic data of the extreme DNA bulks and phenotypic data obtained from gas chromatography-mass spectrometry (GC-MS). A similarity search against the physical maps of Fragaria vesca revealed that FaP1D7 is linked to genes potentially involved in the synthesis of methyl butanoate. A C/T SNP was detected within the feature, which could possibly be converted to a molecular tool for rapid screening of the strawberry accessions for their methyl butanoate production capacity.

  3. Optical properties of lattice matched InxGa1-xP1-yNy heteroepitaxial layers on GaP

    International Nuclear Information System (INIS)

    Imanishi, T.; Wakahara, A.; Kim, S.M.; Yonezu, H.; Furukawa, Y.

    2005-01-01

    Optical constants and band structure of In x Ga 1-x P 1-y N y lattice matched to GaP (100) substrate are investigated. Nitrogen concentration in the film estimated by X-ray diffraction and X-ray photoelectron spectroscopy, was 1.4%, 1.8% and 3.5%. Refractive index and transition critical points E 0 (Γ v to Γ c ), E 1 (L v to L c ) and E 2 (X v to X c ) are evaluated by spectroscopic ellipsometry. When N composition increases from 1.4% to 3.5%, both photoluminescence (PL) peak energy, E PL , and E 0 shift to lower energy, and the energy difference ΔE=E 0 -E PL decrease from 380 meV to 110 meV. The large red-sift of E PL from the E 0 suggest that the luminescence is of defect-related luminescence, and crossover point of indirect band structure estimated by the extrapolation of N-composition dependence of ΔE is estimated to be around in In 0.1 Ga 0.9 P 0.96 N 0.04 . (copyright 2005 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  4. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  5. Constituents of a hit parade: Questioning democracy and listener participation in P4 i P1’s Det elektriske barometer

    Directory of Open Access Journals (Sweden)

    Mette Simonsen Abildgaard

    2014-12-01

    Full Text Available Due to their historically inaccessible nature, public service broadcasters’ media archives have lent themselves primarily to internal refl ection while historical contextualisation of the cultural heritage in these archives has been broadcasters’ prerogative. In this study, digitised material from the Danish youth radio programme P4 i P1’s Det elektriske barometer forms the basis for an experiment into how access to digital archives can inform humanities scholarship. We argue that one important implication of the new digital archives is that they enable approaches independent of broadcasters’ own narratives since they off er the possibility for autonomous study of large quantities of material. The character of listener participation in Det elektriske barometer, which had the slogan ‘the listener-determined hit parade’, is approached from a micro-, meso-, and macro-level employing Carpentier’s concept of participation (2011b, to explore how diff erent approaches to digital archives can provide new answers to media’s self-presentation.

  6. Developmental and cross-modal plasticity in deafness: evidence from the P1 and N1 event related potentials in cochlear implanted children.

    Science.gov (United States)

    Sharma, Anu; Campbell, Julia; Cardon, Garrett

    2015-02-01

    Cortical development is dependent on extrinsic stimulation. As such, sensory deprivation, as in congenital deafness, can dramatically alter functional connectivity and growth in the auditory system. Cochlear implants ameliorate deprivation-induced delays in maturation by directly stimulating the central nervous system, and thereby restoring auditory input. The scenario in which hearing is lost due to deafness and then reestablished via a cochlear implant provides a window into the development of the central auditory system. Converging evidence from electrophysiologic and brain imaging studies of deaf animals and children fitted with cochlear implants has allowed us to elucidate the details of the time course for auditory cortical maturation under conditions of deprivation. Here, we review how the P1 cortical auditory evoked potential (CAEP) provides useful insight into sensitive period cut-offs for development of the primary auditory cortex in deaf children fitted with cochlear implants. Additionally, we present new data on similar sensitive period dynamics in higher-order auditory cortices, as measured by the N1 CAEP in cochlear implant recipients. Furthermore, cortical re-organization, secondary to sensory deprivation, may take the form of compensatory cross-modal plasticity. We provide new case-study evidence that cross-modal re-organization, in which intact sensory modalities (i.e., vision and somatosensation) recruit cortical regions associated with deficient sensory modalities (i.e., auditory) in cochlear implanted children may influence their behavioral outcomes with the implant. Improvements in our understanding of developmental neuroplasticity in the auditory system should lead to harnessing central auditory plasticity for superior clinical technique. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Polymorphic expression of glutathione transferases A1, M1, P1 and T1 in epithelial ovarian cancer: a Serbian case-control study.

    Science.gov (United States)

    Pljesa, Igor; Berisavac, Milica; Simic, Tatjana; Pekmezovic, Tatjana; Coric, Vesna; Suvakov, Sonja; Stamatovic, Ljiljana; Matic, Marija; Gutic, Bojana; Milenkovic, Sanja; Pljesa-Ercegovac, Marija; Savic-Radojevic, Ana

    2017-01-01

    Since several studies have proposed that epithelial ovarian cancer should not be considered as a single disease entity and that it results from an accumulation of genetic changes, we aimed to assess the polymorphic expression of major cytosolic glutathione S-transferases (GSTM1, T1, A1 and P1) with respect to ovarian cancer susceptibility and aggressiveness. This case-control study was conducted on 93 newly diagnosed epithelial ovarian cancer patients and 178 healthy matched controls. The multiplex polymerase chain reaction (PCR) was used to detect homozygous deletions of GSTM1 and GSTT1 genes. Analysis of the single nucleotide polymorphism (SNP) GSTA1 C69T was performed using PCR-restriction fragment length polymorphism (RFLP), while for SNP GSTP1 Ile105Val real-time PCR was used. No significant association to ovarian cancer risk was found for individual GSTM1, GSTA1 and GSTP1 genotypes (p>0.05). However, the carriers of GSTT1-active genotype were at 2-fold higher risk of ovarian cancer development (95%CI: 1.00-4.01, p=0.049), which was even more elevated in the subgroup of patients with positive family history of cancer. Moreover, the frequency of all three GST genotypes that might be associated to ovarian cancer risk (GSTT1-active, GSTA1-active and GSTP1-referent) was significantly higher in patients than in the control group (p=0.042). Even more, patients who were carriers of combination of these three genotypes represented over 64% of the total number of patients within any of the International Federation of Gynecology and Obstetrics (FIGO) stages of ovarian cancer. This study provides supportive evidence that GSTs might affect both susceptibility and progression of ovarian cancer.

  8. Glutathione S-transferase P1, gene-gene interaction, and lung cancer susceptibility in the Chinese population: An updated meta-analysis and review

    Directory of Open Access Journals (Sweden)

    Xue-Ming Li

    2015-01-01

    Full Text Available Aim of Study: To assess the impact of glutathione S-transferase P1 (GSTP1 Ile105Val polymorphism on the risk of lung cancer in the Chinese population, an updated meta-analysis and review was performed. Materials and Methods: Relevant studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure, and Chinese Biology Medicine published through January 22, 2015. The odds ratios (ORs and 95% confidence intervals (CIs were calculated to estimate the strength of the associations. Results: A total of 13 case-control studies, including 2026 lung cancer cases and 2451 controls, were included in this meta-analysis. Overall, significantly increased lung cancer risk was associated with the variant genotypes of GSTP1 polymorphism in the Chinese population (GG vs. AA: OR = 1.36, 95% CI = 1.01-1.84. In subgroup analyses stratified by geographic area and source of controls, the significant results were found in population-based studies (GG vs. AA: OR = 1.62, 95% CI: 1.13-2.31; GG vs. AG: OR = 1.49, 95% CI: 1.03-2.16; GG vs. AA + AG: OR = 1.55, 95% CI: 1.12-2.26. A gene-gene interaction analysis showed that there was an interaction for individuals with combination of GSTM1 (or GSTT1 null genotype and GSTP1 (AG + GG mutant genotype for lung cancer risk in Chinese. Conclusion: This meta-analysis suggests that GSTP1 Ile105Val polymorphism may increase the risk of lung cancer in the Chinese population.

  9. Association between Glutathione S-Transferase GSTM1-T1 and P1 Polymorphisms with Metabolic Syndrome in Zoroastrians in Yazd, Iran

    Science.gov (United States)

    AFRAND, Mohammadhosain; BASHARDOOST, Nasrollah; SHEIKHHA, Mohammad Hasan; AFKHAMI-ARDEKANI, Mohammad

    2015-01-01

    Background: The aim of this study was to assess the possible association between genetic polymorphisms of the glutathione S-transferase (GST) gene family and the risk of the development of metabolic syndrome (MS) in Zoroastrian females in Yazd, Iran. Methods: In this case-control study, GSTM1, T1, and P1 polymorphisms were genotyped in 51 randomly selected MS patients and 50 randomly selected healthy controls on February 2014 among Zoroastrian females whose ages ranged from 40 to 70 yr. DNA was extracted from peripheral blood. Data were analyzed with SPSS version 17. Results: We observed a significant association of GSTP1-I/V (Isoleucine/Valine) allele and GSTP1-V/V (Valine / Valine) allele with MS (P = 0.047 and P = 0.044, respectively). The combined analysis of the two genotypes, the present genotype of GSTT1, I/V and V/V alleles of GSTP1 genotype demonstrated a decrease in the risk of acquiring MS (OR = 0.246, P = 0.031). The null genotype of GSTM1, I/V, and V/V alleles of the GSTP1 genotype showed a lower risk in double combinations (OR = 0.15, P = 0.028 and OR = 0.13, P = 0.013, respectively). The combinations of the GSTM1 null genotypes and GSTT1 present genotypes and the GSTP1 I/V and V/V alleles together were associated with decreased risk of having MS in triple combinations (OR = 0.071, P = 0.039 and OR = 0.065, P = 0.022, respectively). Conclusion: GSTP1-I/V and V/V alleles, alone or in association with GSTM1 null and GSTT1 present genotypes, are related with decreased susceptibility to the development of MS in Zoroastrian females. PMID:26284209

  10. Cigarette smoking, physical activity, and alcohol consumption as predictors of cancer incidence among women at high risk of breast cancer in the NSABP P-1 trial.

    Science.gov (United States)

    Land, Stephanie R; Liu, Qing; Wickerham, D Lawrence; Costantino, Joseph P; Ganz, Patricia A

    2014-05-01

    NSABP P-1 provides an opportunity to examine the association of behavioral factors with prospectively monitored cancer incidence and interactions with tamoxifen. From 1992 to 1997, 13,388 women with estimated 5-year breast cancer risk greater than 1.66% or a history of lobular carcinoma in situ (87% younger than age 65; 67% postmenopausal) were randomly assigned to tamoxifen versus placebo. Invasive breast cancer, lung cancer, colon cancer, and endometrial cancer were analyzed with Cox regression. Predictors were baseline cigarette smoking, leisure-time physical activity, alcohol consumption, and established risk factors. At median 7 years follow-up, we observed 395, 66, 35, and 74 breast cancer, lung cancer, colon cancer, and endometrial cancer, respectively. Women who had smoked were at increased risk of breast cancer (P = 0.007; HR = 1.3 for 15-35 years smoking, HR = 1.6 for ≥ 35 years), lung cancer (P cancer (P breast cancer risk only among women assigned to placebo (P = 0.021 activity main effect, P = 0.013 activity-treatment interaction; HR = 1.4 for the placebo group) and endometrial cancer among all women (P = 0.026, HR = 1.7). Moderate alcohol (>0-1 drink/day) was associated with decreased risk of colon cancer (P = 0.019; HR = 0.35) versus no alcohol. There were no other significant associations between these behaviors and cancer risk. Among women with elevated risk of breast cancer, smoking has an even greater impact on breast cancer risk than observed in past studies in the general population. Women who smoke or are inactive should be informed of the increased risk of multiple types of cancer. ©2014 AACR.

  11. Local structure and morphological evolution of ZnTPP molecules grown on Fe(001)-p(1 × 1)O studied by STM and NEXAFS

    Science.gov (United States)

    Picone, Andrea; Giannotti, Dario; Brambilla, Alberto; Bussetti, Gianlorenzo; Calloni, Alberto; Yivlialin, Rossella; Finazzi, Marco; Duò, Lamberto; Ciccacci, Franco; Goldoni, Andrea; Verdini, Alberto; Floreano, Luca

    2018-03-01

    When used as substrates, thin metal-oxide (MO) layers can perturb the physical and chemical properties of molecules in contact with the surface. To study the molecule-MO layer interaction, we focused our investigation on a prototypical interface, namely zinc tetraphenylporphyrin (ZnTPP) film on Fe(001)-p(1 × 1)O. In a previous study, we found that no significant change of the electronic structure takes place at the monolayer (ML) coverage either in the core level photoemission spectra or in the highest occupied molecular orbitals (HOMOs). However, molecules showed the occurrence of a commensurate (5 × 5) diffraction pattern that indicates a certain degree of interaction with the substrate. In order to better understand the effective molecule/metal decoupling operated by the FeO layer, we performed a combined investigation based on a scanning tunneling microscopy (STM) study of the self-assembled ZnTPP molecular layer and on a near edge X-ray absorption fine structure spectroscopy (NEXAFS). Molecules are found to lie almost parallel to the substrate, even if the central macrocycle displays a characteristic small saddle-like distortion (symmetry from D4h to D2h drives the azimuthal orientation with respect to the substrate and determines the co-existence of four equivalent (5 × 5) ZnTPP domains, following the substrate four-fold symmetry. The comparison with films of increasing thickness shows that, beyond the second layer, the molecules gradually tilt-off the surface (by at least 40°) ordering into 3D islands. The NEXAFS resonances of the lowest unoccupied orbitals (LUMOs) do not display significant changes from the monolayer to the multilayer thickness range, apart from minor modification of the LUMOs relative intensity. The latter variation may be associated with the change of spatial spread of the molecular orbitals in the contact layer due to the saddle-like distortion.

  12. The peroxisomal Lon protease LonP2 in aging and disease: functions and comparisons with mitochondrial Lon protease LonP1.

    Science.gov (United States)

    Pomatto, Laura C D; Raynes, Rachel; Davies, Kelvin J A

    2017-05-01

    Peroxisomes are ubiquitous eukaryotic organelles with the primary role of breaking down very long- and branched-chain fatty acids for subsequent β-oxidation in the mitochondrion. Like mitochondria, peroxisomes are major sites for oxygen utilization and potential contributors to cellular oxidative stress. The accumulation of oxidatively damaged proteins, which often develop into inclusion bodies (of oxidized, aggregated, and cross-linked proteins) within both mitochondria and peroxisomes, results in loss of organelle function that may contribute to the aging process. Both organelles possess an isoform of the Lon protease that is responsible for degrading proteins damaged by oxidation. While the importance of mitochondrial Lon (LonP1) in relation to oxidative stress and aging has been established, little is known regarding the role of LonP2 and aging-related changes in the peroxisome. Recently, peroxisome dysfunction has been associated with aging-related diseases indicating that peroxisome maintenance is a critical component of 'healthy aging'. Although mitochondria and peroxisomes are both needed for fatty acid metabolism, little work has focused on understanding the relationship between these two organelles including how age-dependent changes in one organelle may be detrimental for the other. Herein, we summarize findings that establish proteolytic degradation of damaged proteins by the Lon protease as a vital mechanism to maintain protein homeostasis within the peroxisome. Due to the metabolic coordination between peroxisomes and mitochondria, understanding the role of Lon in the aging peroxisome may help to elucidate cellular causes for both peroxisome and mitochondrial dysfunction. © 2016 Cambridge Philosophical Society.

  13. The 2P1/2 → 2P3/2 laser transition in atomic iodine and the problem of search for signals from extraterrestrial intelligence

    International Nuclear Information System (INIS)

    Kutaev, Yu F; Mankevich, S K; Nosach, O Yu; Orlov, E P

    2007-01-01

    It is proposed to search for signals from extraterrestrial intelligence (ETI) at a wavelength of 1.315 μm of the laser 2 P 1/2 → 2 P 3/2 transition in the atomic iodine, which can be used for this purpose as the natural frequency reference. The search at this wavelength is promising because active quantum filters (AQFs) with the quantum sensitivity limit have been developed for this wavelength, which are capable of receiving laser signals, consisting of only a few photons, against the background of emission from a star under study. In addition, high-power iodine lasers emitting diffraction-limited radiation at 1.315 μm have been created, which highly developed ETI also can have. If a ETI sends in our direction a diffraction-limited 10-ns, 1-kJ laser pulse with the beam diameter of 10 m, a receiver with an AQF mounted on a ten-meter extra-atmospheric optical telescope can detect this signal at a distance of up to 300 light years, irrespective of the ETI position on the celestial sphere. The realisation of the projects for manufacturing optical telescopes of diameter 30 m will increase the research range up to 2700 light years. A weak absorption of the 1.315-μm radiation in the Earth atmosphere (the signal is attenuated by less than 20%) allows the search for ETI signals by using ground telescopes equipped with adaptive optical systems. (laser applications and other topics in quantum electronics)

  14. Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration.

    Science.gov (United States)

    Skogh, Anna; Lesniak, Anna; Gaugaz, Fabienne Z; Svensson, Richard; Lindeberg, Gunnar; Fransson, Rebecca; Nyberg, Fred; Hallberg, Mathias; Sandström, Anja

    2017-11-15

    Substance P 1-7 (SP 1-7 , Arg 1 -Pro 2 -Lys 3 -Pro 4 -Gln 5 -Gln 6 -Phe 7 ) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP 1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP 1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP 1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP 1-7 amide 1 (Arg 1 -Pro 2 -Lys 3 -Pro 4 -Gln 5 -Gln 6 -Phe 7 -NH 2 ) was previously shown to be superior to the endogenous SP 1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP 1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys 3 ]SP 1-7 amide (3) and the [MeGln 5 ]SP 1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg 1 and C-terminal Phe 7 , anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP 1-7 amide (1), the [MeLys 3 ]SP 1-7 amide (3) amide and the [MeGln 5 ]SP 1-7 amide (4) are all considerably more effective

  15. Investigations into the corrosion resistance of copper aluminium alloys. Effect of phosphorus as corrosion resistant third alloying element in the ternary system CuAl20P1

    International Nuclear Information System (INIS)

    Allwardt, A.

    1997-01-01

    The effect of phosphorus on the corrosion resistance of Al-bronzes is studied in detail in this work. A literature review showed that there are a lot of things known about the microstructure and the mechanical properties of Al-bronzes. In spite of their corrosion resistance the corrosion properties and the structure of the protective oxide films of Al-bronzes were seldom a matter of interest. Systematic studies of the influence of different alloying elements on the oxide film and the corrosion properties are rare. Therefore, it is not possible to predict the corrosion resistance of Al-bronzes, made by alloying particular elements. The high corrosion resistance of the new alloy CuAl 20 P 1 was the reason to investigate the influence of phosphorus on the corrosion properties of Al-bronzes in more detail. A systematic study of the microstructure and the corrosion properties of Cu, CuP x , CuAl 20 and CuAl 20 P x offers an insight into the effect of aluminium and phosphorus on the formation of the oxide film on Al-bronzes. It was found that there exists a critical amount of 1 at.-% of phosphorus. Above and below this amount the corrosion resistance becomes worse. This behaviour could be explained by XPS-and electrochemical measurements. Although there are still some questions about the influence of phosphorus on the corrosion resistance of Al-bronzes, this work has produced some important results, which in the future may be helpful to develop new high corrosion resistant Al-bronzes more efficiently: - on clean surface Al-bronze, the oxidation of Al and Cu takes place simultaneously, - Al promotes the formation of Cu 2 O but impedes the formation of Cu(II)-oxide/-hydride in neutral solutions, - P impedes the formation of Cu 2 O and as a consequence promotes the formation of aluminium oxide. This results in a higher amount of Al in the oxide film on the surface of the alloy, which leads to a better corrosion resistance. (author) figs., tabs., 106 refs

  16. Construction of strain engineered for expression of porcine β-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity

    Directory of Open Access Journals (Sweden)

    Jian Xu

    2017-04-01

    Full Text Available Objective To generate recombinant Bacillus subtilis (B. subtilis engineered for expression of porcine β-defensin-2 (pBD-2 and cecropin P1 (CP1 fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli. The weight gain and diarrhea incidence of piglets were measured after challenge. Results The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis and gram-positive bacteria (Staphylococcus aureus. Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial

  17. The three youngest Plinian eruptions of Mt Pelée, Martinique (P1, P2 and P3): Constraining the eruptive conditions from field and experimental studies.

    Science.gov (United States)

    Kueppers, Ulrich; Uhlig, Joan; Carazzo, Guillaume; Kaminski, Edouard; Perugini, Diego; Tait, Steve; Clouard, Valérie

    2015-04-01

    Mt Pelée on Martinique, French Lesser Indies, is infamous for the last big Pelean (i.e., dome forming) eruption in 1902 AD that destroyed agricultural land and the city of Saint Pierre by pyroclastic density currents. Beside such mostly valley-confined deposits, the geological record shows thick fall deposits of at least three Plinian eruptions during the past 2000 years. In an attempt to describe and understand systematic eruptive behaviours as well as the associated variability of eruptive scenarios of Plinian eruptions in Martinique, we have investigated approx. 50 outcrops belonging to the P1 (1315 AD), P2 (345 AD) and P3 (4 AD) eruptions (Traineau et al., JVGR 1989) and collected bulk samples as well as >100 mm pumiceous clasts. All samples are andesitic, contain plagioclase and pyroxene in a glassy matrix and range in porosity between 55 and 69 vol.% with individual bubbles rarely larger than 1 mm. Our approach was two-fold: 1) Loose bulk samples have been subject to dry mechanical sieving in order to quantively describe the grain-size distribution and the fractal dimension. 2) From large clasts, 60*25 mm cylinders have been drilled for fragmentation experiments following the sudden decompression of gas in the sample's pore space. The used experimental set-up allowed for precisely controllable and repeatable conditions (5, 10 and 15 MPa, 25 °C) and the complete sampling of the generated pyroclasts. These experimentally generated clasts were analysed for their grain-size distribution and fractal dimension. For both natural samples and experimental populations, we find we find that the grain-size distribution follows a power-law, with an exponent between 2,5 and 3,7. Deciphering eruption conditions from deposits alone is challenging because of the complex interplay of dynamic volcanic processes and transport-related sorting. We use the quantified values of fractal dimension for a comparison of the power law exponents among the three eruptions and the

  18. Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus elicit a protective response in guinea pigs.

    Science.gov (United States)

    Pan, Li; Zhang, Yongguang; Wang, Yonglu; Wang, Baoqin; Wang, Wenxiu; Fang, Yuzhen; Jiang, Shoutian; Lv, Jianliang; Wang, Wei; Sun, Yuan; Xie, Qingge

    2008-01-15

    The expression of recombinant antigens in transgenic plants is increasingly used as an alternative method of producing experimental immunogens. In this report, we describe the production of transgenic tomato plants that express the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease (FMDV). P1-2A3C was inserted into the plant binary vector, pBin438, and transformed into tomato plants using Agrobacterium tumefaciens strain, GV3101. The presence of P1-2A3C was confirmed by PCR, transcription was verified by RT-PCR, and recombinant protein expression was confirmed by sandwich-ELISA and Western blot analyses. Guinea pigs immunized intramuscularly with foliar extracts from P1-2A3C-transgenic tomato plants were found to develop a virus-specific antibody response against FMDV. Vaccinated guinea pigs were fully protected against a challenge infection, while guinea pigs injected with untransformed plant extracts failed to elicit an antibody response and were not protected against challenge. These results demonstrate that transgenic tomato plants expressing the FMDV structural polyprotein, P1-2A, and the protease, 3C, can be used as a source of recombinant antigen for vaccine production.

  19. The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation

    Directory of Open Access Journals (Sweden)

    Andy Chevigné

    2017-05-01

    Full Text Available The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

  20. Synthesis of P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate for the investigation of biosynthesis of O-antigenic polysaccharides in Pseudomonas aeruginosa and Escherichia coli O104.

    Science.gov (United States)

    Torgov, Vladimir; Danilov, Leonid; Utkina, Natalia; Veselovsky, Vladimir; Brockhausen, Inka

    2017-12-01

    Two new phenoxyundecyl diphosphate sugars were synthesized for the first time: P 1 -(11-phenoxyundecyl)-P 2 - (2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate and P 1 -(11-phenoxyundecyl)-P 2 -(2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate to study the third step of biosynthesis of the repeating units of O-antigenic polysaccharides in Pseudomonas aeruginosa and E.coli O104 respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Structural requirements for the flavonoid-mediated modulation of glutathione S-transferase P1-1 and GS-X pump activity in MCF7 breast cancer cells

    NARCIS (Netherlands)

    Zanden, van J.J.; Geraets, L.; Wortelboer, H.M.; Bladeren, van P.J.; Rietjens, I.M.C.M.; Cnubben, N.H.P.

    2004-01-01

    The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1

  2. Construction of All-Solid-State Batteries based on a Sulfur-Graphene Composite and Li9.54Si1.74P1.44S11.7Cl0.3Solid Electrolyte.

    Science.gov (United States)

    Xu, Ruochen; Wu, Zhang; Zhang, Shenzhao; Wang, Xiuli; Xia, Yan; Xia, Xinhui; Huang, Xiaohua; Tu, Jiangping

    2017-10-09

    Herein an effective way for construction of all-solid-state lithium-sulfur batteries (LSBs) with sulfur/reduced graphene oxide (rGO) and Li 9.54 Si 1.74 P 1.44 S 11.7 Cl 0.3 solid electrolyte is reported. In the composite cathode, the Li 9.54 Si 1.74 P 1.44 S 11.7 Cl 0.3 powder is homogeneously mixed with the S/rGO composite to enhance the ionic conductivity. Coupled with a metallic Li anode and solid electrolyte, the designed S/rGO-Li 9.54 Si 1.74 P 1.44 S 11.7 Cl 0.3 composite cathode exhibits a high specific capacity and good cycling stability. A high initial discharge capacity of 969 mAh g -1 is achieved at a current density of 80 mA g -1 at room temperature and the cell retains a reversible capacity of over 827 mAh g -1 after 60 cycles. The enhanced performance is attributed to the intimate contact between the S/rGO and Li 9.54 Si 1.74 P 1.44 S 11.7 Cl 0.3 electrolyte, and high electrical conductivity of rGO and high ionic conductivity of the solid electrolyte. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling.

    Science.gov (United States)

    Pereañez, Jaime Andrés; Patiño, Arley Camilo; Rey-Suarez, Paola; Núñez, Vitelbina; Henao Castañeda, Isabel Cristina; Rucavado, Alexandra

    2013-09-01

    Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC₅₀ of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²⁺ ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²⁺, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. NMR solution structure of the acylphosphatase from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Pagano, Katiuscia [University of Udine, Department of Biomedical Sciences and Technologies (Italy); Ramazzotti, Matteo [University of Florence, Department of Biochemical Sciences (Italy); Viglino, Paolo; Esposito, Gennaro [University of Udine, Department of Biomedical Sciences and Technologies (Italy); Degl' Innocenti, Donatella; Taddei, Niccolo [University of Florence, Department of Biochemical Sciences (Italy); Corazza, Alessandra [University of Udine, Department of Biomedical Sciences and Technologies (Italy)], E-mail: acorazza@mail.dstb.uniud.it

    2006-11-15

    The solution structure of Escherichia coli acylphosphatase (E. coli AcP), a small enzyme catalyzing the hydrolysis of acylphosphates, was determined by {sup 1}H and {sup 15}N NMR and restrained modelling calculation. In analogy with the other members of AcP family, E. coli AcP shows an {alpha}/{beta} sandwich domain composed of four antiparallel and one parallel {beta}-strand, assembled in a five-stranded {beta}-sheet facing two antiparallel {alpha}-helices. The pairwise RMSD values calculated for the backbone atoms of E. coli and Sulfolobus solfataricus AcP, Bovine common type AcP and Horse muscle AcP are 2.18, 5.31 and 5.12 A, respectively. No significant differences are present in the active site region and the catalytic residue side chains are consistently positioned in the structures.

  5. Proteolysis in hyperthermophilic microorganisms

    Directory of Open Access Journals (Sweden)

    Donald E. Ward

    2002-01-01

    Full Text Available Proteases are found in every cell, where they recognize and break down unneeded or abnormal polypeptides or peptide-based nutrients within or outside the cell. Genome sequence data can be used to compare proteolytic enzyme inventories of different organisms as they relate to physiological needs for protein modification and hydrolysis. In this review, we exploit genome sequence data to compare hyperthermophilic microorganisms from the euryarchaeotal genus Pyrococcus, the crenarchaeote Sulfolobus solfataricus, and the bacterium Thermotoga maritima. An overview of the proteases in these organisms is given based on those proteases that have been characterized and on putative proteases that have been identified from genomic sequences, but have yet to be characterized. The analysis revealed both similarities and differences in the mechanisms utilized for proteolysis by each of these hyperthermophiles and indicated how these mechanisms relate to proteolysis in less thermophilic cells and organisms.

  6. Extensive Lysine Methylation in Hyperthermophilic Crenarchaea: Potential Implications for Protein Stability and Recombinant Enzymes

    Directory of Open Access Journals (Sweden)

    Catherine H. Botting

    2010-01-01

    Full Text Available In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in α-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

  7. Dicty_cDB: Contig-U09490-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 91_1( AY499191 |pid:none) Synthetic construct paraoxonase PO... 47 0.002 CP001399...6641 |pid:none) Sulfolobus solfataricus P2, com... 44 0.015 ( P27170 ) RecName: Full=Serum paraoxonase/aryle..... 43 0.025 AY499189_1( AY499189 |pid:none) Synthetic construct paraoxonase PO... 43 0.025 U32684_1( U32684 ...|pid:none) Mus musculus serum paraoxonase (Pon) m... 42 0.033 FP236842_3562( FP236842 |pid:none) Erwinia pyr...ifoliae strain Ep1/9... 42 0.033 AY499193_1( AY499193 |pid:none) Synthetic construct paraoxonase

  8. [Decontamination of organophosphorus compounds: Towards new alternatives].

    Science.gov (United States)

    Poirier, L; Jacquet, P; Elias, M; Daudé, D; Chabrière, E

    2017-05-01

    Organophosphorus coumpounds (OP) are toxic chemicals mainly used for agricultural purpose such as insecticides and were also developed and used as warfare nerve agents. OP are inhibitors of acetylcholinesterase, a key enzyme involved in the regulation of the central nervous system. Chemical, physical and biological approaches have been considered to decontaminate OP. This review summarizes the current and emerging strategies that are investigated to tackle this issue with a special emphasis on enzymatic remediation methods. During the last decade, many studies have been dedicated to the development of biocatalysts for OP removal. Among these, recent reports have pointed out the promising enzyme SsoPox isolated from the archaea Sulfolobus solfataricus. Considering both its intrinsic stability and activity, this hyperthermostable enzyme is highly appealing for the decontamination of OP. Copyright © 2017 Académie Nationale de Pharmacie. All rights reserved.

  9. Dicty_cDB: Contig-U04013-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available pid:none) Flavobacterium psychrophilum JIP... 98 3e-19 EU678894_1( EU678894 |pid:none) Bacillus intermedius ...46 3e-04 2 ( EH672780 ) CCIL10412.b1_G11.ab1 CCI(LMS) chicory Cichorium i... 46 3e-04 2 ( EJ368820 ) 1092963715450 Global-Ocean-Sampl...41 |pid:none) Sulfolobus solfataricus P2, com... 89 2e-16 AH1770( AH1770 ) conserved hypothetical protein lin2710 [imported...AY653733 ) Acanthamoeba polyphaga mimivirus, complete genome. 62 3e-05 1 ( EH678404 ) CCIL3410.b1_D14.ab1 CC...ngs S... 44 6.8 1 >( AC116963 ) Dictyostelium discoideum chromosome 2 map 4657875-4914984 strain AX4, compl

  10. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    Energy Technology Data Exchange (ETDEWEB)

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  11. Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.

    Science.gov (United States)

    Xu, Weiliang; Jiang, Wenjun; Wang, Jiaxing; Yu, Linping; Chen, Ji; Liu, Xianyu; Liu, Lei; Zhu, Ting F

    2017-01-01

    Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.

  12. Every OGT Is Illuminated … by Fluorescent and Synchrotron Lights.

    Science.gov (United States)

    Miggiano, Riccardo; Valenti, Anna; Rossi, Franca; Rizzi, Menico; Perugino, Giuseppe; Ciaramella, Maria

    2017-12-05

    O ⁶-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs) are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus , respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins.

  13. HYPERDIRE. HYPERgeometric functions DIfferential REduction. MATHEMATICA based packages for differential reduction of generalized hypergeometric functions {sub p}F{sub p-1}, F{sub 1}, F{sub 2}, F{sub 3}, F{sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Bytev, Vladimir V.; Kalmykov, Mikhail Yu. [Hamburg Univ. (Germany). 2. Inst. fuer Theoretische Physik; Joint Institute for Nuclear Research, Dubna (Russian Federation); Kniehl, Bernd A. [Hamburg Univ. (Germany). 2. Inst. fuer Theoretische Physik

    2013-05-15

    HYPERDIRE is a project devoted to the creation of a set of Mathematica based programs for the differential reduction of hypergeometric functions. The current version includes two parts: one, pfq, is relevant for manipulations of hypergeometric functions {sub p+1}F{sub p}, and the second one, AppellF1F4, for manipulations with Appell hypergeometric functions F{sub 1}, F{sub 2}, F{sub 3}, F{sub 4} of two variables.

  14. Sampling system for pulsed signals. Study of the radioactive lifetimes of excited 32P1/2 and 32P3/2 states of Na, excited by a tunable dye laser

    International Nuclear Information System (INIS)

    Thomas, P.; Campos, J.

    1979-01-01

    A system for sampling and averaging repetitive signals in the order of nanoseconds is discussed. The system uses as storage memory a multichannel analyzer operating in multi scaling mode. This instrument is employed for the measurement of atomic level lifetimes using a dye laser to excite the atoms and is applied to the study of lifetimes of the 3 2 P1/2 and 3 2 P3/2 states of sodium. (Author) 32 refs

  15. A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413.

    Science.gov (United States)

    Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui; Yedidi, Ravikiran S; Das, Debananda; Bulut, Haydar; Delino, Nicole S; Sheri, Venkata Reddy; Ghosh, Arun K; Mitsuya, Hiroaki

    2016-12-01

    We report here that GRL-10413, a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a hydroxyethylamine sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC 50 ] of 0.00035 to 0.0018 μM), with minimal cytotoxicity (50% cytotoxic concentration [CC 50 ] = 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1 NL4-3 variants selected by use of atazanavir, lopinavir, or amprenavir (APV) at concentrations of up to 5 μM (EC 50 = 0.0021 to 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multidrug-resistant clinical HIV-1 variants isolated from patients who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that against APV. In addition, GRL-10413 showed favorable central nervous system (CNS) penetration properties as assessed with an in vitro blood-brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multidrug-resistant HIV-1 variants, with favorable CNS penetration capability, and that the newly modified P1 moiety may confer desirable features in designing novel anti-HIV-1 PIs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. Serum and Salivary IgE, IgA, and IgG4 Antibodies to Dermatophagoides pteronyssinus and Its Major Allergens, Der p1 and Der p2, in Allergic and Nonallergic Children

    Directory of Open Access Journals (Sweden)

    Diego O. Miranda

    2011-01-01

    Full Text Available Allergic rhinitis (AR is a public health problem with high prevalence worldwide. We evaluated levels of specific IgE, IgA, and IgG4 antibodies to the Dermatophagoides pteronyssinus (Dpt house dust mite and to its major allergens (Der p1 and Der p2 in serum and saliva samples from allergic and nonallergic children. A total of 86 children were analyzed, from which 72 had AR and 14 were nonallergic healthy children. Serum IgE and serum/salivary IgG4 levels to Dpt, Der p1, and Der p2 were higher in allergic children whereas serum/salivary IgA levels to all allergens were higher in nonallergic children. IgE levels positively correlated with IgG4 and IgA to all allergens in allergic children, while IgA levels negatively correlated with IgG4 to Dpt and Der p1 in nonallergic children. In conclusion, mite-specific IgA antibodies predominate in the serum and saliva of nonallergic children whereas mite-specific IgE and IgG4 are prevalent in allergic children. The presence of specific IgA appears to have a key role for the healthy immune response to mucosal allergens. Also, specific IgA measurements in serum and/or saliva may be useful for monitoring activation of tolerance-inducing mechanisms during allergen specific immunotherapeutic procedures, especially sublingual immunotherapy.

  17. Sampling system for pulsed signals. Study of the radioactive lifetimes of excited 3{sup 2}P1/2 and 3{sup 2}P3/2 states of Na, excited by a tunable dye laser; Sistema de muestreo para senales pulsadas. Estudio de vidas medias de niveles 3{sup 2} P1/2 y 3{sup 2}P3/2 excitados por un laser de colorantes pulsado

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, P.; Campos, J.

    1979-07-01

    A system for sampling and averaging repetitive signals in the order of nanoseconds is discussed. The system uses as storage memory a multichannel analyzer operating in multi scaling mode. This instrument is employed for the measurement of atomic level lifetimes using a dye laser to excite the atoms and is applied to the study of lifetimes of the 3{sup 2}P1/2 and 3{sup 2}P3/2 states of sodium. (Author) 32 refs.

  18. Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE).

    Science.gov (United States)

    Lintner, Nathanael G; Kerou, Melina; Brumfield, Susan K; Graham, Shirley; Liu, Huanting; Naismith, James H; Sdano, Matthew; Peng, Nan; She, Qunxin; Copié, Valérie; Young, Mark J; White, Malcolm F; Lawrence, C Martin

    2011-06-17

    In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea.

  19. Structural and opto-electronic properties of InP1-xBix bismide alloys for MID-infrared optical devices: A DFT + TB-mBJ study

    Science.gov (United States)

    Assali, Abdenacer; Bouslama, M.; Chaabane, L.; Mokadem, A.; Saidi, F.

    2017-12-01

    Using full-potential linearized augmented plane wave (FP-LAPW) method within density functional theory (DFT), we have studied the structural and opto-electronic properties of zinc blende InP1-xBix bismide alloys (0 ≤ x ≤ 0.5). The bowing lattice parameter exhibits a weak composition dependence on InP1-xBix alloys with b = 0.02834 Å. The band gap decreases with Bi composition by about 1.285 meV for x = 0.25 covering the middle (MID) and far-wavelength infrared region [0.88-10.5 μm]. From DOS, the decrease of band gap can be attributed to the both upper shifts of the valence band VB and the downward shifts of the conduction band CB, due to the resonance interaction of the Bi-p orbitals at the top of the VB and hybridization of the occupied s/p orbitals of In/P/Bi atoms at the bottom of the CB, with increasing Bi composition. The dielectric functions (ε1(ω), ε2(ω)) and optical constants such as n(ω), k(ω), α(ω) and R(ω) for InP1-xBix alloys are determined for radiation up to 8 eV in excellent agreement with the measured data. The energies of the critical-point (CP) are also identified agree well with the experimental data. The InPBi material appears as a promising material to realize novel optical devices as Laser Diodes and detectors operating in the MID-Infrared spectrum region.

  20. IncP-1β plasmids of Comamonas sp. and Delftia sp. strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet.

    Science.gov (United States)

    Stolze, Yvonne; Eikmeyer, Felix; Wibberg, Daniel; Brandis, Gerrit; Karsten, Christina; Krahn, Irene; Schneiker-Bekel, Susanne; Viehöver, Prisca; Barsch, Aiko; Keck, Matthias; Top, Eva M; Niehaus, Karsten; Schlüter, Andreas

    2012-08-01

    The application of toxic triphenylmethane dyes such as crystal violet (CV) in various industrial processes leads to large amounts of dye-contaminated sludges that need to be detoxified. Specific bacteria residing in wastewater treatment plants (WWTPs) are able to degrade triphenylmethane dyes. The objective of this work was to gain insights into the genetic background of bacterial strains capable of CV degradation. Three bacterial strains isolated from a municipal WWTP harboured IncP-1β plasmids mediating resistance to and decolorization of CV. These isolates were assigned to the genera Comamonas and Delftia. The CV-resistance plasmid pKV29 from Delftia sp. KV29 was completely sequenced. In addition, nucleotide sequences of the accessory regions involved in conferring CV resistance were determined for plasmids pKV11 and pKV36 from the other two isolates. Plasmid pKV29 contains typical IncP-1β backbone modules that are highly similar to those of previously sequenced IncP-1β plasmids that confer antibiotic resistance, degradative capabilities or mercury resistance. The accessory regions located between the conjugative transfer (tra) and mating pair formation modules (trb) of all three plasmids analysed share common modules and include a triphenylmethane reductase gene, tmr, that is responsible for decolorization of CV. Moreover, these accessory regions encode other enzymes that are dispensable for CV degradation and hence are involved in so-far-unknown metabolic pathways. Analysis of plasmid-mediated degradation of CV in Escherichia coli by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight MS revealed that leuco crystal violet was the first degradation product. Michler's ketone and 4-dimethylaminobenzaldehyde appeared as secondary degradation metabolites. Enzymes encoded in the E. coli chromosome seem to be responsible for cleavage of leuco crystal violet. Plasmid-mediated degradation of triphenylmethane dyes such as CV

  1. Human IgG response to a salivary peptide, gSG6-P1, as a new immuno-epidemiological tool for evaluating low-level exposure to Anopheles bites

    Directory of Open Access Journals (Sweden)

    Simondon François

    2009-08-01

    Full Text Available Abstract Background Human populations exposed to low malaria transmission present particular severe risks of malaria morbidity and mortality. In addition, in a context of low-level exposure to Anopheles vector, conventional entomological methods used for sampling Anopheles populations are insufficiently sensitive and probably under-estimate the real risk of malaria transmission. The evaluation of antibody (Ab responses to arthropod salivary proteins constitutes a novel tool for estimating exposure level to insect bites. In the case of malaria, a recent study has shown that human IgG responses to the gSG6-P1 peptide represented a specific biomarker of exposure to Anopheles gambiae bites. The objective of this study was to investigate if this biomarker can be used to estimate low-level exposure of individuals to Anopheles vector. Methods The IgG Ab level to gSG6-P1 was evaluated at the peak and at the end of the An. gambiae exposure season in children living in Senegalese villages, where the Anopheles density was estimated to be very low by classical entomological trapping but where malaria transmission occurred during the studied season. Results Specific IgG responses to gSG6-P1 were observed in children exposed to very low-level of Anopheles bites. In addition, a significant increase in the specific IgG Ab level was observed during the Anopheles exposure season whereas classical entomological data have reported very few or no Anopheles during the studied period. Furthermore, this biomarker may also be applicable to evaluate the heterogeneity of individual exposure. Conclusion The results strengthen the hypothesis that the evaluation of IgG responses to gSG6-P1 during the season of exposure could reflect the real human contact with anthropophilic Anopheles and suggest that this biomarker of low exposure could be used at the individual level. This promising immuno-epidemiological marker could represent a useful tool to assess the risk to very low

  2. Does occupational exposure to solvents and pesticides in association with glutathione S-transferase A1, M1, P1, and T1 polymorphisms increase the risk of bladder cancer? The Belgrade case-control study.

    Science.gov (United States)

    Matic, Marija G; Coric, Vesna M; Savic-Radojevic, Ana R; Bulat, Petar V; Pljesa-Ercegovac, Marija S; Dragicevic, Dejan P; Djukic, Tatjana I; Simic, Tatjana P; Pekmezovic, Tatjana D

    2014-01-01

    We investigated the role of the glutathione S-transferase A1, M1, P1 and T1 gene polymorphisms and potential effect modification by occupational exposure to different chemicals in Serbian bladder cancer male patients. A hospital-based case-control study of bladder cancer in men comprised 143 histologically confirmed cases and 114 age-matched male controls. Deletion polymorphism of glutathione S-transferase M1 and T1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of glutathione S-transferase A1 and P1 was identified by restriction fragment length polymorphism method. As a measure of effect size, odds ratio (OR) with corresponding 95% confidence interval (95%CI) was calculated. The glutathione S-transferase A1, T1 and P1 genotypes did not contribute independently toward the risk of bladder cancer, while the glutathione S-transferase M1-null genotype was overrepresented among cases (OR = 2.1, 95% CI = 1.1-4.2, p = 0.032). The most pronounced effect regarding occupational exposure to solvents and glutathione S-transferase genotype on bladder cancer risk was observed for the low activity glutathione S-transferase A1 genotype (OR = 9.2, 95% CI = 2.4-34.7, p = 0.001). The glutathione S-transferase M1-null genotype also enhanced the risk of bladder cancer among subjects exposed to solvents (OR = 6,5, 95% CI = 2.1-19.7, p = 0.001). The risk of bladder cancer development was 5.3-fold elevated among glutathione S-transferase T1-active patients exposed to solvents in comparison with glutathione S-transferase T1-active unexposed patients (95% CI = 1.9-15.1, p = 0.002). Moreover, men with glutathione S-transferase T1-active genotype exposed to pesticides exhibited 4.5 times higher risk in comparison with unexposed glutathione S-transferase T1-active subjects (95% CI = 0.9-22.5, p = 0.067). Null or low-activity genotypes of the glutathione S-transferase A1, T1, and P1 did not contribute

  3. Does occupational exposure to solvents and pesticides in association with glutathione S-transferase A1, M1, P1, and T1 polymorphisms increase the risk of bladder cancer? The Belgrade case-control study.

    Directory of Open Access Journals (Sweden)

    Marija G Matic

    Full Text Available OBJECTIVE: We investigated the role of the glutathione S-transferase A1, M1, P1 and T1 gene polymorphisms and potential effect modification by occupational exposure to different chemicals in Serbian bladder cancer male patients. PATIENTS AND METHODS: A hospital-based case-control study of bladder cancer in men comprised 143 histologically confirmed cases and 114 age-matched male controls. Deletion polymorphism of glutathione S-transferase M1 and T1 was identified by polymerase chain reaction method. Single nucleotide polymorphism of glutathione S-transferase A1 and P1 was identified by restriction fragment length polymorphism method. As a measure of effect size, odds ratio (OR with corresponding 95% confidence interval (95%CI was calculated. RESULTS: The glutathione S-transferase A1, T1 and P1 genotypes did not contribute independently toward the risk of bladder cancer, while the glutathione S-transferase M1-null genotype was overrepresented among cases (OR = 2.1, 95% CI = 1.1-4.2, p = 0.032. The most pronounced effect regarding occupational exposure to solvents and glutathione S-transferase genotype on bladder cancer risk was observed for the low activity glutathione S-transferase A1 genotype (OR = 9.2, 95% CI = 2.4-34.7, p = 0.001. The glutathione S-transferase M1-null genotype also enhanced the risk of bladder cancer among subjects exposed to solvents (OR = 6,5, 95% CI = 2.1-19.7, p = 0.001. The risk of bladder cancer development was 5.3-fold elevated among glutathione S-transferase T1-active patients exposed to solvents in comparison with glutathione S-transferase T1-active unexposed patients (95% CI = 1.9-15.1, p = 0.002. Moreover, men with glutathione S-transferase T1-active genotype exposed to pesticides exhibited 4.5 times higher risk in comparison with unexposed glutathione S-transferase T1-active subjects (95% CI = 0.9-22.5, p = 0.067. CONCLUSION: Null or low-activity genotypes of the

  4. Avaliação da atividade antiinflamatória do extrato padronizado de própolis P1 e de seu principal constituinte ativo, Artepillin C

    OpenAIRE

    Paulino, Niraldo

    2005-01-01

    Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Farmacologia A própolis é uma resina natural produzida pelas abelhas e utilizada popularmente para o tratamento de inúmeras doenças, entre elas os processos inflamatórios. O objetivo geral deste trabalho foi avaliar, através de estudos in vitro e in vivo, usando técnicas farmacológicas e bioquímicas, a atividade antiinflamatória da própolis padronizada do sul do Brasil (P1...

  5. Modeling H2O and CO2 in Optically Thick Comets Using Asymmetric Spherical Coupled Escape Probability and Application to Comet C/2009 P1 Garradd Observations of CO, H2O, and CO2

    Science.gov (United States)

    Gersch, Alan M.; Feaga, Lori M.; A’Hearn, Michael F.

    2018-02-01

    We have adapted Coupled Escape Probability, a new exact method of solving radiative transfer problems, for use in asymmetrical spherical situations for use in modeling optically thick cometary comae. Here we present the extension of our model and corresponding results for two additional primary volatile species of interest, H2O and CO2, in purely theoretical comets. We also present detailed modeling and results for the specific examples of CO, H2O, and CO2 observations of C/2009 P1 Garradd by the Deep Impact flyby spacecraft.

  6. Crossed molecular beam-tunable laser determination of velocity dependence of intramultiplet mixing: K(4p2P1/2)+He →K(4p2P3/2)+He

    International Nuclear Information System (INIS)

    Anderson, R.W.; Goddard, T.P.; Parravano, C.; Warner, J.

    1976-01-01

    The velocity dependence of intramultiplet mixing, K(4p 2 P 1 / 2 ) +He→K(4p 2 P 3 / 2 )+He, has been measured over the relative velocity range v=1.3--3.4 km/sec. The cross section appears to fit a linear function Q (v) =A (v-v 0 ), where a=6.3 x 10 -4 A 2 and v 0 = 7.9 x 10 4 cm/sec. The value of A is obtained by normalization to the literature thermal average cross section. The intramultiplet mixing theory of Nikitin is modified to yield Q (v) for the process. The modified theory correctly exhibits detailed balancing, and it is normalized to provide a very good fit to the observed Q (v). The magnitude of the normalization factor, however, is larger than that predicted from recent pseudopotential calculations of the excited state potentials. The temperature dependence of intramultiplet mixing is predicted. The use of laser polarization to determine the m/subj/ dependence of the process K(4p 2 P 3 / 2 +He→K(4p 2 P 1 / 2 )+He and other collision processes of excited 2 P 3 / 2 states is examined

  7. Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

    Science.gov (United States)

    Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

    2016-01-27

    Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

  8. Expression of the glycoprotein of viral haemorrhagic septicaemia virus (VHSV) on the surface of the fish cell line RTG-P1 induces type 1 interferon expression in neighbouring cells

    DEFF Research Database (Denmark)

    Acosta, F.; Collet, B.; Lorenzen, Niels

    2006-01-01

    In the present study using a luciferase/Mx promoter reporter system, it was shown that the rainbow trout gonad cell line (RTG-P1), a fibroblastic cell line, produces IFN when transfected with a plasmid encoding the glycoprotein of VHSV but not with plasmid vector alone. Only a small percentage...... of the cells expressed the G protein on the surface membrane as indicated by immunostaining of transfected cells. When transfection was performed in the presence of monoclonal antibodies (Mab) to the glycoprotein, the production of interferon mRNA transcripts was reduced by over 50%. This indicates...... that the surface expression of G protein was the major mechanism of interferon induction and that most of the interferon was being expressed by cells neighbouring the transfected cells. Crown...

  9. Electron-impact excitation of multiply-charged ions using energy loss in merged beams: e + Si3+(3s2S1/2) → e + Si3+(3p2P1/2,3/2)

    International Nuclear Information System (INIS)

    Wahlin, E.K.; Thompson, J.S.; Dunn, G.H.; Phaneuf, R.A.; Gregory, D.C.; Smith, A.C.H.

    1990-01-01

    For the first time absolute total cross sections for electron-impact excitation of a multiply-charged ion have been measured using an electron-energy-loss technique. Measurements were made near threshold for the process e + Si 3+ (3s 2 S 1/2 ) → e + Si 3+ (3p 2 P 1/2 , 3/2 ) -- 8.88 eV. The 10 -15 cm 2 measured cross section agrees with results of 7-state close coupling calculations to better than the ±20% (90% CL) total uncertainty of the measurements. Convoluting the theoretical curve with a Gaussian energy distribution indicates an energy width of 0.15 approx-lt ΔE approx-lt 0.20 eV. 12 refs., 2 figs

  10. Association between long-term neuro-toxicities in testicular cancer survivors and polymorphisms in glutathione-s-transferase-P1 and -M1, a retrospective cross sectional study

    Directory of Open Access Journals (Sweden)

    Brydøy Marianne

    2007-12-01

    Full Text Available Abstract Background To assess the impact of polymorphisms in Glutathione S-transferase (GST -P1, -M1, and -T1 on self-reported chemotherapy-induced long-term toxicities in testicular cancer survivors (TCSs. Methods A total of 238 TCSs, who had received cisplatin-based chemotherapy at median twelve years earlier, had participated in a long-term follow-up survey which assessed the prevalence of self-reported paresthesias in fingers/toes, Raynaud-like phenomena in fingers/toes, tinnitus, and hearing impairment. From all TCSs lymphocyte-derived DNA was analyzed for the functional A→G polymorphism at bp 304 in GSTP1, and deletions in GST-M1 and GST-T1. Evaluation of associations between GST polymorphisms and self-reported toxicities included adjustment for prior treatment. Results All six evaluated toxicities were significantly associated with the cumulative dose of cisplatin and/or bleomycin. Compared to TCSs with either GSTP1-AG or GSTP1-AA, the 37 TCSs with the genotype GSTP1-GG, were significantly less bothered by paresthesias in fingers and toes (p = 0.039, OR 0.46 [0.22–0.96] and p = 0.023, OR 0.42 [0.20–0.88], respectively, and tinnitus (p = 0.008, OR 0.33 [0.14–0.74]. Furthermore, absence of functional GSTM1 protected against hearing impairment (p = 0.025, OR 1.81 [1.08–3.03]. Conclusion In TCSs long-term self-reported chemotherapy-induced toxicities are associated with functional polymorphisms in GSTP1 and GSTM1. Hypothetically, absence of GST-M1 leaves more glutathione as substrate for the co-expressed GST-P1. Also intracellular inactivation of pro-apoptotic mediators represents a possible explanation of our findings. Genotyping of these GSTs might be a welcomed step towards a more individualized treatment of patients with metastatic testicular cancer.

  11. Semiclassical three-valley Monte Carlo simulation analysis of steady-state and transient electron transport within bulk InAsxP1-x, InAs and InP

    Directory of Open Access Journals (Sweden)

    Hadi Arabshahi

    2010-04-01

    Full Text Available We have studied how electrons, initially in thermal equilibrium, drift under the action of an applied electric field within bulk zincblende InAsxP1-x, InAs and InP. Calculations are made using a non-parabolic effective-mass energy band model. Monte Carlo simulation includes all of the major scattering mechanisms. The band parameters used in the simulation are extracted from optimised pseudo-potential band calculations to ensure excellent agreement with experimental information and ab-initio band models. The effects of alloy scattering on the electron transport physics are examined. For all materials, it is found that electron velocity overshoot only occurs when the electric field is increased to a value above a certain critical field, unique to each material. This critical field is strongly dependent on the material parameters. Transient velocity overshoot has also been simulated, with the sudden application of fields up to 1600 kVm-1, appropriate to the gate-drain fields expected within an operational field-effect transistor. The electron drift velocity relaxes to the saturation value of about 1.5105 ms-1 within 4 pico-seconds for all crystal structures. The steady-state and transient velocity overshoot characteristics are in fair agreement with other recent calculations.

  12. Co-evolutionary dynamics of the bacteria Vibrio sp. CV1 and phages V1G, V1P1, and V1P2: implications for phage therapy.

    Science.gov (United States)

    Barbosa, Camilo; Venail, Patrick; Holguin, Angela V; Vives, Martha J

    2013-11-01

    Bacterial infections are the second largest cause of mortality in shrimp hatcheries. Among them, bacteria from the genus Vibrio constitute a major threat. As the use of antibiotics may be ineffective and banned from the food sector, alternatives are required. Historically, phage therapy, which is the use of bacteriophages, is thought to be a promising option to fight against bacterial infections. However, as for antibiotics, resistance can be rapidly developed. Since the emergence of resistance is highly undesirable, a formal characterization of the dynamics of its acquisition is mandatory. Here, we explored the co-evolutionary dynamics of resistance between the bacteria Vibrio sp. CV1 and the phages V1G, V1P1, and V1P2. Single-phage treatments as well as a cocktail composed of the three phages were considered. We found that in the presence of a single phage, bacteria rapidly evolved resistance, and the phages decreased their infectivity, suggesting that monotherapy may be an inefficient treatment to fight against Vibrio infections in shrimp hatcheries. On the contrary, the use of a phage cocktail considerably delayed the evolution of resistance and sustained phage infectivity for periods in which shrimp larvae are most susceptible to bacterial infections, suggesting the simultaneous use of multiple phages as a serious strategy for the control of vibriosis. These findings are very promising in terms of their consequences to different industrial and medical scenarios where bacterial infections are present.

  13. Impact of the Ile105Val Polymorphism of the Glutathione S-transferase P1 (GSTP1) Gene on Obesity and Markers of Cardiometabolic Risk in Young Adult Population.

    Science.gov (United States)

    Chielle, E O; Trott, A; da Silva Rosa, B; Casarin, J N; Fortuna, P C; da Cruz, I B M; Moretto, M B; Moresco, R N

    2017-05-01

    The aim of the study was to investigate the association between Glutathione S-transferase P1 (GSTP1) gene polymorphism with obesity and markers of cardiometabolic risk. A cross-sectional study was carried out in individuals aged≥18 and ≤30 years. The study included 54 normal weight, 27 overweight and 68 obese volunteers. Anthropometric measurements and biochemical parameters were evaluated, the DNA was extracted from blood samples and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to measure GSTP1 Ile 105 Val gene polymorphism of the study participants. Also, biochemical analysis and hormone assays were carried out. A positive association between GSTP1 polymorphism and obesity was observed on subjects carrying at least one G allele (AG and GG). GG genotype was found only in the obese group. The G allele carriers presented 2.4 times higher chance of obesity when compared to those with the AA genotype. These results were independent of sex and age. We suggest that despite a study in population regional (south of Brazil), the GSTP1 gene polymorphism may play a significant role in the increase of susceptibility of obesity and contribute to identify the cardiovascular risk in young adults. © Georg Thieme Verlag KG Stuttgart · New York.

  14. Fabrication of Zr2WP2O12/ZrV0.6P1.4O7 composite with a nearly zero-thermal-expansion property.

    Science.gov (United States)

    Yanase, Ikuo; Sakai, Hiroshi; Kobayashi, Hidehiko

    2017-11-15

    Sintered bodies of Zr 2 WP 2 O 12 (ZWP) and ZrV 0.6 P 1.4 O 7 (ZVP) were fabricated, and their linear thermal expansion coefficients (TEC) were found to be -2.92 × 10 -6 and 3.27 × 10 -6  °C -1 , respectively, in the range 25-500 °C. In an attempt to fabricate composites with a zero-thermal-expansion property, sintered ZWP/ZVP composites with ZVP/ZWP volume ratios of 0.5/0.5, 0.53/0.47, 0.55/0.45, and 0.6/0.4 were fabricated. Scanning electron microscopy revealed that sintering of ZVP/ZWP composites progressed well compared with that of ZWP. A porous ZVP/ZWP composite with a relative density of ca. 83% was fabricated at a ZVP/ZWP volume ratio of 0.53/0.47. X-ray diffractometry and energy dispersive X-ray spectrometry clarified that the ZVP/ZWP composite mainly consisted of ZWP and ZVP grains. Thermomechanical analysis confirmed that the ZVP/ZWP composite exhibited very low thermal expansion with a slight hysteresis with a TEC of -0.29 × 10 -7  °C -1 in the range 25-500 °C.

  15. Two types of genetic carrier, the IncP genomic island and the novel IncP-1β plasmid, for the aac(2')-IIa gene that confers kasugamycin resistance in Acidovorax avenae ssp. avenae.

    Science.gov (United States)

    Yoshii, Atsushi; Omatsu, Tsutomu; Katayama, Yukie; Koyama, Satoshi; Mizutani, Tetsuya; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2015-04-01

    A unique aminoglycoside antibiotic, kasugamycin (KSM), has been used to control many plant bacterial and fungal diseases in several countries. The emergence of KSM-resistant Acidovorax avenae ssp. avenae and Burkholderia glumae, which cause rice bacterial brown stripe and rice bacterial grain and seedling rot, respectively, is a serious threat for the effective control of these diseases. Previously, we have identified the aac(2')-IIa gene, encoding a KSM 2'-N-acetyltransferase, from both KSM-resistant pathogens. Although all KSM-resistant isolates from both species possess the aac(2')-IIa gene, only A. avenae strain 83 showed higher resistance than other strains. In this research, kinetic analysis indicates that an amino acid substitution from serine to threonine at position 146 of AAC(2')-IIa in strain 83 is not involved in this increased resistance. Whole draft genome analysis of A. avenae 83 shows that the aac(2')-IIa gene is carried by the novel IncP-1β plasmid pAAA83, whereas the genetic carrier of other strains, the IncP genomic island, is inserted into their chromosomes. The difference in the nucleotides of the promoter region of aac(2')-IIa between strain 83 and other strains indicates an additional transcription start site and results in the increased transcription of aac(2')-IIa in strain 83. Moreover, biological characterization of pAAA83 demonstrates that it can be transferred by conjugation and maintained in the host cells. These results demonstrate that acquisition of the aac(2')-IIa gene takes place in at least two ways and that the gene module, which includes aac(2')-IIa and the downstream gene, may be an important unit for the dissemination of antibiotic resistance. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  16. [Anaphylaxis due to peach with negative ImmunoCAP result to peach allergens, including rPru p 1, rPru p 3, AND rPru p 4: a report of two cases].

    Science.gov (United States)

    Maeda, Nobuko; Inomata, Naoko; Morita, Akiko; Kirino, Mio; Moriyama, Tatsuya; Ikezawa, Zenro

    2009-02-01

    We report two cases of anaphylactic reactions to peach with negative result of ImmunoCAP to peach. Case 1 is a 35-year-old man, who felt an itch in his oral cavity immediately after ingesting a whole fresh peach. He rapidly developed generalized urticaria, dyspnea, vomiting, and loss of consciousness. He recovered after treatment at a local hospital, thereafter he was referred to our hospital because ImmunoCAP conducted for screening allergens revealed a negative test result to peach and the cause of anaphylaxis remained unclear. He had a history of pollinosis. He reported that he previously felt an itch on his oral cavity after ingesting melon, watermelon, apple, and strawberry. Serum total IgE was 436 IU/ml. CAP-RAST revealed negative results to peach, strawberry and kiwi. Skin prick tests (SPTs) with raw peach pulp, canned peach pulp, strawberry and kiwi were positive. Case 2 is a 30-year-old woman who felt an itch on her oral cavity accompanied by blepharedema, rhinorrhea, generalized urticaria, nausea, abdominal pain and diarrhea after eating peach. She had a history of pollinosis. She reported that she previously developed urticaria after ingesting an apple. Serum total IgE was 85 IU/ml. ImmunoCAP revealed negative results to peach and apple. SPTs with canned yellow peach, strawberry and apple were positive. Consequently, the two patients were diagnosed with anaphylaxis due to peach, and allergic symptoms have never recurred since they avoided ingesting peach. Furthermore, in two patients ImmunoCAP to rPru p 1, rPru p 3, and rPru p 4 were negative. However, in IgE-immunoblotting of peach, serum IgE antibodies of two patients were bound to approximately 10 kDa proteins. Meanwhile, the cross-reactivity between Rosaceae fruits, such as peach, apple, apricot, and plum, has been reported. These results suggest that in patients, who are suspected of having peach anaphylaxis and show a negative ImmunoCAP result to peach, the additional testing, such as SPT with

  17. Entrapment by magnetic microcapsules of the protein pyrolysates IQ, PhIP and Glu-P-1, and alteration of IQ metabolite exposure within the rat gastrointestinal tract by risk-modulating components of the human diet.

    Science.gov (United States)

    O'Neill, I; Ohgaki, H; Ellul, A; Turesky, R J

    1992-12-01

    The entrapment of heterocyclic aromatic amine gastrointestinal (GI) carcinogens (HAAs), by retrievable semipermeable magnetic polyethylenimine (PEI) microcapsules was investigated in vitro and in vivo as an approach for human biomonitoring. Previous studies showed that PEI microcapsules successfully entrapped benzo[a]pyrene (B[]P) and its metabolites in the GI tract of rodents. In this study, we have shown that 14C-labelled 2-amino-3-methylimidazo[4,5f]quinoline (IQ), 2-amino-1-methylphenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3'2'-d]imidazole (Glu-P-1) are adsorbed to PEI microcapsules in vitro and can be desorbed by treatment with ammoniac methanol. Binding of HAAs to PEI microcapsules containing copper phthalocyanine (TCPTS), a moiety which reversibly binds chemicals with aromatic planar structures, was 2- to 4-fold higher than with unmodified PEI microcapsules. PEI microcapsules also acted as a nucleophile and trapped the proximate carcinogenic metabolite of IQ, 2-hydroxy-amino-3-methyl-imidazo[4,5f]quinoline (N-hydroxy-IQ). The entrapment of 14C-labelled IQ and PhIP by microcapsules was investigated in vivo in male F344 rats fed a conventional chow diet or a human diet with varying amounts of fat and beef intake typically consumed in the UK. Animals were adapted to human diets which were either high (H) or low (L) in fat (F), beef protein (B) and dietary fibre non-starch polysaccharide (NSP). Microcapsule entrapment of IQ and metabolites was 0.5-2.0% of the dose and 4-fold higher in rats consuming a HF/HB/LNSP than those consuming a LF/LB/HNSP diet, these being respectively putatative high- and low-risk-associated diets. In the HF/HB/LNSP diet group, a higher amount of IQ metabolites were detected in the microcapsules; a lower proportion of covalently bound metabolites could be removed by acid hydrolysis. Urinary excretion was 2-fold greater and analysis of the urinary metabolites showed there to be lower sulfotransferase activity

  18. Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Zheng, Tao; Huang, Qihong; Zhang, Changyi

    2012-01-01

    be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic....... islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (¿herA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could...

  19. Studies on DNA Damage Response in Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Han, Wenyuan

    global reactions known as DNA damage response (DDR). In Bacteria and Eukaryotes, the global reactions include a series of transcription regulations and protein post-translation modifications, which can activate DNA repair machineries, suppress cell division and delay DNA replication, and induce......) and methyl methanesulfonate (MMS), and hydroxyurea (HU) that may not introduce DNA lesions directly. Comparison of the effects of the three drugs on S. islandicus cells showed that NQO and MMS led to DNA-less cell formation, while HU did not. In addition, the DNA-less cells were featured with increased side...... scattered light, damaged cell membrane and electron-dense area. During NQO and MMS treatment, degradation of chromatin proteins was coincided with DNA-less cell formation, suggesting their roles in protecting genomic DNA from massive degradation. Further, HU inhibited NQO-induced DSB formation and DNA...

  20. In vivo and in vitro protein imaging in thermophilic archaea by exploiting a novel protein tag.

    Science.gov (United States)

    Visone, Valeria; Han, Wenyuan; Perugino, Giuseppe; Del Monaco, Giovanni; She, Qunxin; Rossi, Mosè; Valenti, Anna; Ciaramella, Maria

    2017-01-01

    Protein imaging, allowing a wide variety of biological studies both in vitro and in vivo, is of great importance in modern biology. Protein and peptide tags fused to proteins of interest provide the opportunity to elucidate protein location and functions, detect protein-protein interactions, and measure protein activity and kinetics in living cells. Whereas several tags are suitable for protein imaging in mesophilic organisms, the application of this approach to microorganisms living at high temperature has lagged behind. Archaea provide an excellent and unique model for understanding basic cell biology mechanisms. Here, we present the development of a toolkit for protein imaging in the hyperthermophilic archaeon Sulfolobus islandicus. The system relies on a thermostable protein tag (H5) constructed by engineering the alkylguanine-DNA-alkyl-transferase protein of Sulfolobus solfataricus, which can be covalently labeled using a wide range of small molecules. As a suitable host, we constructed, by CRISPR-based genome-editing technology, a S. islandicus mutant strain deleted for the alkylguanine-DNA-alkyl-transferase gene (Δogt). Introduction of a plasmid-borne H5 gene in this strain led to production of a functional H5 protein, which was successfully labeled with appropriate fluorescent molecules and visualized in cell extracts as well as in Δogt live cells. H5 was fused to reverse gyrase, a peculiar thermophile-specific DNA topoisomerase endowed with positive supercoiling activity, and allowed visualization of the enzyme in living cells. To the best of our knowledge, this is the first report of in vivo imaging of any protein of a thermophilic archaeon, filling an important gap in available tools for cell biology studies in these organisms.

  1. Global analysis of viral infection in an archaeal model system

    Directory of Open Access Journals (Sweden)

    Walid S. Maaty

    2012-12-01

    Full Text Available The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes.. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled 6 times over a 72 hr period. More than 700 proteins were identified throughout the course of the experiments. Seventy one host proteins were found to change by nearly two-fold (p<0.05 with 40 becoming more abundant and 31 less abundant. The modulated proteins represent 30 different cell pathways and 14 COG groups. 2D gel analysis showed that changes in post translational modifications were a common feature of the affected proteins. The results from these studies showed that the prokaryotic antiviral adaptive immune system CRISPR associated proteins (CAS proteins were regulated in response to the virus infection. It was found that regulated proteins come from mRNAs with a shorter than average half-life. In addition, activity-based protein profiling (ABPP profiling on 2D gels showed caspase, hydrolase and tyrosine phosphatase enzyme activity labeling at the protein isoform level. Together, this data provides a more detailed global view of archaeal cellular responses to viral infection, demonstrates the

  2. Improved Preparation of Beta-D-ManNAc-(1-4)-D-Glc and Beta-d-TalNAc-(1-4)-D-Glc Disaccharides and Evaluation of Their Activating Properties on the Natural Killer Cells NKR-P1 and CD69 Receptors

    Czech Academy of Sciences Publication Activity Database

    Attolino, E.; Bonaccorsi, F.; Catelani, G.; D'Andrea, F.; Křenek, Karel; Bezouška, Karel; Křen, Vladimír

    2008-01-01

    Roč. 27, č. 3 (2008), s. 156-171 ISSN 0732-8303 R&D Projects: GA AV ČR IAA400200503; GA MŠk(CZ) LC06010; GA MŠk OC 136 Grant - others:GA MŠk(CZ) COST D34 Institutional research plan: CEZ:AV0Z50200510 Keywords : reductions * nkr-p1 receptor * nk cells Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.114, year: 2008

  3. A Modified P1 Moiety Enhances in vitro Antiviral Activity against Various Multi-Drug-Resistant HIV-1 Variants and in vitro CNS Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui; Yedidi, Ravikiran S.; Das, Debananda; Bulut, Haydar; Delino, Nicole S.; Sheri, Venkata Reddy; Ghosh, Arun K.; Mitsuya, Hiroaki (Kumamoto); (NIH); (Purdue)

    2016-09-12

    We here report that GRL-10413, a novel non-peptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.00035 - 0.0018 μM) with minimal cytotoxicity (CC50: 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1NL4-3variants selected by up to 5 μM concentrations of atazanavir, lopinavir, or amprenavir (EC50: 0.0021 - 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to various antiviral regimens after long-term antiretroviral therapy. The development of resistance against GRL-10413 was significantly delayed compared to that of APV. In addition, GRL-10413 showed a favorable central nervous system (CNS) penetration property as assessed with anin vitroblood brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active-site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants with favorable CNS-penetration capability and that the newly modified P1-moiety may confer desirable features in designing novel anti-HIV-1 PIs.

  4. Prevalence of IgE antibodies to an extract from rubber tree (Hevea brasiliensis latex and recombinant pollen allergens (Phl P 1, Phl P 2, Phl P 5, Bet v 1 and Bet v 2 in the sera of Italian atopic patients

    Directory of Open Access Journals (Sweden)

    Renato E. Rossi

    2001-01-01

    Conclusions: The findings of the present study may support the concept that a high proportion of sera containing IgE to rBet v 2, rPhl p 1 and rPhl p 5 simultaneously contain antilatex IgE. Therefore, patients with specific IgE to these recombinant allergens with no history of current latex exposure may need additional evaluation.

  5. RESEARCH ARTICLE Glutathione S-Transferase P1 Gene ...

    Indian Academy of Sciences (India)

    Navya

    2017-03-13

    Mar 13, 2017 ... and environmental backgrounds, the variations in population structures may accord for such differences highlighting the value of ethnic-specific studies for population stratification. Considering the g.341C>T SNP and CAD risk, the C/T genotype conferred 5.8-fold increased risk for developing CAD and has ...

  6. The association between glutathione S-transferase P1 ...

    African Journals Online (AJOL)

    Methods: Fifty patients with bronchial asthma and fifty normal control subjects were enrolled in this study and were subjected to asthma questionnaire, spirometric studies, conventional polymerase chain reaction (PCR) with enzyme digestion to determine GSTP1 genotype, serum immunoglobulin E (IgE) measurement and ...

  7. Molecular architecture of mouse activating NKR-P1 receptors

    Czech Academy of Sciences Publication Activity Database

    Kolenko, Petr; Rozbeský, Daniel; Vaněk, Ondřej; Kopecký, V. Jr.; Hofbauerová, Kateřina; Novák, Petr; Pompach, Petr; Hašek, Jindřich; Skálová, Tereza; Bezouška, Karel; Dohnálek, Jan

    2011-01-01

    Roč. 175, č. 3 (2011), s. 434-441 ISSN 1047-8477 R&D Projects: GA MŠk 1M0505; GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR GA305/07/1073; GA ČR GAP207/10/1040; GA AV ČR KJB101120805 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z50200510 Keywords : NK cell * C-type lectin-like receptor * X-ray structure Subject RIV: CE - Biochemistry Impact factor: 3.406, year: 2011

  8. PODAAC-AQR50-3P1DS

    Data.gov (United States)

    National Aeronautics and Space Administration — Aquarius Level 3 sea surface spiciness standard mapped image data contains gridded 1 degree spatial resolution spice data averaged over daily, 7 day, monthly, and...

  9. Status report of the KAERI/NDEL. P1

    International Nuclear Information System (INIS)

    Chang, Jonghwa

    2001-01-01

    KAERI/NDEL has successfully finished a 4 year national nuclear Research and Development project, Establishment of Nuclear Data System, and started a new 4 year project, Evaluation of Nuclear Data for Nuclear Research and development Projects. KAERI/NDEL is providing nuclear data on-line service targeting non-nuclear data experts. Three new facilities have been added for nuclear data measurement capability of Korea, namely, E-Linac based TOF; Tandem VDG based n-source; and Cyclotron based Vacuum chamber. Main directions of nuclear data development on next 4 years are the Fission Products data for Transmutation, the data for Thorium cycle, the Photon-production data for in-core detector, and the Intermediate energy data for ADS

  10. Zhao et al. (2017) Chem. Geol. v. 474 p.1

    Data.gov (United States)

    U.S. Environmental Protection Agency — The dataset provides information on chromium concentrations extracted from rock samples collected at the Garfield SF site in New Jersey (USA). The data are discussed...

  11. Metabolic regulation in the facultative methylotroph arthrobacter P1

    NARCIS (Netherlands)

    1985-01-01

    Many microorganisms are able to utilize C1 compounds, i.e. compounds which do not contain carbon-carbon bonds, as carbon- and energy sources for growth. In order to synthesize cell constituents from these C1 compounds special metabolic pathways are employed by such organisms. Although a great deal

  12. P1-10: The Association between Colors and Emotions

    Directory of Open Access Journals (Sweden)

    Yen-Yu Chen

    2012-10-01

    Full Text Available Wexner (1954 Journal of Applied Psychology 38 432–435 demonstrated colors were associated with specific emotions. Colors have many meanings, such as that red can lead to induce positive or negative emotions (Kaya & Epps, 2011 College Student Journal 38 396–406. This study extends previous findings and aims to investigate two questions, (1 whether colors are associated with the emotions of pictures in IAPS (International Affective Picture System and (2 whether perceiving IAPS consciously and unconsciously has different emotion associations. We replaced facial expressions with pictures of IAPS as stimuli. Five colors (black, red, yellow, blue, and white and 3 categories of IAPS (fear, awe, and amusement were manipulated in this study. Pictures were displayed in different durations to manipulate conscious (250 ms and unconscious (33 ms visual stimuli (Manuel & Pedro, 2009 Behavior Research Method 41 184–191 in two experiments. Participants were required to select the most suitable color at their first glance when the pictures of IAPS were presented, and vice versa. Results showed that overall association between colors and pictures of IAPS was weak but the association pattern was meaningful. It concluded (1 black and red colors are associated with the fearful emotion, and blue color is associated with the happy emotion; (2 white colors do not associate with the fear emotion, which is incongruent with the previous study (Osvaldo & Paul, 2007 Colour: Design & Creativity 1 1–20; (3 participants did not perform differently between conscious and unconscious stimulus conditions.

  13. P1-19: Horizontal Vertical Illusion by Touch

    Directory of Open Access Journals (Sweden)

    Yoshinari Kinoshita

    2012-10-01

    Full Text Available Revesz (1934 Zeitschrift fur PsychologieBd. 1, Kap 20 and Bean (1938 Journal of Experimental Psychology 22 283–289. reported almost all the geometrical optical illusions existed in a tactual mode. Such a study can examine theories of visual illusions with modality-free theories. A number of articles have been devoted to the theory that repeated judgments decline the magnitude of visual illusion. In the current study, we examine whether repeated judgments decline the magnitude of geometrical haptic illusion. The Fick illusion (i.e., a horizontal vertical illusion was investigated. A graphics Braille display with 32×48 dots was used to present an inverted T haptically without vision. The horizontal line was consistently 49.2 mm long, and the vertical line was varied in each trial. Three subjects with normal sight participated. They judged which line was longer than the other. The point of subjective equality at which the subject perceives the two lines to be the same was measured using the method of constant stimuli. In the first session the mean PSE was about 13%; to compensate for the illusion, the vertical line must be set physically shorter than the horizontal line. We found that repeated judgments produced a reduction in illusion magnitude and dissolved the illusion entirely.

  14. Glutathione S-transferase P1 gene polymorphisms and susceptibility ...

    Indian Academy of Sciences (India)

    matched and ethnicity-matched healthy controls (n = 200) were genotyped for polymorphisms in GSTP1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotype distribution of g.313A>G and ...

  15. Glutathione S-transferase P1 gene polymorphisms and susceptibility ...

    Indian Academy of Sciences (India)

    M. A. Bhat

    2017-11-28

    Nov 28, 2017 ... diet, advanced age, smoking, hypertension, diabetes mel- litus and dyslipidemia are associated with increased risk of CAD. In addition, oxidative stress has been regarded as one of the well-established patho-physiological mecha- nisms that contribute to the pathogenesis and progression of CAD (Dhalla ...

  16. Glutathione S-transferase P1 gene polymorphisms and susceptibility ...

    Indian Academy of Sciences (India)

    M. A. Bhat

    2017-11-28

    Nov 28, 2017 ... products of oxidative stress) with glutathione, and, by doing so mitigate oxidative stress and prevent cell injury. (Liet al.2000; Hayeset al.2005). Increased vulnerability to oxidative stress can, therefore, result from decreased GST activity and ... ily history of CAD or any other disease participated voluntarily ...

  17. RESEARCH ARTICLE Glutathione S-Transferase P1 Gene ...

    Indian Academy of Sciences (India)

    Navya

    2017-03-13

    Mar 13, 2017 ... endogeneously-produced products of oxidative stress) with glutathione, and in this manner mitigate ... oxidative stress can therefore result from decreased GST activity and increase susceptibility to ... age-, sex-and ethnicity-matched controls with no present or past family history of CAD or any other disease ...

  18. PODAAC-AQR50-3P1AS

    Data.gov (United States)

    National Aeronautics and Space Administration — Aquarius Level 3 sea surface spiciness standard mapped image data contains gridded 1 degree spatial resolution spice data averaged over daily, 7 day, monthly, and...

  19. PODAAC-AQR50-3P1CS

    Data.gov (United States)

    National Aeronautics and Space Administration — Aquarius Level 3 sea surface spiciness standard mapped image data contains gridded 1 degree spatial resolution spice data averaged over daily, 7 day, monthly, and...

  20. The association between glutathione S-transferase P1 ...

    African Journals Online (AJOL)

    Mahmoud I. Mahmoud

    2011-08-10

    Aug 10, 2011 ... ase chain reaction (PCR) with enzyme digestion to determine GSTP1 genotype, serum immuno- globulin E (IgE) measurement and .... conjunctivitis and (2) total serum IgE level > 100 IU/ml. Pa- tients were diagnosed as ... coded data for typing and spelling mistakes. Finally, analysis and interpretation of ...

  1. Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

    Science.gov (United States)

    Novais, Ângela; Cantón, Rafael; Valverde, Aránzazu; Machado, Elisabete; Galán, Juan-Carlos; Peixe, Luísa; Carattoli, Alessandra; Baquero, Fernando; Coque, Teresa M.

    2006-01-01

    This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing blaCTX-M-9, and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1α (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing blaCTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified blaCTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of blaCTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae. PMID:16870767

  2. Continuous and intermittent exposure of neonatal rat calvarial cells to PTHrP (1-36 inhibits bone nodule mineralization in vitro by downregulating bone sialoprotein expression via the cAMP signaling pathway [v2; ref status: indexed, http://f1000r.es/18x

    Directory of Open Access Journals (Sweden)

    Suzan A Kamel

    2013-06-01

    Full Text Available The development and growth of the skeleton in the absence of parathyroid-hormone-related protein (PTHrP is abnormal.  The shortening of appendicular bones in PTHrP gene null mice is explained by an effect of PTHrP on endochondral bone growth.  Whether or not PTHrP influences intramembranous ossification is less clear.  The purpose of this study was to determine the effect of exogenous PTHrP on intramembranous ossification in vitro.  Neonatal rat calvarial cells maintained in primary cell culture conditions that permit spontaneous formation of woven bone nodules by intramembranous ossification were studied. The expression of PTHrP, parathyroid hormone 1 receptor (PTH1R, and alkaline phosphatase (AP by osteogenic cells in developing nodules and the effects of PTHrP (1-36 on nodule development was determined over 3-18 days. PTHrP and PTH1R were detected colonies of osteogenic cells on culture day three, and AP was detected on day six. PTHrP and its receptor were localized in pre-osteoblasts, osteoblasts, and osteocytes, and AP activity was detected in pre-osteoblasts and osteoblasts but not osteocytes. Continuous and intermittent exposure to PTHrP (1-36 decreased the number of mineralized bone nodules and bone sialoprotein (BSP mRNA and protein, but had no effect on the number of AP-positive osteogenic cell colonies, cell proliferation, apoptosis, or osteopontin (OPN mRNA. These results demonstrate that osteogenic cells that participate in the formation of woven bone nodules in vitro exhibit PTHrP and PTH1R before they demonstrate AP activity. Exogenous PTHrP (1-36 inhibits the mineralization of woven bone deposited during bone nodule formation in vitro, possibly by reducing the expression of BSP.

  3. Hyperthermophilic endoglucanase for in planta lignocellulose conversion

    Directory of Open Access Journals (Sweden)

    Klose Holger

    2012-08-01

    Full Text Available Abstract Background The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. Results Here we show that a cellulase gene (sso1354 isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade α-cellulose or even complex cell wall preparations under those pretreatment conditions. Conclusion The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

  4. Improving small-angle X-ray scattering data for structural analyses of the RNA world.

    Science.gov (United States)

    Rambo, Robert P; Tainer, John A

    2010-03-01

    Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA(val), and yeast tRNA(phe) that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

  5. Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria.

    Science.gov (United States)

    Shcherbakov, D; Dontsova, M; Tribus, M; Garber, M; Piendl, W

    2006-01-01

    The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called 'L12 stalk' on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the 'L12 stalk'. The 'L12 stalk' plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12(4) complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.

  6. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  7. Characterization of Fatty Acids in Crenarchaeota by GC-MS and NMR

    Directory of Open Access Journals (Sweden)

    Timothy Hamerly

    2015-01-01

    Full Text Available Lipids composed of condensed isoprenyl units connected to glycerol backbones by ether linkages are a distinguishing feature of Archaea. Data suggesting that fatty acids with linear hydrocarbon chains are present in some Archaea have been available for decades. However, lack of genomic and biochemical evidence for the metabolic machinery required to synthesize and degrade fatty acids has left the field unclear on this potentially significant biochemical aspect. Because lipids are energy currency and cell signaling molecules, their presence in Archaea is significant for understanding archaeal biology. A recent large-scale bioinformatics analysis reignited the debate as to the importance of fatty acids in Archaea by presenting genetic evidence for the presence of enzymes required for anabolic and catabolic fatty acid metabolism across the archaeal domain. Here, we present direct biochemical evidence from gas chromatography-mass spectrometry (GC-MS and nuclear magnetic resonance (NMR spectroscopy for the presence of fatty acids in two members of the Crenarchaeota, Sulfolobus solfataricus and Ignicoccus hospitalis. This is the first report providing biochemical data for the existence of fatty acids in these Crenarchaeota, opening new discussions on energy balance and the potential for the discovery of new thermostable enzymes for industry.

  8. Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins

    Science.gov (United States)

    Chen, Li-Tzu; Ko, Tzu-Ping; Chang, Yuan-Chih; Lin, Kuei-An; Chang, Chia-Seng; Wang, Andrew H.-J.; Wang, Ting-Fang

    2007-01-01

    The RecA family of proteins mediates homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. RecA proteins are thought to facilitate DNA strand exchange reactions as closed-rings or as right-handed helical filaments. Here, we report the crystal structure of a left-handed Sulfolobus solfataricus RadA helical filament. Each protomer in this left-handed filament is linked to its neighbour via interactions of a β-strand polymerization motif with the neighbouring ATPase domain. Immediately following the polymerization motif, we identified an evolutionarily conserved hinge region (a subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the AMP–PNP right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. Additional structural and functional analyses of wild-type and mutant proteins confirmed that the subunit rotation motif is crucial for enzymatic functions of RecA family proteins. These observations support the hypothesis that RecA family protein filaments may function as rotary motors. PMID:17329376

  9. Every OGT Is Illuminated … by Fluorescent and Synchrotron Lights

    Directory of Open Access Journals (Sweden)

    Riccardo Miggiano

    2017-12-01

    Full Text Available O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus, respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins.

  10. A thermostable d-polymerase for mirror-image PCR.

    Science.gov (United States)

    Pech, Andreas; Achenbach, John; Jahnz, Michael; Schülzchen, Simone; Jarosch, Florian; Bordusa, Frank; Klussmann, Sven

    2017-04-20

    Biological evolution resulted in a homochiral world in which nucleic acids consist exclusively of d-nucleotides and proteins made by ribosomal translation of l-amino acids. From the perspective of synthetic biology, however, particularly anabolic enzymes that could build the mirror-image counterparts of biological macromolecules such as l-DNA or l-RNA are lacking. Based on a convergent synthesis strategy, we have chemically produced and characterized a thermostable mirror-image polymerase that efficiently replicates and amplifies mirror-image (l)-DNA. This artificial enzyme, dubbed d-Dpo4-3C, is a mutant of Sulfolobus solfataricus DNA polymerase IV consisting of 352 d-amino acids. d-Dpo4-3C was reliably deployed in classical polymerase chain reactions (PCR) and it was used to assemble a first mirror-image gene coding for the protein Sso7d. We believe that this d-polymerase provides a valuable tool to further investigate the mysteries of biological (homo)chirality and to pave the way for potential novel life forms running on a mirror-image genome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Characterization of a coupled DNA replication and translesion synthesis polymerase supraholoenzyme from archaea.

    Science.gov (United States)

    Cranford, Matthew T; Chu, Aurea M; Baguley, Joshua K; Bauer, Robert J; Trakselis, Michael A

    2017-08-21

    The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits. In addition to specific PCNA and polymerase interactions (PIP site), we have now identified and characterized a novel protein contact between the Y-family DNA polymerase and the B-family replication polymerase (YB site) bound to PCNA and DNA from Sulfolobus solfataricus. These YB contacts are essential in forming and stabilizing a supraholoenzyme (SHE) complex on DNA, effectively increasing processivity of DNA synthesis. The SHE complex can not only coordinate polymerase exchange within the complex but also provides a mechanism for recruitment of polymerases from solution based on multiequilibrium processes. Our results provide evidence for an archaeal PCNA 'tool-belt' recruitment model of multienzyme function that can facilitate both high fidelity and translesion synthesis within the replisome during DNA replication. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. A Novel Family of Sequence-specific Endoribonucleases Associated with the Clustered Regularly Interspaced Short Palindromic Repeats

    Energy Technology Data Exchange (ETDEWEB)

    Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D.; Proudfoot, Michael; Makarova, Kira S.; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V.; Edwards, Aled M.; Savchenko, Alexei; Yakunin, Alexander F. (NIH); (Toronto); (UV)

    2008-07-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6{angstrom} resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

  13. A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats.

    Science.gov (United States)

    Beloglazova, Natalia; Brown, Greg; Zimmerman, Matthew D; Proudfoot, Michael; Makarova, Kira S; Kudritska, Marina; Kochinyan, Samvel; Wang, Shuren; Chruszcz, Maksymilian; Minor, Wladek; Koonin, Eugene V; Edwards, Aled M; Savchenko, Alexei; Yakunin, Alexander F

    2008-07-18

    Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.

  14. Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    Fei Wang

    Full Text Available The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR.

  15. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    and lysosomes of glycans originating from e.g. terminally misfolded proteins. A divalent metal ion typically resides in the active site, and is essential for enzyme activity. Recently, the α-mannosidase II from Sulfolobus solfataricus (ManA) was purified and characterized as a Zn2+-containing enzyme, but also...... displayed metal ion promiscuity. The present thesis (Manuscript 1) describes the steady state kinetics of ManA in complex with various activating divalent metal ions and substrates. The Co2+-enzyme was superior on both para-nitrophenyl-α-Dmannoside and on 1,2-, 1,3-, 1,4- and 1,6-α-mannobiose. On para......-nitrophenyl-α-Dmannoside the Co2+ substituted ManA displayed a 6-fold higher kcat/KM compared to ManA substituted with Cd2+ and Zn2+. On the α-mannobioses the difference was even more pronounced, with the Co2+substituted ManA being the only metal ion enzyme complex, which allowed the determination of individual KM and kcat...

  16. Identification of replication origins in archaeal genomes based on the Z-curve method

    Directory of Open Access Journals (Sweden)

    Ren Zhang

    2005-01-01

    Full Text Available The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes.

  17. A genome-scale metabolic model of Methanococcus maripaludis S2 for CO2 capture and conversion to methane.

    Science.gov (United States)

    Goyal, Nishu; Widiastuti, Hanifah; Karimi, I A; Zhou, Zhi

    2014-05-01

    Methane is a major energy source for heating and electricity. Its production by methanogenic bacteria is widely known in nature. M. maripaludis S2 is a fully sequenced hydrogenotrophic methanogen and an excellent laboratory strain with robust genetic tools. However, a quantitative systems biology model to complement these tools is absent in the literature. To understand and enhance its methanogenesis from CO2, this work presents the first constraint-based genome-scale metabolic model (iMM518). It comprises 570 reactions, 556 distinct metabolites, and 518 genes along with gene-protein-reaction (GPR) associations, and covers 30% of open reading frames (ORFs). The model was validated using biomass growth data and experimental phenotypic studies from the literature. Its comparison with the in silico models of Methanosarcina barkeri, Methanosarcina acetivorans, and Sulfolobus solfataricus P2 shows M. maripaludis S2 to be a better organism for producing methane. Using the model, genes essential for growth were identified, and the efficacies of alternative carbon, hydrogen and nitrogen sources were studied. The model can predict the effects of reengineering M. maripaludis S2 to guide or expedite experimental efforts.

  18. Transcription start site associated RNAs (TSSaRNAs are ubiquitous in all domains of life.

    Directory of Open Access Journals (Sweden)

    Livia S Zaramela

    Full Text Available A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq and a primary-transcript enriched (dRNA-seq strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome. Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

  19. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    Energy Technology Data Exchange (ETDEWEB)

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  20. Production of galactooligosaccharides using a hyperthermophilic β-galactosidase in permeabilized whole cells of Lactococcus lactis.

    Science.gov (United States)

    Yu, L; O'Sullivan, D J

    2014-02-01

    Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using β-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable β-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.