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Sample records for mediate heterochromatin formation

  1. Regulation of Neurospora Catalase-3 by global heterochromatin formation and its proximal heterochromatin region.

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    Wang, Yajun; Dong, Qing; Ding, Zhaolan; Gai, Kexin; Han, Xiaoyun; Kaleri, Farah Naz; He, Qun; Wang, Ying

    2016-10-01

    Catalase-3 (CAT-3) constitutes the main catalase activity in growing hyphae of Neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. Although extensive progress has been made to identify catalase regulators, the regulation mechanism of CAT-3 at the chromatin level still remains unclear. Here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. We found that CAT-3 protein levels increased in mutants defective in proper global heterochromatin formation. Bioinformatics analysis identified a 5-kb AT-rich sequence adjacent to the cat-3 promoter as a heterochromatin region because of its enrichment of H3K9me3 and HP1. Expression of CAT-3 was induced by H 2 O 2 treatment in wild-type and such change occurred along with the accumulation of histone H3 acetylation at 5-kb heterochromatin boundaries and cat-3 locus, but without alteration of its H3K9me3 repressive modification. Moreover, disruption of 5-kb heterochromatin region results in elevated cat-3 expression, and higher levels of cat-3 expression were promoted by the combination with global heterochromatin defective mutants. Interestingly, the molecular weight and activity bands of CAT-3 protein are different in heterochromatin defective mutants compared with those in wild-type, suggesting that its N-terminal processing and modification may be altered. Our study indicates that the local chromatin structure creates a heterochromatin repressive environment to repress nearby gene expression. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

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    Saveliev, Alexander; Everett, Christopher; Sharpe, Tammy; Webster, Zoë; Festenstein, Richard

    2003-04-24

    Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreich's ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

  3. Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation.

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    Thibaut Josse

    2007-09-01

    Full Text Available The study of P-element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE, a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. Here, we report that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. We show that this silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var205 encoding Heterochromatin Protein 1 and Su(var3-7 and the repeat-associated small interfering RNA (or rasiRNA silencing pathway (aubergine, homeless, armitage, and piwi. In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway.

  4. Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

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    Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

    2007-01-01

    Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

  5. The molecular basis for stability of heterochromatin-mediated silencing in mammals.

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    Hiragami-Hamada, Kyoko; Xie, Sheila Q; Saveliev, Alexander; Uribe-Lewis, Santiago; Pombo, Ana; Festenstein, Richard

    2009-11-04

    The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

  6. The molecular basis for stability of heterochromatin-mediated silencing in mammals

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    Hiragami-Hamada Kyoko

    2009-11-01

    Full Text Available Abstract The archetypal epigenetic phenomenon of position effect variegation (PEV in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3, DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac and lysine 4 di or tri methylation (H3K4me2/3 are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

  7. Correlation of LNCR rasiRNAs Expression with Heterochromatin Formation during Development of the Holocentric Insect Spodoptera frugiperda

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    Stanojcic, Slavica; Gimenez, Sylvie; Permal, Emmanuelle; Cousserans, François; Quesneville, Hadi; Fournier, Philippe; d'Alençon, Emmanuelle

    2011-01-01

    Repeat-associated small interfering RNAs (rasiRNAs) are derived from various genomic repetitive elements and ensure genomic stability by silencing endogenous transposable elements. Here we describe a novel subset of 46 rasiRNAs named LNCR rasiRNAs due to their homology with one long non-coding RNA (LNCR) of Spodoptera frugiperda. LNCR operates as the intermediate of an unclassified transposable element (TE-LNCR). TE-LNCR is a very invasive transposable element, present in high copy numbers in the S. frugiperda genome. LNCR rasiRNAs are single-stranded RNAs without a prominent nucleotide motif, which are organized in two distinct, strand-specific clusters. The expression of LNCR and LNCR rasiRNAs is developmentally regulated. Formation of heterochromatin in the genomic region where three copies of the TE-LNCR are embedded was followed by chromatin immunoprecipitation (ChIP) and we observed this chromatin undergo dynamic changes during development. In summary, increased LNCR expression in certain developmental stages is followed by the appearance of a variety of LNCR rasiRNAs which appears to correlate with subsequent accumulation of a heterochromatic histone mark and silencing of the genomic region with TE-LNCR. These results support the notion that a repeat-associated small interfering RNA pathway is linked to heterochromatin formation and/or maintenance during development to establish repression of the TE-LNCR transposable element. This study provides insights into the rasiRNA silencing pathway and its role in the formation of fluctuating heterochromatin during the development of one holocentric organism. PMID:21980354

  8. The fission yeast heterochromatin protein Rik1 is required for telomere clustering during meiosis

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    Tuzon, Creighton T; Borgstrøm, Britta; Weilguny, Dietmar

    2004-01-01

    Telomeres share the ability to silence nearby transcription with heterochromatin, but the requirement of heterochromatin proteins for most telomere functions is unknown. The fission yeast Rik1 protein is required for heterochromatin formation at centromeres and the mating-type locus, as it recrui...... meiosis. However, Rik1 is dispensable for the protective roles of telomeres in preventing chromosome end-fusion. Thus, a Swi6-independent heterochromatin function distinct from that at centromeres and the mating-type locus operates at telomeres during sexual differentiation....

  9. PREditOR: a synthetic biology approach to removing heterochromatin from cells.

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    Molina, Oscar; Carmena, Mar; Maudlin, Isabella E; Earnshaw, William C

    2016-12-01

    It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex.

  10. Mammalian ChlR1 has a role in heterochromatin organization

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    Inoue, Akira; Hyle, Judith; Lechner, Mark S.; Lahti, Jill M.

    2011-01-01

    The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11 -/- embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1β. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11 -/- embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects. -- Highlights: → New role for ChlR1 (DDX11), a cohesinopathy gene, in heterochromatin organization. → Loss of ChlR1 altered heterochromatin localization and centromere clustering. → Reduced ChlR1 levels also reduced HP1α and H3K9-me3 binding to pericentric DNA. → Decreased DNA methylation was found in pericentric repeats of Ddx11 -/- embryos. → These findings will aid in understanding the pathogenesis of Warsaw breakage syndrome.

  11. Arabidopsis HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1.

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    Taiko Kim To; Jong-Myong Kim; Akihiro Matsui; Yukio Kurihara; Taeko Morosawa; Junko Ishida; Maho Tanaka; Takaho Endo; Tetsuji Kakutani; Tetsuro Toyoda; Hiroshi Kimura; Shigeyuki Yokoyama; Kazuo Shinozaki; Motoaki Seki

    2011-01-01

    Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that t...

  12. RNAi and heterochromatin repress centromeric meiotic recombination

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    Ellermeier, Chad; Higuchi, Emily C; Phadnis, Naina

    2010-01-01

    During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essen......During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes....... Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between...... types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis....

  13. Genome mapping and characterization of the Anopheles gambiae heterochromatin

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    Sharakhova Maria V

    2010-08-01

    Full Text Available Abstract Background Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized. Results To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1 and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin. Conclusions Our results demonstrate that Anopheles polytene chromosomes

  14. Retroelements (LINEs and SINEs) in vole genomes: differential distribution in the constitutive heterochromatin.

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    Acosta, M J; Marchal, J A; Fernández-Espartero, C H; Bullejos, M; Sánchez, A

    2008-01-01

    The chromosomal distribution of mobile genetic elements is scarcely known in Arvicolinae species, but could be of relevance to understand the origin and complex evolution of the sex chromosome heterochromatin. In this work we cloned two retrotransposon sequences, L1 and SINE-B1, from the genome of Chionomys nivalis and investigated their chromosomal distribution on several arvicoline species. Our results demonstrate first that both retroelements are the most abundant repeated DNA sequences in the genome of these species. L1 elements, in most species, are highly accumulated in the sex chromosomes compared to the autosomes. This favoured L1 insertion could have played an important role in the origin of the enlarged heterochromatic blocks existing in the sex chromosomes of some Microtus species. Also, we propose that L1 accumulation on the X heterochromatin could have been the consequence of different, independent and rapid amplification processes acting in each species. SINE elements, however, were completely lacking from the constitutive heterochromatin, either in autosomes or in the heterochromatic blocks of sex chromosomes. These data could indicate that some SINE elements are incompatible with the formation of heterochromatic complexes and hence are necessarily missing from the constitutive heterochromatin.

  15. Karyotypes and heterochromatin variation (C-bands in Melipona species (Hymenoptera, Apidae, Meliponinae

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    Rocha Marla Piumbini

    1998-01-01

    Full Text Available We describe the karyotypes of eight bee species of the genus Melipona and compare them in terms of heterochromatin content and location (C-banding technique. All species had 2n = 18 (females and n = 9 (males chromosomes, but a wide variation in heterochromatin content was detected among karyotypes. On the basis of these differences, the species were divided into two functional groups, one of them comprising species with a karyotype having a low heterochromatin content (M. bicolor bicolor, M. quadrifasciata, M. marginata, and M. asilvai, and the other species with a high heterochromatin content (M. seminigra fuscopilosa, M. capixaba, M. scutellaris, and M. captiosa. In the species with high heterochromatin content, heterochromatin occupied practically the entire extent of all chromosomes, with euchromatin being limited to the extremities, a fact that prevented observation of the centromere. In contrast, in the species with karyotypes having a low heterochromatin content, heterochromatin was visualized only in some chromosomes. In the chromosomes in which it was present, heterochromatin was located in the centromere or on the short arm. M. bicolor bicolor had the smallest heterochromatin content with only three chromosome pairs presenting heterochromatin in females. Increased heterochromatin content may be explained by interstitial and pericentromeric growth.

  16. Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

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    Pichugin, Andrey; Beaujean, Nathalie; Vignon, Xavier; Vassetzky, Yegor

    2011-01-01

    Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

  17. Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

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    Andrey Pichugin

    Full Text Available BACKGROUND: Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3 and by DAPI counterstaining. METHODS: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH. RESULTS: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. CONCLUSIONS: Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

  18. An Interaction between RRP6 and SU(VAR)3-9 Targets RRP6 to Heterochromatin and Contributes to Heterochromatin Maintenance in Drosophila melanogaster

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    Eberle, Andrea B.; Jordán-Pla, Antonio; Gañez-Zapater, Antoni; Hessle, Viktoria; Silberberg, Gilad; von Euler, Anne; Silverstein, Rebecca A.; Visa, Neus

    2015-01-01

    RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. Here we show that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. We show that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network revealed physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrated that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, our results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin. PMID:26389589

  19. Pairing of heterochromatin in response to cellular stress

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    Abdel-Halim, H.I.; Mullenders, L.H.F.; Boei, J.J.W.A.

    2006-01-01

    We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair

  20. Heterochromatin protein 1 (HP1a positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

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    Lucia Piacentini

    2009-10-01

    Full Text Available Heterochromatin Protein 1 (HP1a is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases methylate the histone H3 at lysine 9 (H3K9me, creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays, and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms

  1. Deacetylation of H4-K16Ac and heterochromatin assembly in senescence

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    Contrepois Kévin

    2012-08-01

    Full Text Available Abstract Background Cellular senescence is a stress response of mammalian cells leading to a durable arrest of cell proliferation that has been implicated in tumor suppression, wound healing, and aging. The proliferative arrest is mediated by transcriptional repression of genes essential for cell division by the retinoblastoma protein family. This repression is accompanied by varying degrees of heterochromatin assembly, but little is known regarding the molecular mechanisms involved. Results We found that both deacetylation of H4-K16Ac and expression of HMGA1/2 can contribute to DNA compaction during senescence. SIRT2, an NAD-dependent class III histone deacetylase, contributes to H4-K16Ac deacetylation and DNA compaction in human fibroblast cell lines that assemble striking senescence-associated heterochromatin foci (SAHFs. Decreased H4-K16Ac was observed in both replicative and oncogene-induced senescence of these cells. In contrast, this mechanism was inoperative in a fibroblast cell line that did not assemble extensive heterochromatin during senescence. Treatment of senescent cells with trichostatin A, a class I/II histone deacetylase inhibitor, also induced rapid and reversible decondensation of SAHFs. Inhibition of DNA compaction did not significantly affect the stability of the senescent state. Conclusions Variable DNA compaction observed during senescence is explained in part by cell-type specific regulation of H4 deacetylation and HMGA1/2 expression. Deacetylation of H4-K16Ac during senescence may explain reported decreases in this mark during mammalian aging and in cancer cells.

  2. Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.

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    He, Bing; Caudy, Amy; Parsons, Lance; Rosebrock, Adam; Pane, Attilio; Raj, Sandeep; Wieschaus, Eric

    2012-12-01

    Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes ("H-probes") for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin.

  3. Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster

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    He, Bing; Caudy, Amy; Parsons, Lance; Rosebrock, Adam; Pane, Attilio; Raj, Sandeep; Wieschaus, Eric

    2012-01-01

    Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding sequences that can be mapped to the assembled euchromatic arms, we identified sequences that are specific to heterochromatin and used them to design heterochromatin specific probes (“H-probes”) for microarray. By comparative genomic hybridization (CGH) analyses of embryos deficient for each chromosome or chromosome arm, we were able to map most of our H-probes to specific chromosome arms. We also positioned sequences mapped to the second and X chromosomes to finer intervals by analyzing smaller deletions with breakpoints in heterochromatin. Using this approach, we were able to map >40% (13.9 Mb) of the previously unmapped heterochromatin sequences assembled by the whole-genome sequencing effort on arm U and arm Uextra to specific locations. We also identified and mapped 110 kb of novel heterochromatic sequences. Subsequent analyses revealed that sequences located within different heterochromatic regions have distinct properties, such as sequence composition, degree of repetitiveness, and level of underreplication in polytenized tissues. Surprisingly, although heterochromatin is generally considered to be transcriptionally silent, we detected region-specific temporal patterns of transcription in heterochromatin during oogenesis and early embryonic development. Our study provides a useful approach to elucidate the molecular organization and function of heterochromatin and reveals region-specific variation of heterochromatin. PMID:22745230

  4. Centromeric heterochromatin: the primordial segregation machine.

    Science.gov (United States)

    Bloom, Kerry S

    2014-01-01

    Centromeres are specialized domains of heterochromatin that provide the foundation for the kinetochore. Centromeric heterochromatin is characterized by specific histone modifications, a centromere-specific histone H3 variant (CENP-A), and the enrichment of cohesin, condensin, and topoisomerase II. Centromere DNA varies orders of magnitude in size from 125 bp (budding yeast) to several megabases (human). In metaphase, sister kinetochores on the surface of replicated chromosomes face away from each other, where they establish microtubule attachment and bi-orientation. Despite the disparity in centromere size, the distance between separated sister kinetochores is remarkably conserved (approximately 1 μm) throughout phylogeny. The centromere functions as a molecular spring that resists microtubule-based extensional forces in mitosis. This review explores the physical properties of DNA in order to understand how the molecular spring is built and how it contributes to the fidelity of chromosome segregation.

  5. Overexpression of the heterochromatinization factor BAHD1 in HEK293 cells differentially reshapes the DNA methylome on autosomes and X chromosome.

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    Emanuele eLibertini

    2015-12-01

    Full Text Available BAH domain-containing protein 1 (BAHD1 is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi, but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1. We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX. We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed BAHD1-DMRs, of which 83,850 mapped on autosomes and 7,508 on the X chromosome (chrX. Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these BAHD1-Associated differentially methylated Domains (BADs overlapped with lamina-associated domains (LADs. Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

  6. Ubiquitin ligase RNF123 mediates degradation of heterochromatin protein 1α and β in lamin A/C knock-down cells.

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    Pankaj Chaturvedi

    Full Text Available The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1. However, the pathways of proteasomal degradation have not been well characterized.To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells.Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.

  7. Border Structure of Intercalary Heterochromatin Bands of Drosophila melanogaster Polytene Chromosomes.

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    Khoroshko, V A; Zykova, T Yu; Popova, O O; Zhimulev, I F

    2018-03-01

    The precise genomic localization of the borders of 62 intercalary heterochromatin bands in Drosophila polytene chromosomes was determined. A new type of bands containing chromatin of different states was identified. This type is a combination of the gray band and the intercalary heterochromatin band, creating a genetic structure that with a light microscope is identified as a continuous band. The border structure of such bands includes the coding regions of genes with ubiquitous activity.

  8. Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

    Science.gov (United States)

    Mosiołek, Magdalena; Pasierbek, Paweł; Malarz, Janusz; Moś, Maria; Joachimiak, Andrzej J

    2005-01-01

    Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.

  9. EG-17SUV420-MEDIATED HETEROCHROMATIN CHANGES IN PEDIATRIC BRAIN CANCERS

    Science.gov (United States)

    Van Meter, Timothy E.; Terry, Jocelyn; Rockwell, Nathan; Goggin, Sarah; Nethala, Priya; Khan, Asadullah

    2014-01-01

    Silencing mechanisms play a role in genomic stability by maintaining condensed, non-active regions of the genome. SUV420 enzymes contain a SET domain conferring methyltransferase activity toward histones. The Histone H4 lysine 20 trimethylation (H4K20me3) mark maintained by SUV420H2 is associated with heterochromatin formation and gene silencing, whereas the dimethylated mark (H4K20me2) is associated with DNA repair. In studies of epigenetic factors in large patient cohorts with ependymoma, it was found that SUV420H2 expression was lost or diminished in patients with reciprocal increases in prognostic markers such as hTERT. To better understand the normal function of Suv4-20H1/H2 enzyme in neural progenitors, and pathological changes in cancers, a variety of differentiation paradigms were used. The NT2D1 neurally restricted cell line, and BGO1V and H9 human embryonic stem cells (ESCs), and differentiated progeny, were used alongside tumors to better understand enzyme targets and functional outcomes (e.g.,lineage, differentiation, regional chromatin modifications). Lineage stages were verified with stage-specific markers by immunofluorescence and qPCR. Suv4-20 H1 and H2 were present in ESCs and neural progenitors and decreased thereafter. RNAi knockdown of SUV420 enzymes led to decreased H4K20 methylation in cancer cells. DNA methylation microarrays and ChIP-PCR suggest 1) that SUV420 is not regulated by DNA methylation in ependymomas; 2) that active chromatin marks such as H3K4 dimethylation are enriched near the transcriptional start site in the SUV420H2 gene, and 3) that hTERT is hyper-methylated at specific CpG islands and histones in a tumor sub-group-specific manner. This data supports the hypothesis that Suv4-20H2 is highly active in progenitor cells and functionally lost in some brain cancers. These studies begin to elucidate coincident mechanisms of gene silencing active in neural progenitors that may be altered in a subset of pediatric brain cancers.

  10. Alteration of terminal heterochromatin and chromosome rearrangements in derivatives of wheat-rye hybrids.

    Science.gov (United States)

    Fu, Shulan; Lv, Zhenling; Guo, Xiang; Zhang, Xiangqi; Han, Fangpu

    2013-08-20

    Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres. Furthermore, a mitotically unstable dicentric chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese Spring-Imperial rye 3R addition line. An unstable multicentric chromosome was found in the progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including movement and disappearance of terminal heterochromatin occurred in the progeny of wheat-rye addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were observed in the progeny of a monosomic 4R addition line, a ditelosomic 5RS addition line and a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric DNA (TTTAGGG)n, derived from a monosomic 4R addition line are stable and transmissible to the next generation. The results indicated that centromeres and terminal heterochromatin can be profoundly altered in wheat-rye hybrid derivatives. Copyright © 2013. Published by Elsevier Ltd.

  11. Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.

    Science.gov (United States)

    Zhang, Weiqi; Li, Jingyi; Suzuki, Keiichiro; Qu, Jing; Wang, Ping; Zhou, Junzhi; Liu, Xiaomeng; Ren, Ruotong; Xu, Xiuling; Ocampo, Alejandro; Yuan, Tingting; Yang, Jiping; Li, Ying; Shi, Liang; Guan, Dee; Pan, Huize; Duan, Shunlei; Ding, Zhichao; Li, Mo; Yi, Fei; Bai, Ruijun; Wang, Yayu; Chen, Chang; Yang, Fuquan; Li, Xiaoyu; Wang, Zimei; Aizawa, Emi; Goebl, April; Soligalla, Rupa Devi; Reddy, Pradeep; Esteban, Concepcion Rodriguez; Tang, Fuchou; Liu, Guang-Hui; Belmonte, Juan Carlos Izpisua

    2015-06-05

    Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging. Copyright © 2015, American Association for the Advancement of Science.

  12. Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

    Science.gov (United States)

    Casas-Delucchi, Corella S; van Bemmel, Joke G; Haase, Sebastian; Herce, Henry D; Nowak, Danny; Meilinger, Daniela; Stear, Jeffrey H; Leonhardt, Heinrich; Cardoso, M Cristina

    2012-01-01

    The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

  13. Heterochromatin Reorganization during Early Mouse Development Requires a Single-Stranded Noncoding Transcript

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    Miguel Casanova

    2013-09-01

    Full Text Available The equalization of pericentric heterochromatin from distinct parental origins following fertilization is essential for genome function and development. The recent implication of noncoding transcripts in this process raises questions regarding the connection between RNA and the nuclear organization of distinct chromatin environments. Our study addresses the interrelationship between replication and transcription of the two parental pericentric heterochromatin (PHC domains and their reorganization during early embryonic development. We demonstrate that the replication of PHC is dispensable for its clustering at the late two-cell stage. In contrast, using parthenogenetic embryos, we show that pericentric transcripts are essential for this reorganization independent of the chromatin marks associated with the PHC domains. Finally, our discovery that only reverse pericentric transcripts are required for both the nuclear reorganization of PHC and development beyond the two-cell stage challenges current views on heterochromatin organization.

  14. Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

    Directory of Open Access Journals (Sweden)

    Andrzej J. Joachimiak

    2011-08-01

    Full Text Available Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw. Stearn roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.

  15. RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Smialowska, Agata; Djupedal, Ingela; Wang, Jingwen; Kylsten, Per; Swoboda, Peter; Ekwall, Karl

    2014-01-01

    Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe

  16. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

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    Varvara A Khoroshko

    Full Text Available Late-replicating domains (intercalary heterochromatin in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW, and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a

  17. Comparative Genomic Analyses Provide New Insights into the Evolutionary Dynamics of Heterochromatin in Drosophila.

    Science.gov (United States)

    Caizzi, Ruggiero; Moschetti, Roberta; Piacentini, Lucia; Fanti, Laura; Marsano, Renè Massimiliano; Dimitri, Patrizio

    2016-08-01

    The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, we have identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. Our results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, we suggest a new scenario whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin.

  18. Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones.

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    Jiro C Yasuhara

    2008-01-01

    Full Text Available Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2 was depleted at the 5' ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var3-9 null mutation, implicating a histone methyltransferase other than SU(VAR3-9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature

  19. The Role of piRNA-Mediated Epigenetic Silencing in the Population Dynamics of Transposable Elements in Drosophila melanogaster.

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    Yuh Chwen G Lee

    2015-06-01

    Full Text Available The piwi-interacting RNAs (piRNA are small RNAs that target selfish transposable elements (TEs in many animal genomes. Until now, piRNAs' role in TE population dynamics has only been discussed in the context of their suppression of TE transposition, which alone is not sufficient to account for the skewed frequency spectrum and stable containment of TEs. On the other hand, euchromatic TEs can be epigenetically silenced via piRNA-dependent heterochromatin formation and, similar to the widely known "Position-effect variegation", heterochromatin induced by TEs can "spread" into nearby genes. We hypothesized that the piRNA-mediated spread of heterochromatin from TEs into adjacent genes has deleterious functional effects and leads to selection against individual TEs. Unlike previously identified deleterious effects of TEs due to the physical disruption of DNA, the functional effect we investigated here is mediated through the epigenetic influences of TEs. We found that the repressive chromatin mark, H3K9me, is elevated in sequences adjacent to euchromatic TEs at multiple developmental stages in Drosophila melanogaster. Furthermore, the heterochromatic states of genes depend not only on the number of and distance from adjacent TEs, but also on the likelihood that their nearest TEs are targeted by piRNAs. These variations in chromatin status probably have functional consequences, causing genes near TEs to have lower expression. Importantly, we found stronger selection against TEs that lead to higher H3K9me enrichment of adjacent genes, demonstrating the pervasive evolutionary consequences of TE-induced epigenetic silencing. Because of the intrinsic biological mechanism of piRNA amplification, spread of TE heterochromatin could result in the theoretically required synergistic deleterious effects of TE insertions for stable containment of TE copy number. The indirect deleterious impact of piRNA-mediated epigenetic silencing of TEs is a previously

  20. Nuclear Architecture of Mouse Spermatocytes: Chromosome Topology, Heterochromatin, and Nucleolus.

    Science.gov (United States)

    Berrios, Soledad

    2017-01-01

    The nuclear organization of spermatocytes in meiotic prophase I is primarily determined by the synaptic organization of the bivalents that are bound by their telomeres to the nuclear envelope and described as arc-shaped trajectories through the 3D nuclear space. However, over this basic meiotic organization, a spermatocyte nuclear architecture arises that is based on higher-ordered patterns of spatial associations among chromosomal domains from different bivalents that are conditioned by the individual characteristics of chromosomes and the opportunity for interactions between their domains. Consequently, the nuclear architecture is species-specific and prone to modification by chromosomal rearrangements. This model is valid for the localization of any chromosomal domain in the meiotic prophase nucleus. However, constitutive heterochromatin plays a leading role in shaping nuclear territories. Thus, the nuclear localization of nucleoli depends on the position of NORs in nucleolar bivalents, but the association among nucleolar chromosomes mainly depends on the presence of constitutive heterochromatin that does not affect the expression of the ribosomal genes. Constitutive heterochromatin and nucleoli form complex nuclear territories whose distribution in the nuclear space is nonrandom, supporting the hypothesis regarding the existence of a species-specific nuclear architecture in first meiotic prophase spermatocytes. © 2017 S. Karger AG, Basel.

  1. Positive Selection Drives the Evolution of rhino, a Member of the Heterochromatin Protein 1 Family in Drosophila.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1, which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.

  2. Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila.

    Directory of Open Access Journals (Sweden)

    Danielle Vermaak

    2005-07-01

    Full Text Available Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1, which is an essential, nonhistone chromosomal protein. Here we investigate the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, we find that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of "junk DNA" in eukaryotic genomes.

  3. Heterochromatin and molecular characterization of DsmarMITE transposable element in the beetle Dichotomius schiffleri (Coleoptera: Scarabaeidae).

    Science.gov (United States)

    Xavier, Crislaine; Cabral-de-Mello, Diogo Cavalcanti; de Moura, Rita Cássia

    2014-12-01

    Cytogenetic studies of the Neotropical beetle genus Dichotomius (Scarabaeinae, Coleoptera) have shown dynamism for centromeric constitutive heterochromatin sequences. In the present work we studied the chromosomes and isolated repetitive sequences of Dichotomius schiffleri aiming to contribute to the understanding of coleopteran genome/chromosomal organization. Dichotomius schiffleri presented a conserved karyotype and heterochromatin distribution in comparison to other species of the genus with 2n = 18, biarmed chromosomes, and pericentromeric C-positive blocks. Similarly to heterochromatin distributional patterns, the highly and moderately repetitive DNA fraction (C 0 t-1 DNA) was detected in pericentromeric areas, contrasting with the euchromatic mapping of an isolated TE (named DsmarMITE). After structural analyses, the DsmarMITE was classified as a non-autonomous element of the type miniature inverted-repeat transposable element (MITE) with terminal inverted repeats similar to Mariner elements of insects from different orders. The euchromatic distribution for DsmarMITE indicates that it does not play a part in the dynamics of constitutive heterochromatin sequences.

  4. Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster

    OpenAIRE

    He, Bing; Caudy, Amy; Parsons, Lance; Rosebrock, Adam; Pane, Attilio; Raj, Sandeep; Wieschaus, Eric

    2012-01-01

    Heterochromatin represents a significant portion of eukaryotic genomes and has essential structural and regulatory functions. Its molecular organization is largely unknown due to difficulties in sequencing through and assembling repetitive sequences enriched in the heterochromatin. Here we developed a novel strategy using chromosomal rearrangements and embryonic phenotypes to position unmapped Drosophila melanogaster heterochromatic sequence to specific chromosomal regions. By excluding seque...

  5. Drugs affecting prelamin A processing: Effects on heterochromatin organization

    International Nuclear Information System (INIS)

    Mattioli, Elisabetta; Columbaro, Marta; Capanni, Cristina; Santi, Spartaco; Maraldi, Nadir M.; D'Apice, M. Rosaria; Novelli, Giuseppe; Riccio, Massimo; Squarzoni, Stefano; Foisner, Roland; Lattanzi, Giovanna

    2008-01-01

    Increasing interest in drugs acting on prelamin A has derived from the finding of prelamin A involvement in severe laminopathies. Amelioration of the nuclear morphology by inhibitors of prelamin A farnesylation has been widely reported in progeroid laminopathies. We investigated the effects on chromatin organization of two drugs inhibiting prelamin A processing by an ultrastructural and biochemical approach. The farnesyltransferase inhibitor FTI-277 and the non-peptidomimetic drug N-acetyl-S-farnesyl-L-cysteine methylester (AFCMe) were administered to cultured control human fibroblasts for 6 or 18 h. FTI-277 interferes with protein farnesylation causing accumulation of non-farnesylated prelamin A, while AFCMe impairs the last cleavage of the lamin A precursor and is expected to accumulate farnesylated prelamin A. FTI-277 caused redistribution of heterochromatin domains at the nuclear interior, while AFCMe caused loss of heterochromatin domains, increase of nuclear size and nuclear lamina thickening. At the biochemical level, heterochromatin-associated proteins and LAP2α were clustered at the nuclear interior following FTI-277 treatment, while they were unevenly distributed or absent in AFCMe-treated nuclei. The reported effects show that chromatin is an immediate target of FTI-277 and AFCMe and that dramatic remodeling of chromatin domains occurs following treatment with the drugs. These effects appear to depend, at least in part, on the accumulation of prelamin A forms, since impairment of prelamin A accumulation, here obtained by 5-azadeoxycytidine treatment, abolishes the chromatin effects. These results may be used to evaluate downstream effects of FTIs or other prelamin A inhibitors potentially useful for the therapy of laminopathies

  6. MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation

    Science.gov (United States)

    Agarwal, Noopur; Hardt, Tanja; Brero, Alessandro; Nowak, Danny; Rothbauer, Ulrich; Becker, Annette; Leonhardt, Heinrich; Cardoso, M. Cristina

    2007-01-01

    There is increasing evidence of crosstalk between epigenetic modifications such as histone and DNA methylation, recognized by HP1 and methyl CpG-binding proteins, respectively. We have previously shown that the level of methyl CpG-binding proteins increased dramatically during myogenesis leading to large-scale heterochromatin reorganization. In this work, we show that the level of HP1 isoforms did not change significantly throughout myogenic differentiation but their localization did. In particular, HP1γ relocalization to heterochromatin correlated with MeCP2 presence. Using co-immunoprecipitation assays, we found that these heterochromatic factors interact in vivo via the chromo shadow domain of HP1 and the first 55 amino acids of MeCP2. We propose that this dynamic interaction of HP1 and MeCP2 increases their concentration at heterochromatin linking two major gene silencing pathways to stabilize transcriptional repression during differentiation. PMID:17698499

  7. Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia).

    Science.gov (United States)

    Kawagoshi, Taiki; Nishida, Chizuko; Ota, Hidetoshi; Kumazawa, Yoshinori; Endo, Hideki; Matsuda, Yoichi

    2008-01-01

    Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30-42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG)( n ) sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.

  8. A spherical harmonics intensity model for 3D segmentation and 3D shape analysis of heterochromatin foci.

    Science.gov (United States)

    Eck, Simon; Wörz, Stefan; Müller-Ott, Katharina; Hahn, Matthias; Biesdorf, Andreas; Schotta, Gunnar; Rippe, Karsten; Rohr, Karl

    2016-08-01

    The genome is partitioned into regions of euchromatin and heterochromatin. The organization of heterochromatin is important for the regulation of cellular processes such as chromosome segregation and gene silencing, and their misregulation is linked to cancer and other diseases. We present a model-based approach for automatic 3D segmentation and 3D shape analysis of heterochromatin foci from 3D confocal light microscopy images. Our approach employs a novel 3D intensity model based on spherical harmonics, which analytically describes the shape and intensities of the foci. The model parameters are determined by fitting the model to the image intensities using least-squares minimization. To characterize the 3D shape of the foci, we exploit the computed spherical harmonics coefficients and determine a shape descriptor. We applied our approach to 3D synthetic image data as well as real 3D static and real 3D time-lapse microscopy images, and compared the performance with that of previous approaches. It turned out that our approach yields accurate 3D segmentation results and performs better than previous approaches. We also show that our approach can be used for quantifying 3D shape differences of heterochromatin foci. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term

    Directory of Open Access Journals (Sweden)

    Brochard Vincent

    2009-02-01

    Full Text Available Abstract Background Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development. Results Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT, supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages. Conclusion Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.

  10. Molecular characterization of constitutive heterochromatin in three species of Trypoxylon (Hymenoptera, Crabronidae, Trypoxylini by CMA3/DAPI staining

    Directory of Open Access Journals (Sweden)

    Rodolpho Menezes

    2011-07-01

    Full Text Available Previous cytogenetic analyses in Trypoxylon Latreille, 1796 have been basically restricted to C-banding. In the present study, base-specific CMA3 and DAPI fluorochrome staining were used to characterize the constitutive heterochromatin in three Trypoxylon species. The heterochromatin was GC-rich in all the species studied; however, in Trypoxylon nitidum F. Smith, 1856 the molecular composition of the heterochromatin was different among chromosome pairs. Conversely, the euchromatin was AT-rich in the three species. These results suggest high conservatism in the euchromatic regions as opposed to the heterochromatic regions that have a high rate of changes. In this study, we report the karyotype of Trypoxylon rugifrons F. Smith, 1873 which has the lowest chromosome number in the genus and other characteristics of the likely ancestral Trypoxylon karyotype.

  11. Genetic and bioinformatic analysis of 41C and the 2R heterochromatin of Drosophila melanogaster: a window on the heterochromatin-euchromatin junction.

    OpenAIRE

    Myster, Steven H; Wang, Fei; Cavallo, Robert; Christian, Whitney; Bhotika, Seema; Anderson, Charles T; Peifer, Mark

    2004-01-01

    Genomic sequences provide powerful new tools in genetic analysis, making it possible to combine classical genetics with genomics to characterize the genes in a particular chromosome region. These approaches have been applied successfully to the euchromatin, but analysis of the heterochromatin has lagged somewhat behind. We describe a combined genetic and bioinformatics approach to the base of the right arm of the Drosophila melanogaster second chromosome, at the boundary between pericentric h...

  12. Amplification of a GC-rich heterochromatin in the freshwater fish Leporinus desmotes (Characiformes, Anostomidae

    Directory of Open Access Journals (Sweden)

    Vladimir Pavan Margarido

    2000-09-01

    Full Text Available This is the first description of the karyotype of Leporinus desmotes. The diploid female number was 2n = 54 meta- and submetacentric chromosomes. The nucleolar organizing regions (NORs were studied by silver nitrate staining and rDNA fluorescence in situ hybridization (FISH and were found to be located in the telomeric region of the long arm of the 9th pair. C-banding revealed centromeric and telomeric heterochromatin segments in most chromosomes. Intercalar blocks of heterochromatin were observed in the long arm of six chromosome pairs. Besides a NOR-adjacent heterochromatin, all of the intercalar heterochromatic segments were brightly fluorescent by mithramycin staining. These data suggest that a unique amplification of a primordial GC-rich heterochromatin, probably NOR-associated, may have taken place in the karyotype diversification of this Leporinus species.Esta é a primeira descrição do cariótipo de Leporinus desmotes fêmea. O número diplóide encontrado foi 2n = 54 cromossomos meta- e submetacêntricos. As regiões organizadoras de nucléolos (NORs foram estudadas através da impregnação pela prata e por hibridização in situ com sondas de DNAr (FISH, e foram localizadas na região telomérica do braço longo do 9º par. Heterocromatinas centromérica e telomérica foram reveladas pelo bandamento C na maioria dos cromossomos. Adicionalmente, grande quantidade de heterocromatina intercalar ou subtelomérica foi também observada. Diferenciação composicional na maior parte da heterocromatina identificada em L. desmotes pode ser inferida através da coloração pela mitramicina, caracterizando um caso peculiar de amplificação de segmentos heterocromáticos ricos em bases GC neste grupo de peixes.

  13. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases

    DEFF Research Database (Denmark)

    Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath

    2011-01-01

    organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well......, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci....

  14. Mediator structure and rearrangements required for holoenzyme formation.

    Science.gov (United States)

    Tsai, Kuang-Lei; Yu, Xiaodi; Gopalan, Sneha; Chao, Ti-Chun; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Murakami, Kenji; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

    2017-04-13

    The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

  15. Herpesviral ICP0 Protein Promotes Two Waves of Heterochromatin Removal on an Early Viral Promoter during Lytic Infection

    Directory of Open Access Journals (Sweden)

    Jennifer S. Lee

    2016-01-01

    Full Text Available Herpesviruses must contend with host cell epigenetic silencing responses acting on their genomes upon entry into the host cell nucleus. In this study, we confirmed that unchromatinized herpes simplex virus 1 (HSV-1 genomes enter primary human foreskin fibroblasts and are rapidly subjected to assembly of nucleosomes and association with repressive heterochromatin modifications such as histone 3 (H3 lysine 9-trimethylation (H3K9me3 and lysine 27-trimethylation (H3K27me3 during the first 1 to 2 h postinfection. Kinetic analysis of the modulation of nucleosomes and heterochromatin modifications over the course of lytic infection demonstrates a progressive removal that coincided with initiation of viral gene expression. We obtained evidence for three phases of heterochromatin removal from an early gene promoter: an initial removal of histones and heterochromatin not dependent on ICP0, a second ICP0-dependent round of removal of H3K9me3 that is independent of viral DNA synthesis, and a third phase of H3K27me3 removal that is dependent on ICP0 and viral DNA synthesis. The presence of ICP0 in transfected cells is also sufficient to promote removal of histones and H3K9me3 modifications of cotransfected genes. Overall, these results show that ICP0 promotes histone removal, a reduction of H3K9me3 modifications, and a later indirect reduction of H3K27me3 modifications following viral early gene expression and DNA synthesis. Therefore, HSV ICP0 promotes the reversal of host epigenetic silencing mechanisms by several mechanisms.

  16. Heterochromatin heterogeneity in Hypostomus prope unae (Steindachner, 1878 (Siluriformes, Loricariidae from Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Jamille Bitencourt

    2011-11-01

    Full Text Available Cytogenetic analyses using C-banding and chromosomal digestion by several restriction enzymes were carried out in four populations (named A, B, C and D of Hypostomus prope unae (Loricariidae, Hypostominae from Contas river basin, northeastern Brazil. These populations share 2n=76 and single NORs on the second metacentric pair but exclusive karyotype forms for each locality. Populations A and B presented conspicuous terminal and interstitial heterochromatic blocks on most of acrocentric chromosomes and equivalent to NORs with differences in both position and bearing pair. Population D showed evident marks at interstitial regions and interspersed with nucleolar region while population C presented interstitial and terminal heterochromatin segments, non-coincident with NORs. The banding pattern after digestion with the endonucleases Alu I, Bam HI, Hae III and Dde I revealed a remarkable heterogeneity within heterochromatin, allowing the identification of distinctive clusters of repeated DNA in the studied populations, besides specific patterns along euchromatic regions. The analysis using restriction enzymes has proved to be highly informative, characterizing population differences and peculiarities in the genome organization of H. prope unae.

  17. Translocations of chromosome end-segments and facultative heterochromatin promote meiotic ring formation in evening primroses.

    Science.gov (United States)

    Golczyk, Hieronim; Massouh, Amid; Greiner, Stephan

    2014-03-01

    Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories.

  18. Heterochromatin marks HP1γ, HP1α and H3K9me3, and DNA damage response activation in human testis development and germ cell tumours

    DEFF Research Database (Denmark)

    Bartkova, J; Moudry, P; Hodny, Z

    2011-01-01

    activation in CIS; and enhanced HP1α in teratoma structures with epithelial and neuronal differentiation. Differential expression of the three heterochromatin markers suggests their partly non-overlapping roles, and separation of heterochromatinization from DDR activation highlights distinct responses...

  19. Heterochromatin analysis in the fish species Liposarcus anisitsi (siluriformes and Leporinus elongatus (characiformes

    Directory of Open Access Journals (Sweden)

    Roberto Ferreira Artoni

    1999-03-01

    Full Text Available The chromosomes of two neotropical freshwater fish species, namely Liposarcus anisitsi (Siluriformes, Loricariidae and Leporinus elongatus (Characiformes, Anostomidae, were investigated by means of C-banding, Ag-NORs, fluorochrome staining and banding by hot saline solution (HSS treatment, to reveal patterns of heterochromatin differentiation. The karyotype of L. anisitsi is described for the first time. Staining with the GC-specific fluorescent antibiotic mithramycin (MM revealed bright signals in some C-banded blocks in both species, suggesting that these MM+ heterochromatin contains GC-rich DNA. Banding by denaturation employing HSS, followed by Giemsa staining, yielded corresponding results documenting the thermal stability of GC-rich DNA part of heterochromatin positive after C-banding. In L. elongatus the Ag-NOR also followed the above banding patterns. However, in L. anisitsi the Ag-NOR was MM+ but negatively stained after C-banding and HSS treatment. L. elongatus also showed C-banded segments that were negative for mithramycin staining and HSS treatment. The results obtained evidence the heterochromatin heterogeneity in these fish species.Cromossomos mitóticos de duas especies de peixes neotropicais, Leporinus elongatus (Characiformes e Liposarcus anisitsi (Siluriformes, foram estudados por diferentes métodos de bandamentos, com o intuito de investigar a diferenciação da heterocromatina. Enquanto que a macroestrutura cariotípica de L. elongatus já foi objeto de estudos anteriores, o cariótipo de L. anisitsi está sendo apresentado pela primeira vez. Em ambas as espécies, a coloração dos cromossomos com a mitramicina (MM, fluorocromo GC específico, evidenciou sinais brilhantes em alguns segmentos heterocromáticos também positivos ao bandamento C, sugerindo ser esta fração da heterocromatina rica em seqüências de bases GC. O tratamento dos cromossomos com solução salina aquecida e posterior coloração com Giemsa

  20. Karyotype, heterochromatin distribution and meiosis of Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae)

    International Nuclear Information System (INIS)

    Correa, Ronan X.; Santos, Igor S.; Silva, Janisete G.; Costa, Marco A.; Pompolo, Silvia G.

    2008-01-01

    Zabrotes subfasciatus (Boh.) has been extensively studied in its agronomic and biochemical aspects due to its importance as a damaging insect to leguminous grains during storage. The few cytogenetic studies published on this species yielded conflicting results. In this study, the karyotype was analyzed in order to accurately describe the chromosome C-banding patterns and meiosis. The brain ganglion at the pre pupa and the adult and pupal testes were analyzed. All individuals had 26 chromosomes in both brain ganglion and spermatogonic mitotic metaphases. These chromosomes were classified as follows: the 12 th pair and the Y chromosome were telocentric; the X chromosome was acrocentric; the 4 th and 5 th pairs were sub metacentric; and the remaining pairs were all metacentric. One of the members of the 5 th pair presented a secondary constriction. All chromosomes presented pericentromeric heterochromatin. The large arms of the pairs 5, 9 and X presented heterochromatin. The X chromosome showed to be heteropyknotic throughout the prophase of the fi rst meiotic division. The sub phases of prophase I were atypical and meiosis II was rarely identified. Testes of all males showed a few cells; the bivalents were rod-like shaped in metaphase I. Karyological formulae were 2n = 24 + XX in females and 2n = 24 + XYp and either n = 12 + X or n = 12 + Y in males. (author)

  1. Karyotype, heterochromatin distribution and meiosis of Zabrotes subfasciatus (Bohemann) (Coleoptera: Chrysomelidae, Bruchinae)

    Energy Technology Data Exchange (ETDEWEB)

    Correa, Ronan X.; Santos, Igor S.; Silva, Janisete G.; Costa, Marco A. [Universidade Estadual de Santa Cruz, Ilheus, BA (Brazil). Dept. de Ciencias Biologicas; Pompolo, Silvia G. [Universidade Federal de Vicosa, MG (Brazil). Dept. de Biologia Geral

    2008-09-15

    Zabrotes subfasciatus (Boh.) has been extensively studied in its agronomic and biochemical aspects due to its importance as a damaging insect to leguminous grains during storage. The few cytogenetic studies published on this species yielded conflicting results. In this study, the karyotype was analyzed in order to accurately describe the chromosome C-banding patterns and meiosis. The brain ganglion at the pre pupa and the adult and pupal testes were analyzed. All individuals had 26 chromosomes in both brain ganglion and spermatogonic mitotic metaphases. These chromosomes were classified as follows: the 12{sup th} pair and the Y chromosome were telocentric; the X chromosome was acrocentric; the 4{sup th} and 5{sup th} pairs were sub metacentric; and the remaining pairs were all metacentric. One of the members of the 5{sup th} pair presented a secondary constriction. All chromosomes presented pericentromeric heterochromatin. The large arms of the pairs 5, 9 and X presented heterochromatin. The X chromosome showed to be heteropyknotic throughout the prophase of the fi rst meiotic division. The sub phases of prophase I were atypical and meiosis II was rarely identified. Testes of all males showed a few cells; the bivalents were rod-like shaped in metaphase I. Karyological formulae were 2n = 24 + XX in females and 2n = 24 + XYp and either n = 12 + X or n = 12 + Y in males. (author)

  2. Theoretical Propositions for the Citizen Formation Mediated by Technology

    Directory of Open Access Journals (Sweden)

    Tania Margarita Martínez de Padrón

    2018-02-01

    Full Text Available The research presented emerges from the connotations concerning the formation of the citizenship by the information technology and communication. Be part of the conception of the current citizen and the influence that has emerged through the utility of the various social networks, as well as Canaima education project and the guidance provided to schoolchildren in this sense. The general objective was to generate propositions theoretical that guide the formation of citizenship mediated by ICT in the primary schools of Santa Teresa del Tuy. The methodology used was the qualitative paradigm, based on the interpretative phenomenological approach of Heidegger, which is interested in discovering and understanding the meanings, habits and practices of the human being. Castle (2000: 5. The researcher approached the field object of study to observe, describe and interpret a reality. As an instrument used the interview in depth. The information obtained was recorded in pictures which allowed to comply with the development of specific objectives through triangulation. On whose findings prevailed deviating from the use of technological tools and how these have formed the formation of citizenship in school children in their behavior and actions. At the same time, allowed know from educational technological approach, that sparing the teacher provides guidance that redirect the formation of citizenship. Also was the stated objective as it was the theoretical propositions that guide the formation of citizens ICT-mediated.

  3. Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin

    International Nuclear Information System (INIS)

    Chiolo, Irene; Tang, Jonathan; Georgescu, Walter; Costes, Sylvain V.

    2013-01-01

    Repair of double strand breaks (DSBs) is essential for cell survival and genome integrity. While much is known about the molecular mechanisms involved in DSB repair and checkpoint activation, the roles of nuclear dynamics of radiation-induced foci (RIF) in DNA repair are just beginning to emerge. Here, we summarize results from recent studies that point to distinct features of these dynamics in two different chromatin environments: heterochromatin and euchromatin. We also discuss how nuclear architecture and chromatin components might control these dynamics, and the need of novel quantification methods for a better description and interpretation of these phenomena. These studies are expected to provide new biomarkers for radiation risk and new strategies for cancer detection and treatment

  4. Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin

    Energy Technology Data Exchange (ETDEWEB)

    Chiolo, Irene; Tang, Jonathan; Georgescu, Walter; Costes, Sylvain V.

    2013-10-01

    Repair of double strand breaks (DSBs) is essential for cell survival and genome integrity. While much is known about the molecular mechanisms involved in DSB repair and checkpoint activation, the roles of nuclear dynamics of radiation-induced foci (RIF) in DNA repair are just beginning to emerge. Here, we summarize results from recent studies that point to distinct features of these dynamics in two different chromatin environments: heterochromatin and euchromatin. We also discuss how nuclear architecture and chromatin components might control these dynamics, and the need of novel quantification methods for a better description and interpretation of these phenomena. These studies are expected to provide new biomarkers for radiation risk and new strategies for cancer detection and treatment.

  5. The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements

    Czech Academy of Sciences Publication Activity Database

    Evtushenko, E.V.; Levitsky, V.G.; Elisafenko, E.A.; Gunbin, K.V.; Belousov, A.I.; Šafář, Jan; Doležel, Jaroslav; Vershinin, A.V.

    2016-01-01

    Roč. 17, MAY 4 (2016), s. 337 ISSN 1471-2164 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Tandem repeats * Transposable elements * Subtelomeric heterochromatin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.729, year: 2016

  6. Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins

    Energy Technology Data Exchange (ETDEWEB)

    Montoya-Durango, Diego E. [Department of Biochemistry and Molecular Biology and Center for Genetics and Molecular Medicine, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40202 (United States); Liu, Yongqing [James Graham Brown Cancer Center and Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40202 (United States); Teneng, Ivo; Kalbfleisch, Ted; Lacy, Mary E.; Steffen, Marlene C. [Department of Biochemistry and Molecular Biology and Center for Genetics and Molecular Medicine, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40202 (United States); Ramos, Kenneth S., E-mail: kenneth.ramos@louisville.edu [Department of Biochemistry and Molecular Biology and Center for Genetics and Molecular Medicine, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40202 (United States)

    2009-06-01

    Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

  7. Epigenetic control of mammalian LINE-1 retrotransposon by retinoblastoma proteins

    International Nuclear Information System (INIS)

    Montoya-Durango, Diego E.; Liu, Yongqing; Teneng, Ivo; Kalbfleisch, Ted; Lacy, Mary E.; Steffen, Marlene C.; Ramos, Kenneth S.

    2009-01-01

    Long interspersed nuclear elements (LINEs or L1 elements) are targeted for epigenetic silencing during early embryonic development and remain inactive in most cells and tissues. Here we show that E2F-Rb family complexes participate in L1 elements epigenetic regulation via nucleosomal histone modifications and recruitment of histone deacetylases (HDACs) HDAC1 and HDAC2. Our experiments demonstrated that (i) Rb and E2F interact with human and mouse L1 elements, (ii) L1 elements are deficient in both heterochromatin-associated histone marks H3 tri methyl K9 and H4 tri methyl K20 in Rb family triple knock out (Rb, p107, and p130) fibroblasts (TKO), (iii) L1 promoter exhibits increased histone H3 acetylation in the absence of HDAC1 and HDAC2 recruitment, (iv) L1 expression in TKO fibroblasts is upregulated compared to wild type counterparts, (v) L1 expression increases in the presence of the HDAC inhibitor TSA. On the basis of these findings we propose a model in which L1 sequences throughout the genome serve as centers for heterochromatin formation in an Rb family-dependent manner. As such, Rb proteins and L1 elements may play key roles in heterochromatin formation beyond pericentromeric chromosomal regions. These findings describe a novel mechanism of L1 reactivation in mammalian cells mediated by failure of corepressor protein recruitment by Rb, loss of histone epigenetic marks, heterochromatin formation, and increased histone H3 acetylation.

  8. Translocations of Chromosome End-Segments and Facultative Heterochromatin Promote Meiotic Ring Formation in Evening Primroses[W][OPEN

    Science.gov (United States)

    Golczyk, Hieronim; Massouh, Amid; Greiner, Stephan

    2014-01-01

    Due to reciprocal chromosomal translocations, many species of Oenothera (evening primrose) form permanent multichromosomal meiotic rings. However, regular bivalent pairing is also observed. Chiasmata are restricted to chromosomal ends, which makes homologous recombination virtually undetectable. Genetic diversity is achieved by changing linkage relations of chromosomes in rings and bivalents via hybridization and reciprocal translocations. Although the structural prerequisite for this system is enigmatic, whole-arm translocations are widely assumed to be the mechanistic driving force. We demonstrate that this prerequisite is genome compartmentation into two epigenetically defined chromatin fractions. The first one facultatively condenses in cycling cells into chromocenters negative both for histone H3 dimethylated at lysine 4 and for C-banding, and forms huge condensed middle chromosome regions on prophase chromosomes. Remarkably, it decondenses in differentiating cells. The second fraction is euchromatin confined to distal chromosome segments, positive for histone H3 lysine 4 dimethylation and for histone H3 lysine 27 trimethylation. The end-segments are deprived of canonical telomeres but capped with constitutive heterochromatin. This genomic organization promotes translocation breakpoints between the two chromatin fractions, thus facilitating exchanges of end-segments. We challenge the whole-arm translocation hypothesis by demonstrating why reciprocal translocations of chromosomal end-segments should strongly promote meiotic rings and evolution toward permanent translocation heterozygosity. Reshuffled end-segments, each possessing a major crossover hot spot, can furthermore explain meiotic compatibility between genomes with different translocation histories. PMID:24681616

  9. A computational model for histone mark propagation reproduces the distribution of heterochromatin in different human cell types.

    Science.gov (United States)

    Schwämmle, Veit; Jensen, Ole Nørregaard

    2013-01-01

    Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.

  10. The formation of group norms in computer-mediated communication

    NARCIS (Netherlands)

    Postmes, T; Spears, R; Lea, M

    The formation of group norms in computer-mediated communication (CMC) was examined among students who used e-mail as part of a course. A network analysis of group structures revealed that (a) content and form of communication is normative, group norms defining communication patterns within groups,

  11. Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

    Energy Technology Data Exchange (ETDEWEB)

    Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan; Koury, Mark J.; Ney, Paul A.; Chasis, Joel Anne; Grigoryev, Sergei A.

    2008-08-21

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

  12. Congenital dyserythropoiesis with intererythroblastic chromatin bridges and ultrastructurally-normal erythroblast heterochromatin: a new disorder.

    Science.gov (United States)

    Wickramasinghe, S N; Spearing, R L; Hill, G R

    1998-12-01

    Two non-anaemic subjects, a father and daughter, with a new form of congenital dyserythropoiesis are reported. The features of their disorder are: (1) an abnormal blood film with basophilic stippling of red cells and oval macrocytes, (2) various dysplastic changes in the erythroblasts, including internuclear chromatin bridges, (3) ultrastructurally-normal erythroblast heterochromatin, (4) normal serum thymidine kinase activity, and (5) a probable autosomal dominant inheritance. The last three features distinguish this disorder from CDA type I.

  13. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

    Directory of Open Access Journals (Sweden)

    Enders Greg H

    2010-06-01

    Full Text Available Abstract Background Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF, that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts. Results We show that mouse embryo fibroblasts (MEFs and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells. Conclusions In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types to become immortalized and transformed, compared to human cells.

  14. Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA

    NARCIS (Netherlands)

    Camacho, Oscar Velazquez; Galan, Carmen; Swist-Rosowska, Kalina; Ching, Reagan; Gamalinda, Michael; Karabiber, Fethullah; La Rosa-Velazquez, De Inti; Engist, Bettina; Koschorz, Birgit; Shukeir, Nicholas; Onishi-Seebacher, Megumi; De Nobelen, Van Suzanne; Jenuwein, Thomas

    2017-01-01

    The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity

  15. Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

    Science.gov (United States)

    Matsubara, Kazumi; Uno, Yoshinobu; Srikulnath, Kornsorn; Seki, Risako; Nishida, Chizuko; Matsuda, Yoichi

    2015-12-01

    Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

  16. Methyl CpG–binding proteins induce large-scale chromatin reorganization during terminal differentiation

    Science.gov (United States)

    Brero, Alessandro; Easwaran, Hariharan P.; Nowak, Danny; Grunewald, Ingrid; Cremer, Thomas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2005-01-01

    Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG–binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG–binding domain (MBD) only. Similar to MeCP2, another methyl CpG–binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation. PMID:15939760

  17. The Mediating Role of Psychosocial Benefits in the Satisfaction Formation Process

    NARCIS (Netherlands)

    O. Prevo; P. Leunisse; H. Roest (Henk)

    1999-01-01

    textabstractIn this paper we investigate the mediating role of psychosocial benefits in the customer satisfaction/dissatisfaction formation process. Most research on this subject deals with the causality direction of psychosocial benefits and satisfaction, sometimes preceded by an overall functional

  18. Age-Associated Decrease of the Histone Methyltransferase SUV39H1 in HSC Perturbs Heterochromatin and B Lymphoid Differentiation

    Directory of Open Access Journals (Sweden)

    Dounia Djeghloul

    2016-06-01

    Full Text Available The capacity of hematopoietic stem cells (HSC to generate B lymphocytes declines with age, contributing to impaired immune function in the elderly. Here we show that the histone methyltransferase SUV39H1 plays an important role in human B lymphoid differentiation and that expression of SUV39H1 decreases with age in both human and mouse HSC, leading to a global reduction in H3K9 trimethylation and perturbed heterochromatin function. Further, we demonstrate that SUV39H1 is a target of microRNA miR-125b, a known regulator of HSC function, and that expression of miR-125b increases with age in human HSC. Overexpression of miR-125b and inhibition of SUV39H1 in young HSC induced loss of B cell potential. Conversely, both inhibition of miR-125 and enforced expression of SUV39H1 improved the capacity of HSC from elderly individuals to generate B cells. Our findings highlight the importance of heterochromatin regulation in HSC aging and B lymphopoiesis.

  19. A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

    Energy Technology Data Exchange (ETDEWEB)

    Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

    2016-09-20

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

  20. Toxoplasma gondii chromodomain protein 1 binds to heterochromatin and colocalises with centromeres and telomeres at the nuclear periphery.

    Directory of Open Access Journals (Sweden)

    Mathieu Gissot

    Full Text Available BACKGROUND: Apicomplexan parasites are responsible for some of the most deadly parasitic diseases afflicting humans, including malaria and toxoplasmosis. These obligate intracellular parasites exhibit a complex life cycle and a coordinated cell cycle-dependant expression program. Their cell division is a coordinated multistep process. How this complex mechanism is organised remains poorly understood. METHODS AND FINDINGS: In this study, we provide evidence for a link between heterochromatin, cell division and the compartmentalisation of the nucleus in Toxoplasma gondii. We characterised a T. gondii chromodomain containing protein (named TgChromo1 that specifically binds to heterochromatin. Using ChIP-on-chip on a genome-wide scale, we report TgChromo1 enrichment at the peri-centromeric chromatin. In addition, we demonstrate that TgChromo1 is cell-cycle regulated and co-localised with markers of the centrocone. Through the loci-specific FISH technique for T. gondii, we confirmed that TgChromo1 occupies the same nuclear localisation as the peri-centromeric sequences. CONCLUSION: We propose that TgChromo1 may play a role in the sequestration of chromosomes at the nuclear periphery and in the process of T. gondii cell division.

  1. Heterochromatin and rDNA sites distribution in the holocentric chromosomes of Cuscuta approximata Bab. (Convolvulaceae).

    Science.gov (United States)

    Guerra, Marcelo; García, Miguel A

    2004-02-01

    Cuscuta is a widely distributed genus of holoparasitic plants. Holocentric chromosomes have been reported only in species of one of its subgenera (Cuscuta subg. Cuscuta). In this work, a representative of this subgenus, Cuscuta approximata, was investigated looking for its mitotic and meiotic chromosome behaviour and the heterochromatin distribution. The mitotic chromosomes showed neither primary constriction nor Rabl orientation whereas the meiotic ones exhibited the typical quadripartite structure characteristic of holocentrics, supporting the assumption of holocentric chromosomes as a synapomorphy of Cuscuta subg. Cuscuta. Chromosomes and interphase nuclei displayed many heterochromatic blocks that stained deeply with hematoxylin, 4',6-diamidino-2-phenylindole (DAPI), or after C banding. The banded karyotype showed terminal or subterminal bands in all chromosomes and central bands in some of them. The single pair of 45S rDNA sites was observed at the end of the largest chromosome pair, close to a DAPI band and a 5S rDNA site. Two other 5S rDNA site pairs were found, both closely associated with DAPI bands. The noteworthy giant nuclei of glandular cells of petals and ovary wall exhibited large chromocentres typical of polytenic nuclei. The chromosomal location of heterochromatin and rDNA sites and the structure of the endoreplicated nuclei of C. approximata seemed to be similar to those known in monocentric nuclei, suggesting that centromeric organization has little or no effect on chromatin organization.

  2. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    International Nuclear Information System (INIS)

    Patheja, Pooja; Sahu, Khageswar

    2017-01-01

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  3. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments

    Energy Technology Data Exchange (ETDEWEB)

    Patheja, Pooja, E-mail: pooja.patheja8@gmail.com [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India); Homi Bhabha National Institute, Training School Complex, Anushaktinagar, Mumbai 400094, Maharashtra (India); Sahu, Khageswar [Laser Biomedical Applications Section, Raja Ramanna Centre for Advanced Technology, Indore 452013, Madhya Pradesh (India)

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MφCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MφCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation.

  4. Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris)

    Czech Academy of Sciences Publication Activity Database

    Kowar, T.; Zakrzewski, F.; Macas, Jiří; Koblížková, Andrea; Viehoever, P.; Weisshaar, B.; Schmidt, T.

    2016-01-01

    Roč. 16, č. 120 (2016) ISSN 1471-2229 R&D Projects: GA ČR GBP501/12/G090 Institutional support: RVO:60077344 Keywords : Centromere * CenH3 * H3K9me2 * Heterochromatin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.964, year: 2016

  5. Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediated release of viable cytoplasmic fragments.

    Science.gov (United States)

    Patheja, Pooja; Sahu, Khageswar

    2017-06-15

    Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Madison Floyd

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also

  7. Structural aspects of the nucleolus and its associated heterochromatin in neurons

    International Nuclear Information System (INIS)

    Pessacq, T.P.; Vidal Rioja, L.A.

    1975-01-01

    The study of the structure of the nucleolus has received considerable attention in recent years. Most data have been provided by a number of electromicroscopical studies which to some extent are confirmatory of the previous opticomicroscopical findings of Estable and Sotelo (1951, 1952, 1955), Davis (1960), Altmann, Stoecker, and Thoenes (1963), O'Donell (1961), Lettre, and Siebs (1961), Bernhard, Bauer, Gropp, Haguenau and Oberling (1955) among others. In a recent work Melker (1970) points out the existence in neurons of a mammal of a particular arrangement of the nucleolus and its associated chromatin very similar to that already established in avian neurons (Pessacq 1969). The research reported here adds some new structural facts on nucleolar-heterochromatinic rapports which although recognizable in a great variety of cellular types appears with schematic clearness only in a limited number of highly specialized cells of some species. (auth.)

  8. Med5(Nut1) and Med17(Srb4) Are Direct Targets of Mediator Histone H4 Tail Interactions

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C.

    2012-01-01

    The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct. PMID:22693636

  9. Systematic Identification of Determinants for Single-Strand Annealing-Mediated Deletion Formation in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Maia Segura-Wang

    2017-10-01

    Full Text Available To ensure genomic integrity, living organisms have evolved diverse molecular processes for sensing and repairing damaged DNA. If improperly repaired, DNA damage can give rise to different types of mutations, an important class of which are genomic structural variants (SVs. In spite of their importance for phenotypic variation and genome evolution, potential contributors to SV formation in Saccharomyces cerevisiae (budding yeast, a highly tractable model organism, are not fully recognized. Here, we developed and applied a genome-wide assay to identify yeast gene knockout mutants associated with de novo deletion formation, in particular single-strand annealing (SSA-mediated deletion formation, in a systematic manner. In addition to genes previously linked to genome instability, our approach implicates novel genes involved in chromatin remodeling and meiosis in affecting the rate of SSA-mediated deletion formation in the presence or absence of stress conditions induced by DNA-damaging agents. We closely examined two candidate genes, the chromatin remodeling gene IOC4 and the meiosis-related gene MSH4, which when knocked-out resulted in gene expression alterations affecting genes involved in cell division and chromosome organization, as well as DNA repair and recombination, respectively. Our high-throughput approach facilitates the systematic identification of processes linked to the formation of a major class of genetic variation.

  10. Constraints on superoxide mediated formation of manganese oxides

    Directory of Open Access Journals (Sweden)

    Deric R. Learman

    2013-09-01

    Full Text Available Manganese (Mn oxides are among the most reactive sorbents and oxidants within the environment, where they play a central role in the cycling of nutrients, metals, and carbon. Recent discoveries have identified superoxide (O2- (both of biogenic and abiogenic origin as an effective oxidant of Mn(II leading to the formation of Mn oxides. Here we examined the conditions under which abiotically produced superoxide led to oxidative precipitation of Mn and the solid-phases produced. Oxidized Mn, as both aqueous Mn(III and Mn(III/IV oxides, was only observed in the presence of active catalase, indicating that hydrogen peroxide, a product of the reaction of O2- with Mn(II, inhibits the oxidation process presumably through the reduction of Mn(III. Citrate and pyrophosphate increased the yield of oxidized Mn but decreased the amount of Mn oxide produced via formation of Mn(III-ligand complexes. While complexing ligands played a role in stabilizing Mn(III, they did not eliminate the inhibition of net Mn(III formation by H2O2. The Mn oxides precipitated were highly disordered colloidal hexagonal birnessite, similar to those produced by biotically generated superoxide. Yet, in contrast to the large particulate Mn oxides formed by biogenic superoxide, abiotic Mn oxides did not ripen to larger, more crystalline phases. This suggests that the deposition of crystalline Mn oxides within the environment requires a biological, or at least organic, influence. This work provides the first direct evidence that, under conditions relevant to natural waters, oxidation of Mn(II by superoxide can occur and lead to formation of Mn oxides. For organisms that oxidize Mn(II by producing superoxide, these findings may also point to other microbially mediated processes, in particular enzymatic hydrogen peroxide degradation and/or production of organic ligand metabolites, that allow for Mn oxide formation.

  11. Exon resequencing of H3K9 methyltransferase complex genes, EHMT1, EHTM2 and WIZ, in Japanese autism subjects

    OpenAIRE

    Balan, Shabeesh; Iwayama, Yoshimi; Maekawa, Motoko; Toyota, Tomoko; Ohnishi, Tetsuo; Toyoshima, Manabu; Shimamoto, Chie; Esaki, Kayoko; Yamada, Kazuo; Iwata, Yasuhide; Suzuki, Katsuaki; Ide, Masayuki; Ota, Motonori; Fukuchi, Satoshi; Tsujii, Masatsugu

    2014-01-01

    Background Histone H3 methylation at lysine 9 (H3K9) is a conserved epigenetic signal, mediating heterochromatin formation by trimethylation, and transcriptional silencing by dimethylation. Defective GLP (Ehmt1) and G9a (Ehmt2) histone lysine methyltransferases, involved in mono and dimethylation of H3K9, confer autistic phenotypes and behavioral abnormalities in animal models. Moreover, EHMT1 loss of function results in Kleefstra syndrome, characterized by severe intellectual disability, dev...

  12. Euchromatin islands in large heterochromatin domains are enriched for CTCF binding and differentially DNA-methylated regions

    Directory of Open Access Journals (Sweden)

    Wen Bo

    2012-10-01

    Full Text Available Abstract Background The organization of higher order chromatin is an emerging epigenetic mechanism for understanding development and disease. We and others have previously observed dynamic changes during differentiation and oncogenesis in large heterochromatin domains such as Large Organized Chromatin K (lysine modifications (LOCKs, of histone H3 lysine-9 dimethylation (H3K9me2 or other repressive histone posttranslational modifications. The microstructure of these regions has not previously been explored. Results We analyzed the genome-wide distribution of H3K9me2 in two human pluripotent stem cell lines and three differentiated cells lines. We identified > 2,500 small regions with very low H3K9me2 signals in the body of LOCKs, which were termed as euchromatin islands (EIs. EIs are 6.5-fold enriched for DNase I Hypersensitive Sites and 8-fold enriched for the binding of CTCF, the major organizer of higher-order chromatin. Furthermore, EIs are 2–6 fold enriched for differentially DNA-methylated regions associated with tissue types (T-DMRs, reprogramming (R-DMRs and cancer (C-DMRs. Gene ontology (GO analysis suggests that EI-associated genes are functionally related to organ system development, cell adhesion and cell differentiation. Conclusions We identify the existence of EIs as a finer layer of epigenomic architecture within large heterochromatin domains. Their enrichment for CTCF sites and DNAse hypersensitive sites, as well as association with DMRs, suggest that EIs play an important role in normal epigenomic architecture and its disruption in disease.

  13. The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin

    Science.gov (United States)

    Papait, Roberto; Pistore, Christian; Grazini, Ursula; Babbio, Federica; Cogliati, Sara; Pecoraro, Daniela; Brino, Laurent; Morand, Anne-Laure; Dechampesme, Anne-Marie; Spada, Fabio; Leonhardt, Heinrich; McBlane, Fraser; Oudet, Pierre

    2008-01-01

    Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH. PMID:18508923

  14. Loss of maternal ATRX results in centromere instability and aneuploidy in the mammalian oocyte and pre-implantation embryo.

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    Claudia Baumann

    2010-09-01

    Full Text Available The α-thalassemia/mental retardation X-linked protein (ATRX is a chromatin-remodeling factor known to regulate DNA methylation at repetitive sequences of the human genome. We have previously demonstrated that ATRX binds to pericentric heterochromatin domains in mouse oocytes at the metaphase II stage where it is involved in mediating chromosome alignment at the meiotic spindle. However, the role of ATRX in the functional differentiation of chromatin structure during meiosis is not known. To test ATRX function in the germ line, we developed an oocyte-specific transgenic RNAi knockdown mouse model. Our results demonstrate that ATRX is required for heterochromatin formation and maintenance of chromosome stability during meiosis. During prophase I arrest, ATRX is necessary to recruit the transcriptional regulator DAXX (death domain associated protein to pericentric heterochromatin. At the metaphase II stage, transgenic ATRX-RNAi oocytes exhibit abnormal chromosome morphology associated with reduced phosphorylation of histone 3 at serine 10 as well as chromosome segregation defects leading to aneuploidy and severely reduced fertility. Notably, a large proportion of ATRX-depleted oocytes and 1-cell stage embryos exhibit chromosome fragments and centromeric DNA-containing micronuclei. Our results provide novel evidence indicating that ATRX is required for centromere stability and the epigenetic control of heterochromatin function during meiosis and the transition to the first mitosis.

  15. Sulphur bacteria mediated formation of Palaeoproterozoic phosphorites

    Science.gov (United States)

    Joosu, Lauri; Lepland, Aivo; Kirsimäe, Kalle

    2014-05-01

    Modern phosphorite formation is typically associated with high productivity in upwelling areas where apatite (Ca-phosphate) precipitation is mediated by sulphur oxidising bacteria [1]. They inhabit the oxic/anoxic interface within the upper few centimetres of sediment column, accumulating phosphate in their cells under oxic conditions and releasing it rapidly when conditions become anoxic. Sulphur bacteria are known to live in close association with a consortium of anaerobic methane oxidising archaea and syntrophic sulphate-reducing bacteria. Paleoproterozoic, c. 2.0 Ga Zaonega Formation in Karelia, Russia contains several P-rich intervals in the upper part of 1500 m thick succession of organic-rich sedimentary rocks interlayered with mafic tuffs and lavas. Apatite in these P-rich intervals forms impure laminae, lenses and round-oval nodules which diameters typically range from 300 to 1000 μm. Individual apatite particles in P-rich laminae and nodules commonly occur as cylinders that are 1-8 μm long and have diameters of 0.5-4 μm. Cross-sections of best preserved cylindrical apatite particles reveal a thin outer rim whereas the internal parts consist of small anhedral elongated crystallites, intergrown with carbonaceous material. During recrystallization the outer rim thickens towards interior and cylinders may attain hexagonal crystal habit, but their size and shape remains largely unchanged [2]. The sizes of Zaonega nodules are similar to giant sulphide-oxidising bacteria known from modern and ancient settings [3, 4]. Individual apatite cylinders and aggregates have shapes and sizes similar to the methanotrophic archaea that inhabit microbial mats in modern seep/vent areas where they operate in close associations with sulphur-oxidising microbial communities [5]. Seep/vent influence during the Zaonega phosphogenesis is indicated by variable, though positive Eu anomaly, expected in magmatically active sedimentary environment experiencing several lava flows

  16. The histone H3K9 methylation and RNAi pathways regulate normalnucleolar and repeated DNA organization by inhibiting formation ofextrachromosomal DNAs

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jamy C.; Karpen, Gary H.

    2006-06-15

    In order to identify regulators of nuclear organization, Drosophila mutants in the Su(var)3-9 histone H3K9 methyltransferase, RNAi pathway components, and other regulators of heterochromatin-mediated gene silencing were examined for altered nucleoli and positioning of repeated DNAs. Animals lacking components of the H3K9 methylation and RNAi pathways contained disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared to wild type. We also observed a substantial increase in extrachromosomal repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 (Lig4), and ecc DNA formation is not induced by removal of cohesin. We conclude that H3K9 methylation of rDNA and satellites, maintained by Su(var)3-9, HP1, and the RNAi pathway, is necessary for the structural stability of repeated DNAs, which is mediated through suppression of non-homologous end joining (NHEJ). These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.

  17. Med5(Nut1 and Med17(Srb4 are direct targets of mediator histone H4 tail interactions.

    Directory of Open Access Journals (Sweden)

    Zhongle Liu

    Full Text Available The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. In addition to its canonical role in transcriptional activation, recent studies have demonstrated that S. cerevisiae Mediator can interact directly with nucleosomes, and their histone tails. Mutations in Mediator subunits have shown that Mediator and certain chromatin structures mutually impact each other structurally and functionally in vivo. We have taken a UV photo cross-linking approach to further delineate the molecular basis of Mediator chromatin interactions and help determine whether the impact of certain Mediator mutants on chromatin is direct. Specifically, by using histone tail peptides substituted with an amino acid analog that is a UV activatible crosslinker, we have identified specific subunits within Mediator that participate in histone tail interactions. Using Mediator purified from mutant yeast strains we have evaluated the impact of these subunits on histone tail binding. This analysis has identified the Med5 subunit of Mediator as a target for histone tail interactions and suggests that the previously observed effect of med5 mutations on telomeric heterochromatin and silencing is direct.

  18. Male and female meiosis in the mountain scorpion Zabius fuscus (Scorpiones, Buthidae): heterochromatin, rDNA and TTAGG telomeric repeats.

    Science.gov (United States)

    Adilardi, Renzo Sebastián; Ojanguren-Affilastro, Andrés Alejandro; Mattoni, Camilo Iván; Mola, Liliana María

    2015-08-01

    All cytogenetically studied scorpions present male achiasmatic meiosis and lack heteromorphic sex chromosomes. In contrast, information about female meiosis in scorpions is scarce due to the difficulty of finding meiotic cells. The genus Zabius includes three described species and no chromosome studies have been performed on it until now. We analyzed the constitutive heterochromatin distribution, NORs and telomeric sequences in mitosis and meiosis of males and females of different populations of Zabius fuscus. All specimens presented 2n = 18 holokinetic chromosomes that gradually decreased in size. Male meiosis presented nine bivalents and a polymorphism for one reciprocal translocation in one population. Telomeric signals were detected at every terminal region, confirming also the presence of a (TTAGG) n motif in Buthidae. Constitutive heterochromatin was found in three chromosome pairs at a terminal region; moreover, NORs were embedded in the heterochromatic region of the largest pair. Chromosome size and landmarks allowed us to propose the chromosomes involved in the rearrangement. In four females, cells at different prophase I stages were analyzed. We describe a diffuse stage and the presence of ring-shaped bivalents. We discuss the possible origin of these bivalents in the framework of chiasmatic or achiasmatic female meiosis. These results contribute to increase the scarce evidence of female meiosis in scorpions and raise new questions about its mechanism.

  19. Copy and paste: the impact of a new non-L1 retroposon on the gonosomal heterochromatin of Microtus agrestis.

    Science.gov (United States)

    Neitzel, H; Kalscheuer, V; Singh, A P; Henschel, S; Sperling, K

    2002-01-01

    Mobile elements are most abundant in the mammalian genome, comprising at least 40-50% of the DNA. They are differentiated into two most prominent families: the LINE elements, which are preferentially located in the G-bands, and SINES, which are clustered in the R-bands. We report here a novel mammalian non-L1-retroposon, which invaded the genome of Microtus agrestis in a very short time from an evolutionary viewpoint. No relevant sequence homology could be demonstrated to known sequences in the NCBI database. However, cross-hybridizing sequences exist in the genomes of all other Microtus species analyzed, but not in Mus musculus, indicating the recent evolutionary origin of this element. This retroposon is enriched in the entire heterochromatin of the X and Y chromosomes, but is also interspersed in autosomal locations in euchromatic portions of the genome. We show that the retroposon is heavily transcribed from the heterochromatin during female meiosis prerequisite for the subsequent retrotransposition. The estimated rate of retrotransposition is at least 1-2 x 10(-2) per generation, which is hundred-fold higher than that of the majority of invertebrate retroposons and also higher than the transposition rate of a murine L1 element, which was calculated to be 3 x 10(-3) per generation. Copyright 2002 S. Karger AG, Basel

  20. CD14 is a key mediator of both lysophosphatidic acid and lipopolysaccharide induction of foam cell formation.

    Science.gov (United States)

    An, Dong; Hao, Feng; Zhang, Fuqiang; Kong, Wei; Chun, Jerold; Xu, Xuemin; Cui, Mei-Zhen

    2017-09-01

    Macrophage uptake of oxidized low-density lipoprotein (oxLDL) plays an important role in foam cell formation and the pathogenesis of atherosclerosis. We report here that lysophosphatidic acid (LPA) enhances lipopolysaccharide (LPS)-induced oxLDL uptake in macrophages. Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages. The role of CD14, one component of the LPS receptor cluster, in LPA-induced biological functions has been unknown. We took several steps to examine the role of CD14 in LPA signaling pathways. Knockdown of CD14 expression nearly completely blocked LPA/LPS-induced oxLDL uptake in macrophages, demonstrating for the first time that CD14 is a key mediator responsible for both LPA- and LPS-induced oxLDL uptake/foam cell formation. To determine the molecular mechanism mediating CD14 function, we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I (SR-AI), which has been implicated in lipid uptake process, and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression. We further showed that the SR-AI-specific antibody, which quenches SR-AI function, blocked LPA- and LPS-induced foam cell formation. Thus, SR-AI is the downstream mediator of CD14 in regulating LPA-, LPS-, and LPA/LPS-induced foam cell formation. Taken together, our results provide the first experimental evidence that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation. The LPA/LPS-CD14-SR-AI nexus might be the new convergent pathway, contributing to the worsening of atherosclerosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae

    Directory of Open Access Journals (Sweden)

    Juliano S. Cabral

    2006-01-01

    Full Text Available We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana in regard to the position of heterochromatin blocks as revealed using chromomycin A3 (CMA and 4'-6-diamidino-2-phenylindole (DAPI fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH. The species showed differences in chromosome number and a diversified pattern of CMA+ and DAPI+ bands, including heteromorphism for CMA+ bands. The 5S and 45S rDNA sites also varied in number and most of them were co-localized with CMA+ bands. The relationship between 5S rDNA sites and CMA+ bands was more evident in M. notylioglossa, in which the brighter CMA+ bands were associated with large 5S rDNA sites. However, not all 5S and 45S rDNA sites were co-localized with CMA+ bands, probably due to technical constraints. We compare these results to banding data from other species and suggest that not all blocks of tandemly repetitive sequences, such as 5S rDNA sites, can be observed as heterochromatin blocks.

  2. Neutral lipids associated with haemozoin mediate efficient and rapid β-haematin formation at physiological pH, temperature and ionic composition

    Directory of Open Access Journals (Sweden)

    Ambele Melvin A

    2012-10-01

    Full Text Available Abstract Background The malaria parasite disposes of host-derived ferrihaem (iron(IIIprotoporphyrin IX, Fe(IIIPPIX by conversion to crystalline haemozoin in close association with neutral lipids. Lipids mediate synthetic haemozoin (β-haematin formation very efficiently. However, the effect on reaction rates of concentrations of lipid, Fe(IIIPPIX and physiologically relevant ions and biomolecules are unknown. Methods Lipid emulsions containing Fe(IIIPPIX were prepared in aqueous medium (pH 4.8, 37°C to mediate β-haematin formation. The reaction was quenched at various times and free Fe(IIIPPIX measured colorimetrically as a pyridine complex and the kinetics and yields analysed. Products were also characterized by FTIR, TEM and electron diffraction. Autofluorescence was also used to monitor β-haematin formation by confocal microscopy. Results At fixed Fe(IIIPPIX concentration, β-haematin yields remained constant with decreasing lipid concentration until a cut-off ratio was reached whereupon efficiency decreased dramatically. For the haemozoin-associated neutral lipid blend (NLB and monopalmitoylglycerol (MPG, this occurred below a lipid/Fe(IIIPPIX (L/H ratio of 0.54. Rate constants were found to increase with L/H ratio above the cut-off. At 16 μM MPG, Fe(IIIPPIX concentration could be raised until the L/H ratio reached the same ratio before a sudden decline in yield was observed. MPG-mediated β-haematin formation was relatively insensitive to biologically relevant cations (Na+, K+, Mg2+, Ca2+, or anions (H2PO4−, HCO3−, ATP, 2,3-diphosphoglycerate, glutathione. Confocal microscopy demonstrated β-haematin formation occurs in association with the lipid particles. Conclusions Kinetics of β-haematin formation have shown that haemozoin-associated neutral lipids alone are capable of mediating β-haematin formation at adequate rates under physiologically realistic conditions of ion concentrations to account for haemozoin formation.

  3. Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

    Science.gov (United States)

    Fornero, Christy; Suo, Bangxia; Zahde, Mais; Juveland, Katelyn; Kirik, Viktor

    2017-11-01

    Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

  4. A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

    Science.gov (United States)

    Anosova, Irina; Melnik, Svitlana; Tripsianes, Konstantinos; Kateb, Fatiha; Grummt, Ingrid; Sattler, Michael

    2015-05-26

    The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Free radical mediated formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters.

    Science.gov (United States)

    Zhang, Xiaowei; Gao, Boyan; Qin, Fang; Shi, Haiming; Jiang, Yuangrong; Xu, Xuebing; Yu, Liangli Lucy

    2013-03-13

    The present study was conducted to test the hypothesis that a free radical was formed and mediated the formation of 3-monochloropropanediol (3-MCPD) fatty acid diesters, a group of food contaminants, from diacylglycerols at high temperature under a low-moisture condition for the first time. The presence of free radicals in a vegetable oil kept at 120 °C for 20 min was demonstrated using an electron spin resonance (ESR) spectroscopy examination with 5,5-dimethylpyrroline-N-oxide (DMPO) as the spin trap agent. ESR investigation also showed an association between thermal treatment degree and the concentration of free radicals. A Fourier transform infrared spectroscopy (FT-IR) analysis of sn-1,2-stearoylglycerol (DSG) at 25 and 120 °C suggested the possible involvement of an ester carbonyl group in forming 3-MCPD diesters. On the basis of these results, a novel free radical mediated chemical mechanism was proposed for 3-MCPD diester formation. Furthermore, a quadrupole-time of flight (Q-TOF) MS/MS investigation was performed and detected the DMPO adducts with the cyclic acyloxonium free radical (CAFR) and its product MS ions, proving the presence of CAFR. Furthermore, the free radical mechanism was validated by the formation of 3-MCPD diesters through reacting DSG with a number of organic and inorganic chlorine sources including chlorine gas at 120 and 240 °C. The findings of this study might lead to the improvement of oil and food processing conditions to reduce the level of 3-MCPD diesters in foods and enhance food safety.

  6. Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

    Science.gov (United States)

    Mendez-Bermudez, Aaron; Lototska, Liudmyla; Bauwens, Serge; Giraud-Panis, Marie-Josèphe; Croce, Olivier; Jamet, Karine; Irizar, Agurtzane; Mowinckel, Macarena; Koundrioukoff, Stephane; Nottet, Nicolas; Almouzni, Genevieve; Teulade-Fichou, Mare-Paule; Schertzer, Michael; Perderiset, Mylène; Londoño-Vallejo, Arturo; Debatisse, Michelle; Gilson, Eric; Ye, Jing

    2018-05-03

    Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings

    OpenAIRE

    Bharti, Niharika; Bhatla, Satish C

    2015-01-01

    Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The...

  8. Contact in Adoption and Adoptive Identity Formation: The Mediating Role of Family Conversation

    Science.gov (United States)

    Korff, Lynn Von; Grotevant, Harold D.

    2012-01-01

    The present study examined adoption-related family conversation as a mediator of the association between adoptive parents’ facilitation of contact with birth relatives and adolescent adoptive identity formation. The sample consisted of 184 adoptive families. Data were collected in two waves from adoptive mothers and fathers, and adoptees (M = 15.68 years at adolescence; M = 24.95 years at emerging adulthood) using semistructured interviews and questionnaires. Structural equation models showed a good fit to sample data, and analyses supported the hypothesized mediation model. Contact with birth relatives is associated with more frequent adoption-related family conversation, which in turn is associated with the development of adoptive identity. These results highlight the importance of supporting activities such as contact that lead to adoption-related family conversation. PMID:21517175

  9. Menadione induces the formation of reactive oxygen species and depletion of GSH-mediated apoptosis and inhibits the FAK-mediated cell invasion.

    Science.gov (United States)

    Kim, Yun Jeong; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Chung Soo

    2014-09-01

    Menadione induces apoptosis in tumor cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to menadione is not clear. In addition, it is unclear whether menadione-induced apoptosis is mediated by the depletion of glutathione (GSH) contents that is associated with the formation of reactive oxygen species. Furthermore, the effect of menadione on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of menadione exposure on apoptosis, cell adhesion, and cell migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that menadione may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of menadione appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Menadione inhibited fetal-bovine-serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase (FAK)-dependent activation of cytoskeletal-associated components. Therefore, menadione might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy.

  10. Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

    Directory of Open Access Journals (Sweden)

    Rachel Deplus

    2014-08-01

    Full Text Available DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.

  11. Heterochromatin diversity and its co-localization with 5S and 45S rDNA sites in chromosomes of four Maxillaria species (Orchidaceae)

    OpenAIRE

    Cabral, Juliano S.; Felix, Leonardo P.; Guerra, Marcelo

    2006-01-01

    We investigated four orchids of the genus Maxillaria (M. discolor, M. acicularis, M. notylioglossa and M. desvauxiana) in regard to the position of heterochromatin blocks as revealed using chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) fluorochrome staining and 5S and 45S rDNA sites using fluorescence in situ hybridization (FISH). The species showed differences in chromosome number and a diversified pattern of CMA+ and DAPI+ bands, including heteromorphism for CMA+ bands. The 5...

  12. Contact in Adoption and Adoptive Identity Formation: The Mediating Role of Family Conversation

    OpenAIRE

    Korff, Lynn Von; Grotevant, Harold D.

    2011-01-01

    The present study examined adoption-related family conversation as a mediator of the association between adoptive parents’ facilitation of contact with birth relatives and adolescent adoptive identity formation. The sample consisted of 184 adoptive families. Data were collected in two waves from adoptive mothers and fathers, and adoptees (M = 15.68 years at adolescence; M = 24.95 years at emerging adulthood) using semistructured interviews and questionnaires. Structural equation models showed...

  13. A general approach for controlling transcription and probing epigenetic mechanisms: application to the CD4 locus.

    Science.gov (United States)

    Wan, Mimi; Kaundal, Ravinder; Huang, Haichang; Zhao, Jiugang; Yang, Xiaojun; Chaiyachati, Barbara H; Li, Sicong; Chi, Tian

    2013-01-15

    Synthetic regulatory proteins such as tetracycline (tet)-controlled transcription factors are potentially useful for repression as well as ectopic activation of endogenous genes and also for probing their regulatory mechanisms, which would offer a versatile genetic tool advantageous over conventional gene targeting methods. In this study, we provide evidence supporting this concept using Cd4 as a model. CD4 is expressed in double-positive and CD4 cells but irreversibly silenced in CD8 cells. The silencing is mediated by heterochromatin established during CD8 lineage development via transient action of the Cd4 silencer; once established, the heterochromatin becomes self-perpetuating independently of the Cd4 silencer. Using a tet-sensitive Cd4 allele harboring a removable Cd4 silencer, we found that a tet-controlled repressor recapitulated the phenotype of Cd4-deficient mice, inhibited Cd4 expression in a reversible and dose-dependent manner, and could surprisingly replace the Cd4 silencer to induce irreversible Cd4 silencing in CD8 cells, thus suggesting the Cd4 silencer is not the (only) determinant of heterochromatin formation. In contrast, a tet-controlled activator reversibly disrupted Cd4 silencing in CD8 cells. The Cd4 silencer impeded this disruption but was not essential for its reversal, which revealed a continuous role of the silencer in mature CD8 cells while exposing a remarkable intrinsic self-regenerative ability of heterochromatin after forced disruption. These data demonstrate an effective approach for gene manipulation and provide insights into the epigenetic Cd4 regulatory mechanisms that are otherwise difficult to obtain.

  14. Contact in adoption and adoptive identity formation: the mediating role of family conversation.

    Science.gov (United States)

    Von Korff, Lynn; Grotevant, Harold D

    2011-06-01

    The present study examined adoption-related family conversation as a mediator of the association between adoptive parents' facilitation of contact with birth relatives and adolescent adoptive identity formation. The sample consisted of 184 adoptive families. Data were collected in two waves from adoptive mothers and fathers, and adoptees (M = 15.68 years at adolescence; M = 24.95 years at emerging adulthood) using semistructured interviews and questionnaires. Structural equation models showed a good fit to sample data, and analyses supported the hypothesized mediation model. Contact with birth relatives is associated with more frequent adoption-related family conversation, which in turn is associated with the development of adoptive identity. These results highlight the importance of supporting activities such as contact that lead to adoption-related family conversation. 2011 APA, all rights reserved

  15. Male meiosis, heterochromatin characterization and chromosomal location of rDNA in Microtomus lunifer (Berg, 1900 (Hemiptera: Reduviidae: Hammacerinae

    Directory of Open Access Journals (Sweden)

    María Poggio

    2011-05-01

    Full Text Available In the present work, we analysed the male meiosis, the content and distribution of heterochromatin and the number and location of nucleolus organizing regions in Microtomus lunifer (Berg, 1900 by means of standard technique, C- and fluorescent bandings, and fluorescent in situ hybridization with an 18S rDNA probe. This species is the second one cytogenetically analysed within the Hammacerinae. Its male diploid chromosome number is 31 (2n=28+X1X2Y, including a minute pair of m-chromosomes. The diploid autosomal number and the presence of m-chromosomes are similar to those reported in M. conspicillaris (Drury, 1782 (2n=28+XY. However, M. lunifer has a multiple sex chromosome system X1X2Y (male that could have originated by fragmentation of the ancestral X chromosome. Taking into account that M. conspicillaris and M. lunifer are the only two species within Reduviidae that possess m-chromosomes, the presence of this pair could be a synapomorphy for the species of this genus. C- and fluorescent bandings showed that the amount of heterochromatin in M. lunifer was small, and only a small CMA3 bright band was observed in the largest autosomal pair at one terminal region. FISH with the 18S rDNA probe demonstrated that ribosomal genes were terminally placed on the largest autosomal pair. Our present results led us to propose that the location of rDNA genes could be associated with variants  of the sex chromosome systems in relation with a kind of the sex chromosome systems within this family. Furthermore, the terminal location of NOR in the largest autosomal pair allowed us to use it as a chromosome marker and, thus, to infer that the kinetic activity of both ends is not a random process, and there is an inversion of this activity.

  16. Unique ζ-chain motifs mediate a direct TCR-actin linkage critical for immunological synapse formation and T-cell activation.

    Science.gov (United States)

    Klieger, Yair; Almogi-Hazan, Osnat; Ish-Shalom, Eliran; Pato, Aviad; Pauker, Maor H; Barda-Saad, Mira; Wang, Lynn; Baniyash, Michal

    2014-01-01

    TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Otani, Satoshi [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Kakinuma, Sei, E-mail: skakinuma.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Kamiya, Akihide [Institute of Innovative Science and Technology, Tokai University, Isehara (Japan); Goto, Fumio; Kaneko, Shun; Miyoshi, Masato; Tsunoda, Tomoyuki; Asano, Yu; Kawai-Kitahata, Fukiko; Nitta, Sayuri; Nakata, Toru; Okamoto, Ryuichi; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Asahina, Yasuhiro [Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo (Japan); Department for Liver Disease Control, Tokyo Medical and Dental University, Tokyo (Japan); Yamaguchi, Tomoyuki [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Koshikawa, Naohiko [Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); Seiki, Motoharu [Medical School, Kanazawa University, Kanazawa (Japan); Nakauchi, Hiromitsu [Division of Stem Cell Therapy, Institute of Medical Science, The University of Tokyo, Tokyo (Japan); and others

    2016-01-22

    Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14–deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14–deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14–deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14–deficient livers. We demonstrate that MMP-14–mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes. - Highlights: • Loss of MMP-14 delayed formation of bile duct-like structures in perinatal liver. • Overexpression of MMP-14 in hepatobalsts promoted the biliary formation in vitro. • Loss of MMP-14 promoted hepatocyte maturation of hepatoblasts in vivo. • MMP-14–mediated signaling regulates terminal differentiation of

  18. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner, and follow expression of p16 (ink4a)

    Czech Academy of Sciences Publication Activity Database

    Košař, Martin; Bartkova, J.; Hubáčková, Soňa; Hodný, Zdeněk; Lukas, J.; Bartek, Jiří

    2011-01-01

    Roč. 10, č. 3 (2011), s. 457-468 ISSN 1538-4101 R&D Projects: GA ČR GA204/08/1418; GA ČR GA301/08/0353 Institutional research plan: CEZ:AV0Z50520514 Keywords : genotoxic and replicative stress * senescence-associated heterochromatin foci * DNA damage response Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.359, year: 2011

  19. Strain-Mediated Interfacial Dynamics during Au–PbS Core–Shell Nanostructure Formation

    KAUST Repository

    Niu, Kai-Yang; Liu, Miao; Persson, Kristin A.; Han, Yu; Zheng, Haimei

    2016-01-01

    An understanding of the hierarchical nanostructure formation is of significant importance for the design of advanced functional materials. Here, we report the in situ study of lead sulfide (PbS) growth on gold (Au) nanorod seeds using liquid cell transmission electron microscopy (TEM). By tracking the formation dynamics of Au-PbS core-shell nanoparticles, we found the preferential heterogeneous nucleation of PbS on the ends of a Au nanorod prior to the development of a complete PdS shell. During PbS shell growth, drastic sulfidation of Au nanorod was observed, leading to large volume shrinkage (up to 50%) of the initial Au nanorod seed. We also captured intriguing wavy interfacial behavior, which can be explained by our DFT calculation results that the local strain gradient at the core-shell interface facilitates the mass transport and mediates reversible phase transitions of Au ↔ Au2S during the PbS shell growth. © 2016 American Chemical Society.

  20. Strain-Mediated Interfacial Dynamics during Au–PbS Core–Shell Nanostructure Formation

    KAUST Repository

    Niu, Kai-Yang

    2016-05-23

    An understanding of the hierarchical nanostructure formation is of significant importance for the design of advanced functional materials. Here, we report the in situ study of lead sulfide (PbS) growth on gold (Au) nanorod seeds using liquid cell transmission electron microscopy (TEM). By tracking the formation dynamics of Au-PbS core-shell nanoparticles, we found the preferential heterogeneous nucleation of PbS on the ends of a Au nanorod prior to the development of a complete PdS shell. During PbS shell growth, drastic sulfidation of Au nanorod was observed, leading to large volume shrinkage (up to 50%) of the initial Au nanorod seed. We also captured intriguing wavy interfacial behavior, which can be explained by our DFT calculation results that the local strain gradient at the core-shell interface facilitates the mass transport and mediates reversible phase transitions of Au ↔ Au2S during the PbS shell growth. © 2016 American Chemical Society.

  1. Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium.

    Science.gov (United States)

    Fu, Panfeng; Ebenezer, David L; Berdyshev, Evgeny V; Bronova, Irina A; Shaaya, Mark; Harijith, Anantha; Natarajan, Viswanathan

    2016-12-30

    Hepatocyte growth factor (HGF) signaling via c-Met is known to promote endothelial cell motility and angiogenesis. We have previously reported that HGF stimulates lamellipodia formation and motility of human lung microvascular endothelial cells (HLMVECs) via PI3K/Akt signal transduction and reactive oxygen species generation. Here, we report a role for HGF-induced intracellular sphingosine-1-phosphate (S1P) generation catalyzed by sphingosine kinase 1 (SphK1), S1P transporter, spinster homolog 2 (Spns2), and S1P receptor, S1P 1 , in lamellipodia formation and perhaps motility of HLMVECs. HGF stimulated SphK1 phosphorylation and enhanced intracellular S1P levels in HLMVECs, which was blocked by inhibition of SphK1. HGF enhanced co-localization of SphK1/p-SphK1 with actin/cortactin in lamellipodia and down-regulation or inhibition of SphK1 attenuated HGF-induced lamellipodia formation in HLMVECs. In addition, down-regulation of Spns2 also suppressed HGF-induced lamellipodia formation, suggesting a key role for inside-out S1P signaling. The HGF-mediated phosphorylation of SphK1 and its localization in lamellipodia was dependent on c-Met and ERK1/2 signaling, but not the PI3K/Akt pathway; however, blocking PI3K/Akt signaling attenuated HGF-mediated phosphorylation of Spns2. Down-regulation of S1P 1 , but not S1P 2 or S1P 3 , with specific siRNA attenuated HGF-induced lamellipodia formation. Further, HGF enhanced association of Spns2 with S1P 1 that was blocked by inhibiting SphK1 activity with PF-543. Moreover, HGF-induced migration of HLMVECs was attenuated by down-regulation of Spns2 . Taken together, these results suggest that HGF/c-Met-mediated lamellipodia formation, and perhaps motility is dependent on intracellular generation of S1P via activation and localization of SphK1 to cell periphery and Spns2-mediated extracellular transportation of S1P and its inside-out signaling via S1P 1 . © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Distinct classical and molecular cytogenetics of Astyanax marionae and A. fasciatus (Characiformes: Characidae): a comparative study of the organization of heterochromatin and repetitive genes.

    Science.gov (United States)

    Piscor, Diovani; Centofante, Liano; Parise-Maltempi, Patricia Pasquali

    2017-09-01

    Genus Astyanax is well distributed in Neotropical freshwater environments and its taxonomic position is uncertain, as is the case with other Characidae genera allocated in the group incertae sedis. This study aimed to analyse the karyotype of different populations of Astyanax fasciatus (Corumbataí River basin) using Giemsa staining, C-band technique, and fluorescence in situ hybridization for the H3 histone and 5S rRNA genes, in addition we describe for the first time the chromosomal organization of H3 histone and 5S rRNAgenes in A. marionae (ParaguayRiver basin). Chromosomes of three A. fasciatus populations were analysed (two with 2n = 50 and one with 2n = 48) and the heterochromatin was organized in two forms (blocks with blurred boundaries and distinct blocks). H3 histone and 5S rRNA genes were observed in all the three populations of A. fasciatus on two chromosome pairs (one metacentric chromosome showing H3 histone and 5S rRNA gene clusters). In A. marionae (2n = 48), H3 histone and 5S rRNA genes were observed in one acrocentric chromosome pair (different pairs). Further, differences between karyotypes and heterochromatin, as well as the chromosomal organization of H3 histone and 5S rRNA genes in Astyanax species, focussing on chromosome evolution in the group are discussed.

  3. The Role of Lactate-Mediated Metabolic Coupling between Astrocytes and Neurons in Long-Term Memory Formation

    Science.gov (United States)

    Steinman, Michael Q.; Gao, Virginia; Alberini, Cristina M.

    2016-01-01

    Long-term memory formation, the ability to retain information over time about an experience, is a complex function that affects multiple behaviors, and is an integral part of an individual’s identity. In the last 50 years many scientists have focused their work on understanding the biological mechanisms underlying memory formation and processing. Molecular studies over the last three decades have mostly investigated, or given attention to, neuronal mechanisms. However, the brain is composed of different cell types that, by concerted actions, cooperate to mediate brain functions. Here, we consider some new insights that emerged from recent studies implicating astrocytic glycogen and glucose metabolisms, and particularly their coupling to neuronal functions via lactate, as an essential mechanism for long-term memory formation. PMID:26973477

  4. The role of lactate-mediated metabolic coupling between astrocytes and neurons in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Michael eSteinman

    2016-03-01

    Full Text Available Long-term memory formation, the ability to retain information over time about an experience, is a complex function that affects multiple behaviors, and is an integral part of an individual’s identity. In the last 50 years many scientists have focused their work on understanding the biological mechanisms underlying memory formation and processing. Molecular studies over the last three decades have mostly investigated, or given attention to, neuronal mechanisms. However, the brain is composed of different cell types that, by concerted actions, cooperate to mediate brain functions. Here we consider some new insights that emerged from recent studies implicating astrocytic glycogen and glucose metabolisms, and particularly their coupling to neuronal functions via lactate, as an essential mechanism for long-term memory formation.

  5. Reaction kinetic model of the surface-mediated formation of PCDD/F from pyrolysis of 2-chlorophenol on a CuP/Silica suface

    Energy Technology Data Exchange (ETDEWEB)

    Lomnicki, S.; Khachatryan, L.; Dellinger, B. [Louisiana State Univ., Baton Rouge (United States). Dept. of Chemistry

    2004-09-15

    One of the major challenges in developing predictive models of the surface mediated pollutant formation and fuel combustion is the construction of reliable reaction kinetic mechanisms and models. While the homogeneous, gas-phase chemistry of various light fuels such as hydrogen and methane is relatively well-known large uncertainties exist in the reaction paths of surface mediated reaction mechanisms for even these very simple species. To date, no detailed kinetic consideration of the surface mechanisms of formation of complex organics such as PCDD/F have been developed. In addition to the complexity of the mechanism, a major difficulty is the lack of reaction kinetic parameters (pre-exponential factor and activation energy) of surface reactions, Consequently, numerical studies of the surface-mediated formation of PCDD/F have often been incorporated only a few reactions. We report the development of a numerical multiple-step surface model based on experimental data of surface mediated (5% CuO/SiO2) conversion of 2-monochlorphenol (2-MCP) to PCDD/F under pyrolytic or oxidative conditions. A reaction kinetic model of the catalytic conversion of 2-MCP on the copper oxide catalyst under pyrolytic conditions was developed based on a detailed multistep surface reaction mechanism developed in our laboratory. The performance of the chemical model is assessed by comparing the numerical predictions with experimental measurements. SURFACE CHEMKIN (version 3.7.1) software was used for modeling. Our results confirm the validity of previously published mechanism of the reaction and provides new insight concerning the formation of PCDD/F formation in combustion processes. This model successfully explains the high yields of PCDD/F at low temperatures that cannot be explained using a purely gas-phase mode.

  6. In vitro antibiofilm efficacy of Piper betle against quorum sensing mediated biofilm formation of luminescent Vibrio harveyi.

    Science.gov (United States)

    Srinivasan, Ramanathan; Santhakumari, Sivasubramanian; Ravi, Arumugam Veera

    2017-09-01

    Vibrio harveyi is a potent biofilm former, which confers resistance to multiple antimicrobials, disinfectants, chemicals and biocides. The prevalence of biofilm mediated antibiotic resistance among aquatic bacterial pathogens stresses the search for novel alternative approach to treat vibriosis in aquaculture. Exploring suitable therapeutics from natural resources could be a novel area of research. Therefore, this work was executed to evaluate the inhibitory effect of Piper betle ethyl acetate extract (PBE) on bioluminescence production and biofilm formation of V. harveyi. Minimal inhibitory concentration (MIC) of PBE against planktonic V. harveyi was found to be 1600 μg ml -1 ; furthermore, PBE inhibited the quorum sensing (QS) mediated bioluminescence production and biofilm formation in V. harveyi upto 98 and 74% respectively, at its sub-MIC concentration of 400 μg ml -1 without affecting their cell viability. Similar results were obtained for exopolysaccharides production and swimming motility related to biofilm formation of V. harveyi, where PBE reduced EPS production upto 64%. Light and confocal laser scanning microscopic analyses further confirmed that the PBE effectively prevented the initial attachment as well as microcolonies formation of V. harveyi biofilm, when compared to their untreated controls. This study demonstrates the promising antibiofilm activity of PBE and confirms the ethnopharmacological potential of this plant against V. harveyi infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Involvement of DNA mismatch repair in the maintenance of heterochromatic DNA stability in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Basanta K Dahal

    2017-10-01

    Full Text Available Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i DNA mismatch repair (MMR is required for the maintenance of heterochromatic DNA stability, (ii MutLα (Mlh1-Pms1 heterodimer, MutSα (Msh2-Msh6 heterodimer, MutSβ (Msh2-Msh3 heterodimer, and Exo1 are involved in MMR at heterochromatin, (iii Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin.

  8. Proportionate Dwarfism in Mice Lacking Heterochromatin Protein 1 Binding Protein 3 (HP1BP3) Is Associated With Alterations in the Endocrine IGF-1 Pathway

    OpenAIRE

    Garfinkel, Benjamin P.; Arad, Shiri; Le, Phuong T.; Bustin, Michael; Rosen, Clifford J.; Gabet, Yankel; Orly, Joseph

    2015-01-01

    Heterochromatin protein 1 binding protein 3 (HP1BP3) is a recently described histone H1-related protein with roles in chromatin structure and transcriptional regulation. To explore the potential physiological role of HP1BP3, we have previously described an Hp1bp3?/? mouse model with reduced postnatal viability and growth. We now find that these mice are proportionate dwarfs, with reduction in body weight, body length, and organ weight. In addition to their small size, microcomputed tomography...

  9. Wrestling with Chromosomes: The Roles of SUMO During Meiosis.

    Science.gov (United States)

    Nottke, Amanda C; Kim, Hyun-Min; Colaiácovo, Monica P

    2017-01-01

    Meiosis is a specialized form of cell division required for the formation of haploid gametes and therefore is essential for successful sexual reproduction. Various steps are exquisitely coordinated to ensure accurate chromosome segregation during meiosis, thereby promoting the formation of haploid gametes from diploid cells. Recent studies are demonstrating that an important form of regulation during meiosis is exerted by the post-translational protein modification known as sumoylation. Here, we review and discuss the various critical steps of meiosis in which SUMO-mediated regulation has been implicated thus far. These include the maintenance of meiotic centromeric heterochromatin , meiotic DNA double-strand break repair and homologous recombination, centromeric coupling, and the assembly of a proteinaceous scaffold between homologous chromosomes known as the synaptonemal complex.

  10. RGM regulates BMP-mediated secondary axis formation in the sea anemone Nematostella vectensis.

    Science.gov (United States)

    Leclère, Lucas; Rentzsch, Fabian

    2014-12-11

    Patterning of the metazoan dorsoventral axis is mediated by a complex interplay of BMP signaling regulators. Repulsive guidance molecule (RGM) is a conserved BMP coreceptor that has not been implicated in axis specification. We show that NvRGM is a key positive regulator of BMP signaling during secondary axis establishment in the cnidarian Nematostella vectensis. NvRGM regulates first the generation and later the shape of a BMP-dependent Smad1/5/8 gradient with peak activity on the side opposite the NvBMP/NvRGM/NvChordin expression domain. Full knockdown of Smad1/5/8 signaling blocks the formation of endodermal structures, the mesenteries, and the establishment of bilateral symmetry, while altering the gradient through partial NvRGM or NvBMP knockdown shifts the boundaries of asymmetric gene expression and the positioning of the mesenteries along the secondary axis. These findings provide insight into the diversification of axis specification mechanisms and identify a previously unrecognized role for RGM in BMP-mediated axial patterning. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Mediator-dependent Nuclear Receptor Functions

    Science.gov (United States)

    Chen, Wei; Roeder, Robert

    2011-01-01

    As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

  12. Super-Resolution Localization Microscopy of γ-H2AX and Heterochromatin after Folate Deficiency.

    Science.gov (United States)

    Bach, Margund; Savini, Claudia; Krufczik, Matthias; Cremer, Christoph; Rösl, Frank; Hausmann, Michael

    2017-08-08

    Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with "high-risk" human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access

  13. Genetic interaction of two abscisic acid signaling regulators, HY5 and FIERY1, in mediating lateral root formation

    KAUST Repository

    Chen, Hao

    2011-01-01

    Root architecture is continuously shaped in a manner that helps plants to better adapt to the environment. Gene regulation at the transcriptional or post-transcriptional levels largely controls this environmental response. Recently, RNA silencing has emerged as an important player in gene regulation and is involved in many aspects of plant development, including lateral root formation. In a recent study, we found that FIERY1, a bifunctional abiotic stress and abscisic acid (ABA) signaling regulator and an endogenous RNA silencing suppressor, mediates auxin response during lateral root formation in Arabidopsis. We proposed that FRY1 regulates lateral root development through its activity on adenosine 3,5-bisphosphate (PAP), a strong inhibitor of exoribonucleases (XRNs). Interestingly, some of the phenotypes of fry1, such as enhanced response to light in repressing hypocotyl elongation and hypersensitivity to ABA in lateral root growth, are opposite to those of another light- and ABA-signaling mutant, hy5. Here we analyzed the hy5 fry1 double mutant for root and hypocotyl growth. We found that the hy5 mutation can suppress the enhanced light sensitivity in fry1 hypocotyl elongation and restore the lateral root formation. The genetic interaction between HY5 and FRY1 indicates that HY5 and FRY1 may act in overlapping pathways that mediate light signaling and lateral root development. © 2011 Landes Bioscience.

  14. Comparative Chromosome Map and Heterochromatin Features of the Gray Whale Karyotype (Cetacea).

    Science.gov (United States)

    Kulemzina, Anastasia I; Proskuryakova, Anastasia A; Beklemisheva, Violetta R; Lemskaya, Natalia A; Perelman, Polina L; Graphodatsky, Alexander S

    2016-01-01

    Cetacean karyotypes possess exceptionally stable diploid numbers and highly conserved chromosomes. To date, only toothed whales (Odontoceti) have been analyzed by comparative chromosome painting. Here, we studied the karyotype of a representative of baleen whales, the gray whale (Eschrichtius robustus, Mysticeti), by Zoo-FISH with dromedary camel and human chromosome-specific probes. We confirmed a high degree of karyotype conservation and found an identical order of syntenic segments in both branches of cetaceans. Yet, whale chromosomes harbor variable heterochromatic regions constituting up to a third of the genome due to the presence of several types of repeats. To investigate the cause of this variability, several classes of repeated DNA sequences were mapped onto chromosomes of whale species from both Mysticeti and Odontoceti. We uncovered extensive intrapopulation variability in the size of heterochromatic blocks present in homologous chromosomes among 3 individuals of the gray whale by 2-step differential chromosome staining. We show that some of the heteromorphisms observed in the gray whale karyotype are due to distinct amplification of a complex of common cetacean repeat and heavy satellite repeat on homologous autosomes. Furthermore, we demonstrate localization of the telomeric repeat in the heterochromatin of both gray and pilot whale (Globicephala melas, Odontoceti). Heterochromatic blocks in the pilot whale represent a composite of telomeric and common repeats, while heavy satellite repeat is lacking in the toothed whale consistent with previous studies. © 2016 S. Karger AG, Basel.

  15. Antimicrobial properties of Kalanchoe blossfeldiana: a focus on drug resistance with particular reference to quorum sensing-mediated bacterial biofilm formation.

    Science.gov (United States)

    Sarkar, Ratul; Mondal, Chaitali; Bera, Rammohan; Chakraborty, Sumon; Barik, Rajib; Roy, Paramita; Kumar, Alekh; Yadav, Kirendra K; Choudhury, Jayanta; Chaudhary, Sushil K; Samanta, Samir K; Karmakar, Sanmoy; Das, Satadal; Mukherjee, Pulok K; Mukherjee, Joydeep; Sen, Tuhinadri

    2015-07-01

    This study attempts to investigate the antimicrobial properties of Kalanchoe blossfeldiana with a particular reference to quorum sensing (QS)-mediated biofilm formation. The methanol extract of K. blossfeldiana leaves (MEKB) was evaluated for antimicrobial properties including QS-controlled production of biofilm (including virulence factor, motility and lactone formation) in Pseudomonas aeruginosa. Methanol extract of K. blossfeldiana was also evaluated for anti-cytokine (tumour necrosis factor-alpha, interleukin-6 and interleukin-1 beta) properties in peripheral blood mononuclear cells (PBMC). Methanol extract of K. blossfeldiana exhibited antimicrobial effect on clinical isolates, as well as standard reference strains. Pseudomonas aeruginosa exposed to MEKB (subminimum inhibitory concentration (MIC)) displayed reduced biofilm formation, whereas supra-MIC produced destruction of preformed biofilms. Methanol extract of K. blossfeldiana reduced the secretion of virulence factors (protease and pyoverdin) along with generation of acyl homoserine lactone (AHL). Confocal laser scanning microscopy images indicate reduction of biofilm thickness. The extract also reduced cytokine formation in lipopolysaccharide-stimulated PBMC. Kalanchoe blossfeldiana was found to interfere with AHL production, which in turn may be responsible for downregulating QS-mediated production of biofilm and virulence. This first report on the antibiofilm and anticytokine properties of this plant may open up new vistas for future exploration of this plant for combating biofilm-related resistant infections. © 2015 Royal Pharmaceutical Society.

  16. Electron Emission by Doppler-Mediated Formation of Doubly Excited H -** in Grazing Incidence of Protons on Clean Al(111)

    Czech Academy of Sciences Publication Activity Database

    Lörinčík, Jan; Šroubek, Zdeněk; Aumayr, H.; Winter, H. P.

    2001-01-01

    Roč. 54, č. 5 (2001), s. 633-639 ISSN 0295-5075 R&D Projects: GA AV ČR IAA1067801 Institutional research plan: CEZ:AV0Z4040901 Keywords : ion scattering from surfaces * Doppler-mediated formation * Al(111) Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.304, year: 2001

  17. Essential loci in centromeric heterochromatin of Drosophila melanogaster. I: the right arm of chromosome 2.

    Science.gov (United States)

    Coulthard, Alistair B; Alm, Christina; Cealiac, Iulia; Sinclair, Don A; Honda, Barry M; Rossi, Fabrizio; Dimitri, Patrizio; Hilliker, Arthur J

    2010-06-01

    With the most recent releases of the Drosophila melanogaster genome sequences, much of the previously absent heterochromatic sequences have now been annotated. We undertook an extensive genetic analysis of existing lethal mutations, as well as molecular mapping and sequence analysis (using a candidate gene approach) to identify as many essential genes as possible in the centromeric heterochromatin on the right arm of the second chromosome (2Rh) of D. melanogaster. We also utilized available RNA interference lines to knock down the expression of genes in 2Rh as another approach to identifying essential genes. In total, we verified the existence of eight novel essential loci in 2Rh: CG17665, CG17683, CG17684, CG17883, CG40127, CG41265, CG42595, and Atf6. Two of these essential loci, CG41265 and CG42595, are synonymous with the previously characterized loci l(2)41Ab and unextended, respectively. The genetic and molecular analysis of the previously reported locus, l(2)41Ae, revealed that this is not a single locus, but rather it is a large region of 2Rh that extends from unextended (CG42595) to CG17665 and includes four of the novel loci uncovered here.

  18. Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus.

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    Renata Stiebler

    Full Text Available Hemozoin (Hz is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus, Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM. Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE and phosphatidylcholine (uPC, with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut.

  19. Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model.

    Science.gov (United States)

    Zheng, Min; Mitra, Rajendra N; Filonov, Nazar A; Han, Zongchao

    2016-03-01

    Previously, we compared the efficacy of nanoparticle (NP)-mediated intron-containing rhodopsin (sgRho) vs. intronless cDNA in ameliorating retinal disease phenotypes in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa. We showed that NP-mediated sgRho delivery achieved long-term expression and phenotypic improvement in RKO mice, but not NP housing cDNA. However, the protein level of the NP-sgRho construct was only 5-10% of wild-type at 8 mo postinjection. To have a better understanding of the reduced levels of long-term expression of the vectors, in the present study, we evaluated the epigenetic changes of subretinal delivering NP-cDNA vs. NP-sgRho in the RKO mouse eyes. Following the administration, DNA methylation and histone status of specific regions (bacteria plasmid backbone, promoter, rhodopsin gene, and scaffold/matrix attachment region) of the vectors were evaluated at various time points. We documented that epigenetic transgene silencing occurred in vector-mediated gene transfer, which were caused by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene. No toxicity or inflammation was found in the treated eyes. Our results suggest that cDNA of the rhodopsin transgene and bacteria backbone interfered with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochromatin-associated modifications. © FASEB.

  20. Effect of Chromatin Structure on the Extent and Distribution of DNA Double Strand Breaks Produced by Ionizing Radiation; Comparative Study of hESC and Differentiated Cells Lines.

    Science.gov (United States)

    Venkatesh, Priyanka; Panyutin, Irina V; Remeeva, Evgenia; Neumann, Ronald D; Panyutin, Igor G

    2016-01-02

    Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC.

  1. Superficial Dsg2 Expression Limits Epidermal Blister Formation Mediated by Pemphigus Foliaceus Antibodies and Exfoliative Toxins

    Directory of Open Access Journals (Sweden)

    Donna Brennan

    2010-01-01

    Full Text Available Cell-cell adhesion mediated by desmosomes is crucial for maintaining proper epidermal structure and function, as evidenced by several severe and potentially fatal skin disorders involving impairment of desmosomal proteins. Pemphigus foliaceus (PF and staphylococcal scalded skin syndrome (SSSS are subcorneal blistering diseases resulting from loss of function of the desmosomal cadherin, desmoglein 1 (Dsg1. To further study the pathomechanism of these diseases and to assess the adhesive properties of Dsg2, we employed a recently established transgenic (Tg mouse model expressing Dsg2 in the superficial epidermis. Neonatal Tg and wild type (WT mice were injected with purified ETA or PF Ig. We showed that ectopic expression of Dsg2 reduced the extent of blister formation in response to both ETA and PF Ig. In response to PF Ig, we observed either a dramatic loss or a reorganization of Dsg1-α, Dsg1-β, and, to a lesser extent, Dsg1-γ, in WT mice. The Inv-Dsg2 Tg mice showed enhanced retention of Dsg1 at the cell-cell border. Collectively, our data support the role for Dsg2 in cell adhesion and suggest that ectopic superficial expression of Dsg2 can increase membrane preservation of Dsg1 and limit epidermal blister formation mediated by PF antibodies and exfoliative toxins.

  2. Formation of a new crystalline form of anhydrous β-maltose by ethanol-mediated crystal transformation.

    Science.gov (United States)

    Verhoeven, Nicolas; Neoh, Tze Loon; Ohashi, Tetsuya; Furuta, Takeshi; Kurozumi, Sayaka; Yoshii, Hidefumi

    2012-04-01

    β-Maltose monohydrate was transformed into an anhydrous form by ethanol-mediated method under several temperatures with agitation. A new stable anhydrous form of β-maltose (Mβ(s)) was obtained, as substantiated by the X-ray diffraction patterns. Mβ(s) obtained by this method presented a fine porous structure, resulting in greater specific surface area compared to those of β-maltose monohydrate and anhydrous β-maltose obtained by vacuum drying (Mβ(h)). The crystal transformation presumably consisted of two steps: dehydration reaction from the hydrous to amorphous forms and crystal formation from the amorphous forms to the noble anhydrous form. The kinetics of these reactions were determined by thermal analysis using Jander's equation and Arrhenius plots. The overall activation energies of the dehydration reaction and the formation of anhydrous maltose were evaluated to be 100 and 90 kJ/mol, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

    Science.gov (United States)

    Kalonia, Cavan K; Heinrich, Frank; Curtis, Joseph E; Raman, Sid; Miller, Maria A; Hudson, Steven D

    2018-03-05

    Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

  4. Quantitative 3D Analysis of Nuclear Morphology and Heterochromatin Organization from Whole-Mount Plant Tissue Using NucleusJ.

    Science.gov (United States)

    Desset, Sophie; Poulet, Axel; Tatout, Christophe

    2018-01-01

    Image analysis is a classical way to study nuclear organization. While nuclear organization used to be investigated by colorimetric or fluorescent labeling of DNA or specific nuclear compartments, new methods in microscopy imaging now enable qualitative and quantitative analyses of chromatin pattern, and nuclear size and shape. Several procedures have been developed to prepare samples in order to collect 3D images for the analysis of spatial chromatin organization, but only few preserve the positional information of the cell within its tissue context. Here, we describe a whole mount tissue preparation procedure coupled to DNA staining using the PicoGreen ® intercalating agent suitable for image analysis of the nucleus in living and fixed tissues. 3D Image analysis is then performed using NucleusJ, an open source ImageJ plugin, which allows for quantifying variations in nuclear morphology such as nuclear volume, sphericity, elongation, and flatness as well as in heterochromatin content and position in respect to the nuclear periphery.

  5. AtlA Mediates Extracellular DNA Release, Which Contributes to Streptococcus mutans Biofilm Formation in an Experimental Rat Model of Infective Endocarditis.

    Science.gov (United States)

    Jung, Chiau-Jing; Hsu, Ron-Bin; Shun, Chia-Tung; Hsu, Chih-Chieh; Chia, Jean-San

    2017-09-01

    Host factors, such as platelets, have been shown to enhance biofilm formation by oral commensal streptococci, inducing infective endocarditis (IE), but how bacterial components contribute to biofilm formation in vivo is still not clear. We demonstrated previously that an isogenic mutant strain of Streptococcus mutans deficient in autolysin AtlA (Δ atlA ) showed a reduced ability to cause vegetation in a rat model of bacterial endocarditis. However, the role of AtlA in bacterial biofilm formation is unclear. In this study, confocal laser scanning microscopy analysis showed that extracellular DNA (eDNA) was embedded in S. mutans GS5 floes during biofilm formation on damaged heart valves, but an Δ atlA strain could not form bacterial aggregates. Semiquantification of eDNA by PCR with bacterial 16S rRNA primers demonstrated that the Δ atlA mutant strain produced dramatically less eDNA than the wild type. Similar results were observed with in vitro biofilm models. The addition of polyanethol sulfonate, a chemical lysis inhibitor, revealed that eDNA release mediated by bacterial cell lysis is required for biofilm initiation and maturation in the wild-type strain. Supplementation of cultures with calcium ions reduced wild-type growth but increased eDNA release and biofilm mass. The effect of calcium ions on biofilm formation was abolished in Δ atlA cultures and by the addition of polyanethol sulfonate. The VicK sensor, but not CiaH, was found to be required for the induction of eDNA release or the stimulation of biofilm formation by calcium ions. These data suggest that calcium ion-regulated AtlA maturation mediates the release of eDNA by S. mutans , which contributes to biofilm formation in infective endocarditis. Copyright © 2017 American Society for Microbiology.

  6. Managing Impression Formation in Computer-Mediated Communication.

    Science.gov (United States)

    Liu, Yuliang; Ginther, Dean

    2001-01-01

    Offers suggestions for online instructors regarding verbal and nonverbal impression management. The recommendations should facilitate computer mediated teacher-student or manager-client interactions and help develop constructive relationships that promote learning and productivity. (EV)

  7. Nuclear morphology, polyploidy, and chromatin elimination in tissue culture of Allium fistulosum L.

    Directory of Open Access Journals (Sweden)

    Andrzej Joachimiak

    2011-01-01

    Full Text Available The morphology of cell nuclei in callus obtained from root-tip meristems of Allium fistulosum L. (Monocotyledoneae, Alliaceae was analysed. The most interesting phenomena observed in long-term callus culture were the different mechanisms of cell polyploidization, enlargement of telomeric segments of heterochromatin, and extensive chromatin elimination, associated with instability of nuclei size and DNA content. Protruding heterochromatin "spikes" were observed on the surface of some di- and polyploid nuclei. The presence of these spikes was connected with the formation of small heterochromatic micronuclei frequently found in the cytoplasm. It is suggested that these micronuclei are produced by direct elimination of heterochromatin from the interphase nuclei. Polyploid cells accumulated with each successive cell collection. The ploidy level attained by highly polyploid cells was 15C-220C. The shape of the nuclei and heterochromatin distribution suggest that polyploid nuclei in A. fistulosum tissue culture are produced by endoreduplication and by restitution cycles.

  8. Nucleosome Positioning and Epigenetics

    Science.gov (United States)

    Schwab, David; Bruinsma, Robijn

    2008-03-01

    The role of chromatin structure in gene regulation has recently taken center stage in the field of epigenetics, phenomena that change the phenotype without changing the DNA sequence. Recent work has also shown that nucleosomes, a complex of DNA wrapped around a histone octamer, experience a sequence dependent energy landscape due to the variation in DNA bend stiffness with sequence composition. In this talk, we consider the role nucleosome positioning might play in the formation of heterochromatin, a compact form of DNA generically responsible for gene silencing. In particular, we discuss how different patterns of nucleosome positions, periodic or random, could either facilitate or suppress heterochromatin stability and formation.

  9. Palladium- and copper-mediated N-aryl bond formation reactions for the synthesis of biological active compounds

    Directory of Open Access Journals (Sweden)

    Burkhard Koenig

    2011-01-01

    Full Text Available N-Arylated aliphatic and aromatic amines are important substituents in many biologically active compounds. In the last few years, transition-metal-mediated N-aryl bond formation has become a standard procedure for the introduction of amines into aromatic systems. While N-arylation of simple aromatic halides by simple amines works with many of the described methods in high yield, the reactions may require detailed optimization if applied to the synthesis of complex molecules with additional functional groups, such as natural products or drugs. We discuss and compare in this review the three main N-arylation methods in their application to the synthesis of biologically active compounds: Palladium-catalysed Buchwald–Hartwig-type reactions, copper-mediated Ullmann-type and Chan–Lam-type N-arylation reactions. The discussed examples show that palladium-catalysed reactions are favoured for large-scale applications and tolerate sterically demanding substituents on the coupling partners better than Chan–Lam reactions. Chan–Lam N-arylations are particularly mild and do not require additional ligands, which facilitates the work-up. However, reaction times can be very long. Ullmann- and Buchwald–Hartwig-type methods have been used in intramolecular reactions, giving access to complex ring structures. All three N-arylation methods have specific advantages and disadvantages that should be considered when selecting the reaction conditions for a desired C–N bond formation in the course of a total synthesis or drug synthesis.

  10. Karyotypes, heterochromatin, and physical mapping of 18S-26S rDNA in Cactaceae.

    Science.gov (United States)

    Las Peñas, M L; Urdampilleta, J D; Bernardello, G; Forni-Martins, E R

    2009-01-01

    Karyotype analyses in members of the four Cactaceae subfamilies were performed. Numbers and karyotype formula obtained were: Pereskioideae = Pereskiaaculeata(2n = 22; 10 m + 1 sm), Maihuenioideae = Maihuenia patagonica (2n = 22, 9 m + 2 sm; 2n = 44, 18 m + 4 sm), Opuntioideae = Cumulopuntia recurvata(2n = 44; 20 m + 2 sm), Cactoideae = Acanthocalycium spiniflorum (2n = 22; 10 m + 1 sm),Echinopsis tubiflora (2n = 22; 10 m + 1 sm), Trichocereus candicans (2n = 22, 22 m). Chromosomes were small, the average chromosome length was 2.3 mum. Diploid species and the tetraploid C. recurvata had one terminal satellite, whereas the remaining tetraploid species showed four satellited chromosomes. Karyotypes were symmetrical. No CMA(-)/DAPI(+) bands were detected, but CMA(+)/DAPI(-) bands associated with NOR were always found. Pericentromeric heterochromatin was found in C. recurvata, A. spiniflorum, and the tetraploid cytotype of M. patagonica. The locations of the 18S-26S rDNA sites in all species coincided with CMA(+)/DAPI(-) bands; the same occurred with the sizes and numbers of signals for each species. This technique was applied for the first time in metaphase chromosomes in cacti. NOR-bearing pair no.1 may be homeologous in all species examined. In Cactaceae, the 18S-26S loci seem to be highly conserved. Copyright 2009 S. Karger AG, Basel.

  11. Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs Formation and Arterial Thrombosis.

    Directory of Open Access Journals (Sweden)

    Daniella M Mizurini

    Full Text Available The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs, a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability.This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method.Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

  12. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

    Science.gov (United States)

    Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

    2016-01-01

    Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

  13. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

    Directory of Open Access Journals (Sweden)

    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  14. Competing to destroy: a fight between two RNA-degradation systems

    DEFF Research Database (Denmark)

    Thon, Genevieve

    2008-01-01

    The Argonaute-1 (Ago1) protein bound to small interfering RNAs (siRNAs) directs heterochromatin formation in fission yeast. A high-throughput sequencing approach reveals that the composition of the Ago1-bound siRNA population is sensitive to the noncanonical poly(A) polymerase Cid14, indicating t...... that the RNA-interference and Cid14-TRAMP RNA-degradation pathways compete for substrates in fission yeast.......The Argonaute-1 (Ago1) protein bound to small interfering RNAs (siRNAs) directs heterochromatin formation in fission yeast. A high-throughput sequencing approach reveals that the composition of the Ago1-bound siRNA population is sensitive to the noncanonical poly(A) polymerase Cid14, indicating...

  15. Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

    Science.gov (United States)

    Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

    2013-01-01

    Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

  16. Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.

    Science.gov (United States)

    Ikeda, Yuka; Kawai, Katsuhisa; Ikawa, Akira; Kawamoto, Kyoko; Egami, Youhei; Araki, Nobukazu

    2017-08-01

    Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup. © 2017. Published by The Company of Biologists Ltd.

  17. Mediating Dynamic Supply Chain Formation by Collaborative Single Machine Earliness/Tardiness Agents in Supply Mesh

    Directory of Open Access Journals (Sweden)

    Hang Yang

    2014-01-01

    Full Text Available Nowadays, a trend of forming dynamic supply chains with different trading partners over different e-marketplaces has emerged. These supply chains, which are called “supply mesh,” generally refer to heterogeneous electronic marketplaces in which dynamic supply chains, as per project (often make-to-order, are formed across different parties. Conceptually, in a supply mesh a dynamic supply chain is formed vertically, mediating several companies for a project. Companies that are on the same level horizontally are either competitors or cohorts. A complex scenario such as this makes it challenging to find the right group of members for a dynamic supply chain. Earlier on, a multiagent model called the collaborative single machine earliness/tardiness (CSET model was proposed for the optimal formation of make-to-order supply chains. This paper contributes the particular agent designs, for enabling the mediation of CSET in a supply mesh, and the possibilities are discussed. It is demonstrated via a computer simulation, based on samples from the U.S. textile industry, that by using intelligent agents under the CSET model it is possible to automatically find an ideal group of trading partners from a supply mesh.

  18. Augmented BMPRIA-mediated BMP signaling in cranial neural crest lineage leads to cleft palate formation and delayed tooth differentiation.

    Directory of Open Access Journals (Sweden)

    Lu Li

    Full Text Available The importance of BMP receptor Ia (BMPRIa mediated signaling in the development of craniofacial organs, including the tooth and palate, has been well illuminated in several mouse models of loss of function, and by its mutations associated with juvenile polyposis syndrome and facial defects in humans. In this study, we took a gain-of-function approach to further address the role of BMPR-IA-mediated signaling in the mesenchymal compartment during tooth and palate development. We generated transgenic mice expressing a constitutively active form of BmprIa (caBmprIa in cranial neural crest (CNC cells that contributes to the dental and palatal mesenchyme. Mice bearing enhanced BMPRIa-mediated signaling in CNC cells exhibit complete cleft palate and delayed odontogenic differentiation. We showed that the cleft palate defect in the transgenic animals is attributed to an altered cell proliferation rate in the anterior palatal mesenchyme and to the delayed palatal elevation in the posterior portion associated with ectopic cartilage formation. Despite enhanced activity of BMP signaling in the dental mesenchyme, tooth development and patterning in transgenic mice appeared normal except delayed odontogenic differentiation. These data support the hypothesis that a finely tuned level of BMPRIa-mediated signaling is essential for normal palate and tooth development.

  19. Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

    Science.gov (United States)

    van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G

    2001-04-15

    Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

  20. Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Wei [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035 (China); Department of Food Science and Technology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (United States); Zhu, Hong; Jia, Zhenquan [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); Li, Jianrong [College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035 (China); Misra, Hara P. [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States); Zhou, Kequan, E-mail: kzhou@wayne.edu [Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202 (United States); Li, Yunbo, E-mail: yli@vcom.vt.edu [Division of Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine, Virginia Tech Corporate Research Center, Blacksburg, VA 24060 (United States)

    2009-12-04

    Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in {phi}X-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 {mu}M SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

  1. Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

    International Nuclear Information System (INIS)

    Chen, Wei; Zhu, Hong; Jia, Zhenquan; Li, Jianrong; Misra, Hara P.; Zhou, Kequan; Li, Yunbo

    2009-01-01

    Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in φX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 μM SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.

  2. Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

    Science.gov (United States)

    Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

    2012-09-01

    IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

  3. Mast cells mediate malignant pleural effusion formation.

    Science.gov (United States)

    Giannou, Anastasios D; Marazioti, Antonia; Spella, Magda; Kanellakis, Nikolaos I; Apostolopoulou, Hara; Psallidas, Ioannis; Prijovich, Zeljko M; Vreka, Malamati; Zazara, Dimitra E; Lilis, Ioannis; Papaleonidopoulos, Vassilios; Kairi, Chrysoula A; Patmanidi, Alexandra L; Giopanou, Ioanna; Spiropoulou, Nikolitsa; Harokopos, Vaggelis; Aidinis, Vassilis; Spyratos, Dionisios; Teliousi, Stamatia; Papadaki, Helen; Taraviras, Stavros; Snyder, Linda A; Eickelberg, Oliver; Kardamakis, Dimitrios; Iwakura, Yoichiro; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Kalomenidis, Ioannis; Blackwell, Timothy S; Agalioti, Theodora; Stathopoulos, Georgios T

    2015-06-01

    Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

  4. Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

    Science.gov (United States)

    Chen, Panpan; Douglas, Steven D.; Meshki, John; Tuluc, Florin

    2012-01-01

    Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing. PMID:23024816

  5. Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle formation in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Panpan Chen

    Full Text Available Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP. We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2-10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

  6. tri-n-butyltin hydride-mediated radical reaction of a 2-iodobenzamide: formation of an unexpected carbon-tin bond

    International Nuclear Information System (INIS)

    Oliveira, Marcelo T.; Alves, Rosemeire B.; Cesar, Amary; Prado, Maria Auxiliadora F.; Alves, Ricardo J.; Queiroga, Carla G.; Santos, Leonardo S.; Eberlin, Marcos N.

    2007-01-01

    The tri-n-butyltin hydride-mediated reaction of methyl 2,3-di-O-benzyl-4-O-trans-cinnamyl- 6-deoxy-6-(2-iodobenzoylamino)-α-D-galactopyranoside afforded an unexpected aryltributyltin compound. The structure of this new tetraorganotin(IV) product has been elucidated by 1 H, 13 C NMR spectroscopy, COSY and HMQC experiments and electrospray ionization mass spectrometry (ESI-MS). The formation of this new compound via a radical coupling reaction and a radical addition-elimination process is discussed. (author)

  7. Interaction mediated by the putative tip regions of MdsA and MdsC in the formation of a Salmonella-specific tripartite efflux pump.

    Directory of Open Access Journals (Sweden)

    Saemee Song

    Full Text Available To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND-type inner membrane transporter (IMT that requires the membrane fusion protein (MFP MdsA, and an outer membrane protein (OMP; MdsC or TolC to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.

  8. Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription.

    Science.gov (United States)

    Ito, Jun; Fukaki, Hidehiro; Onoda, Makoto; Li, Lin; Li, Chuanyou; Tasaka, Masao; Furutani, Masahiko

    2016-06-07

    Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

  9. Alpha 1-adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. Possible parallel activation of phospholipase C and phospholipase A2

    International Nuclear Information System (INIS)

    Slivka, S.R.; Insel, P.A.

    1987-01-01

    alpha 1-Adrenergic receptors mediate two effects on phospholipid metabolism in Madin-Darby canine kidney (MDCK-D1) cells: hydrolysis of phosphoinositides and arachidonic acid release with generation of prostaglandin E2 (PGE2). The similarity in concentration dependence for the agonist (-)-epinephrine in eliciting these two responses implies that they are mediated by a single population of alpha 1-adrenergic receptors. However, we find that the kinetics of the two responses are quite different, PGE2 production occurring more rapidly and transiently than the hydrolysis of phosphoinositides. The antibiotic neomycin selectively decreases alpha 1-receptor-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis without decreasing alpha 1-receptor-mediated arachidonic acid release and PGE2 generation. In addition, receptor-mediated inositol trisphosphate formation is independent of extracellular calcium, whereas release of labeled arachidonic acid is largely calcium-dependent. Moreover, based on studies obtained with labeled arachidonic acid, receptor-mediated generation of arachidonic acid cannot be accounted for by breakdown of phosphatidylinositol monophosphate, phosphatidylinositol bisphosphate, or phosphatidic acid. Further studies indicate that epinephrine produces changes in formation or turnover of several classes of membrane phospholipids in MDCK cells. We conclude that alpha 1-adrenergic receptors in MDCK cells appear to regulate phospholipid metabolism by the parallel activation of phospholipase C and phospholipase A2. This parallel activation of phospholipases contrasts with models described in other systems which imply sequential activation of phospholipase C and diacylglycerol lipase or phospholipase A2

  10. Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Zoltán Németh

    2016-11-01

    Full Text Available Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L−1 ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis.

  11. Formation of Fructose-Mediated Advanced Glycation End Products and Their Roles in Metabolic and Inflammatory Diseases.

    Science.gov (United States)

    Gugliucci, Alejandro

    2017-01-01

    Fructose is associated with the biochemical alterations that promote the development of metabolic syndrome (MetS), nonalcoholic fatty liver disease, and type 2 diabetes. Its consumption has increased in parallel with MetS. It is metabolized by the liver, where it stimulates de novo lipogenesis. The triglycerides synthesized lead to hepatic insulin resistance and dyslipidemia. Fructose-derived advanced glycation end products (AGEs) may be involved via the Maillard reaction. Fructose has not been a main focus of glycation research because of the difficulty in measuring its adducts, and, more importantly, because although it is 10 times more reactive than glucose, its plasma concentration is only 1% of that of glucose. In this focused review, I summarize exogenous and endogenous fructose metabolism, fructose glycation, and in vitro, animal, and human data. Fructose is elevated in several tissues of diabetic patients where the polyol pathway is active, reaching the same order of magnitude as glucose. It is plausible that the high reactivity of fructose, directly or via its metabolites, may contribute to the formation of intracellular AGEs and to vascular complications. The evidence, however, is still unconvincing. Two areas that have been overlooked so far and should be actively explored include the following: 1) enteral formation of fructose AGEs, generating an inflammatory response to the receptor for AGEs (which may explain the strong association between fructose consumption and asthma, chronic bronchitis, and arthritis); and 2) inactivation of hepatic AMP-activated protein kinase by a fructose-mediated increase in methylglyoxal flux (perpetuating lipogenesis, fatty liver, and insulin resistance). If proven correct, these mechanisms would put the fructose-mediated Maillard reaction in the limelight again as a contributing factor in chronic inflammatory diseases and MetS. © 2017 American Society for Nutrition.

  12. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  13. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  14. Nrp2 is sufficient to instruct circuit formation of mitral-cells to mediate odour-induced attractive social responses.

    Science.gov (United States)

    Inokuchi, Kasumi; Imamura, Fumiaki; Takeuchi, Haruki; Kim, Ryang; Okuno, Hiroyuki; Nishizumi, Hirofumi; Bito, Haruhiko; Kikusui, Takefumi; Sakano, Hitoshi

    2017-07-21

    Odour information induces various innate responses that are critical to the survival of the individual and for the species. An axon guidance molecule, Neuropilin 2 (Nrp2), is known to mediate targeting of olfactory sensory neurons (primary neurons), to the posteroventral main olfactory bulb (PV MOB) in mice. Here we report that Nrp2-positive (Nrp2 + ) mitral cells (MCs, second-order neurons) play crucial roles in transmitting attractive social signals from the PV MOB to the anterior part of medial amygdala (MeA). Semaphorin 3F, a repulsive ligand to Nrp2, regulates both migration of Nrp2 + MCs to the PV MOB and their axonal projection to the anterior MeA. In the MC-specific Nrp2 knockout mice, circuit formation of Nrp2 + MCs and odour-induced attractive social responses are impaired. In utero, electroporation demonstrates that activation of the Nrp2 gene in MCs is sufficient to instruct their circuit formation from the PV MOB to the anterior MeA.

  15. New chromosome characteristics of the monozoic tapeworm Caryophyllaeus laticeps (Cestoda, Caryophyllidea

    Directory of Open Access Journals (Sweden)

    Bombarová M.

    2015-12-01

    Full Text Available The karyotype of a caryophyllidean tapeworm Caryophyllaeus laticeps (Pallas, 1781 from the freshwater bream Abramis brama (L. caught in the Slovak part of the River Tisa, was described and originally inspected for amount of heterochromatin and its chromosome localization. The chromosome set comprised nine metacentric and one submetacentric (No. 3 pairs (2n = 20. The chromosomes were up to 12.0 ± 2.5 μm long and the mean total length of haploid genome (TLC reached 80.6 μm that represents one of the highest yet recorded values among tapeworms. C-banding and staining with fl uorescent dyes DAPI and YOYO1 revealed a distinct banding pattern explicitly on chromosomes with centromeric bright heterochromatin bands present on all 10 chromosome pairs; no pair showed any interstitial heterochromatin. A complete course of spermatocyte meiosis and dynamics of nucleolus formation and degradation during meiotic division was described.

  16. The group A streptococcal collagen-like protein 1, Scl1, mediates biofilm formation by targeting the EDA-containing variant of cellular fibronectin expressed in wounded tissue

    Science.gov (United States)

    Oliver-Kozup, Heaven; Martin, Karen H.; Schwegler-Berry, Diane; Green, Brett J.; Betts, Courtney; Shinde, Arti V.; Van De Water, Livingston; Lukomski, Slawomir

    2012-01-01

    Summary Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C′ loop region recognized by the α9β1 integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization. PMID:23217101

  17. Clathrin-Mediated Auxin Efflux and Maxima Regulate Hypocotyl Hook Formation and Light-Stimulated Hook Opening in Arabidopsis.

    Science.gov (United States)

    Yu, Qinqin; Zhang, Ying; Wang, Juan; Yan, Xu; Wang, Chao; Xu, Jian; Pan, Jianwei

    2016-01-04

    The establishment of auxin maxima by PIN-FORMED 3 (PIN3)- and AUXIN RESISTANT 1/LIKE AUX1 (LAX) 3 (AUX1/LAX3)-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism has remained poorly understood. Here, we show that loss of function of clathrin light chain CLC2 and CLC3 genes enhanced auxin maxima and thereby hook curvature, alleviated the inhibitory effect of auxin overproduction on auxin maxima and hook curvature, and delayed blue light-stimulated auxin maxima reduction and hook opening. Moreover, pharmacological experiments revealed that auxin maxima formation and hook curvature in clc2 clc3 were sensitive to auxin efflux inhibitors 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid but not to the auxin influx inhibitor 1-naphthoxyacetic acid. Live-cell imaging analysis further uncovered that loss of CLC2 and CLC3 function impaired PIN3 endocytosis and promoted its lateralization in the cortical cells but did not affect AUX1 localization. Taken together, these results suggest that clathrin regulates auxin maxima and thereby hook formation through modulating PIN3 localization and auxin efflux, providing a novel mechanism that integrates developmental signals and environmental cues to regulate plant skotomorphogenesis and photomorphogenesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  18. Formation of Fructose-Mediated Advanced Glycation End Products and Their Roles in Metabolic and Inflammatory Diseases12

    Science.gov (United States)

    2017-01-01

    Fructose is associated with the biochemical alterations that promote the development of metabolic syndrome (MetS), nonalcoholic fatty liver disease, and type 2 diabetes. Its consumption has increased in parallel with MetS. It is metabolized by the liver, where it stimulates de novo lipogenesis. The triglycerides synthesized lead to hepatic insulin resistance and dyslipidemia. Fructose-derived advanced glycation end products (AGEs) may be involved via the Maillard reaction. Fructose has not been a main focus of glycation research because of the difficulty in measuring its adducts, and, more importantly, because although it is 10 times more reactive than glucose, its plasma concentration is only 1% of that of glucose. In this focused review, I summarize exogenous and endogenous fructose metabolism, fructose glycation, and in vitro, animal, and human data. Fructose is elevated in several tissues of diabetic patients where the polyol pathway is active, reaching the same order of magnitude as glucose. It is plausible that the high reactivity of fructose, directly or via its metabolites, may contribute to the formation of intracellular AGEs and to vascular complications. The evidence, however, is still unconvincing. Two areas that have been overlooked so far and should be actively explored include the following: 1) enteral formation of fructose AGEs, generating an inflammatory response to the receptor for AGEs (which may explain the strong association between fructose consumption and asthma, chronic bronchitis, and arthritis); and 2) inactivation of hepatic AMP-activated protein kinase by a fructose-mediated increase in methylglyoxal flux (perpetuating lipogenesis, fatty liver, and insulin resistance). If proven correct, these mechanisms would put the fructose-mediated Maillard reaction in the limelight again as a contributing factor in chronic inflammatory diseases and MetS. PMID:28096127

  19. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    Science.gov (United States)

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  20. Karyotypes, constitutive heterochromatin, and genomic DNA values in the blowfly genera Chrysomya, Lucilia, and Protophormia (Diptera: Calliphoridae).

    Science.gov (United States)

    Ullerich, Fritz-Helmut; Schöttke, Michael

    2006-06-01

    The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.

  1. Chromatin organization regulated by EZH2-mediated H3K27me3 is required for OPN-induced migration of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Liu, Lingling; Luo, Qing; Sun, Jinghui; Ju, Yang; Morita, Yasuyuki; Song, Guanbin

    2018-03-01

    Osteopontin (OPN) is a chemokine-like extracellular matrix-associated protein involved in the migration of bone marrow-derived mesenchymal stem cells (BMSCs). An increasing number of studies have found that chromatin organization may affect cellular migration. However, whether OPN regulates chromatin organization is not understood, nor are the underlying molecular mechanisms. In this study, we investigated the link between chromatin organization and BMSC migration and demonstrated that OPN-mediated BMSC migration leads to elevated levels of heterochromatin marker histone H3 lysine 27 trimethylation (H3K27me3) through the methyltransferase EZH2. The expression of EZH2 reorganizes the chromatin structure of BMSCs. Pharmacological inhibition or depletion of EZH2 blocks BMSC migration. Moreover, using an atomic force microscope (AFM), we found that chromatin decondensation alters the mechanical properties of the nucleus. In addition, inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signals represses OPN-promoted chromatin condensation and cell migration. Thus, our results identify a mechanism by which ERK1/2 signalling drives specific chromatin modifications in BMSCs, which alters chromatin organization and thereby enables OPN-mediated BMSC migration. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Hydroxyl Radical-Mediated Novel Modification of Peptides: N-Terminal Cyclization through the Formation of α-Ketoamide.

    Science.gov (United States)

    Lee, Seon Hwa; Kyung, Hyunsook; Yokota, Ryo; Goto, Takaaki; Oe, Tomoyuki

    2015-01-20

    The hydroxyl radical-mediated oxidation of peptides and proteins constitutes a large group of post-translational modifications that can result in structural and functional changes. These oxidations can lead to hydroxylation, sulfoxidation, or carbonylation of certain amino acid residues and cleavage of peptide bonds. In addition, hydroxyl radicals can convert the N-terminus of peptides to an α-ketoamide via abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate. In the present study, we identified N-terminal cyclization as a novel modification mediated by a hydroxyl radical. The reaction of angiotensin (Ang) II (DRVYIHPF) and the hydroxyl radical generated by the Cu(II)/ascorbic acid (AA) system or UV/hydrogen peroxide system produced N-terminal cyclized-Ang II (Ang C) and pyruvamide-Ang II (Ang P, CH3COCONH-RVYIHPF). The structure of Ang C was confirmed by mass spectrometry and comparison to an authentic standard. The subsequent incubation of isolated Ang P in the presence of Cu(II)/AA revealed that Ang P was the direct precursor of Ang C. The proposed mechanism involves the formation of a nitrogen-centered (aminyl) radical, which cyclizes to form a five-membered ring containing the alkoxy radical. The subsequent β-scission reaction of the alkoxyl radical results in the cleavage of the terminal CH3CO group. The initial aminyl radical can be stabilized by chelation to the Cu(II) ions. The affinity of Ang C toward the Ang II type 1 receptor was significantly lower than that of Ang II or Ang P. Ang C was not further metabolized by aminopeptidase A, which converts Ang II to Ang III. Hydroxyl radical-mediated N-terminal cyclization was also observed in other Ang peptides containing N-terminal alanine, arginine, valine, and amyloid β 1-11 (DAEFRHDSGYE).

  3. Nitric oxide mediates strigolactone signaling in auxin and ethylene-sensitive lateral root formation in sunflower seedlings.

    Science.gov (United States)

    Bharti, Niharika; Bhatla, Satish C

    2015-01-01

    Strigolactones (SLs) play significant role in shaping root architecture whereby auxin-SL crosstalk has been observed in SL-mediated responses of primary root elongation, lateral root formation and adventitious root (AR) initiation. Whereas GR24 (a synthetic strigolactone) inhibits LR and AR formation, the effect of SL biosynthesis inhibitor (fluridone) is just the opposite (root proliferation). Naphthylphthalamic acid (NPA) leads to LR proliferation but completely inhibits AR development. The diffusive distribution of PIN1 in the provascular cells in the differentiating zone of the roots in response to GR24, fluridone or NPA treatments further indicates the involvement of localized auxin accumulation in LR development responses. Inhibition of LR formation by GR24 treatment coincides with inhibition of ACC synthase activity. Profuse LR development by fluridone and NPA treatments correlates with enhanced [Ca(2+)]cyt in the apical region and differentiating zones of LR, indicating a critical role of [Ca(2+)] in LR development in response to the coordinated action of auxins, ethylene and SLs. Significant enhancement of carotenoid cleavage dioxygenase (CCD) activity (enzyme responsible for SL biosynthesis) in tissue homogenates in presence of cPTIO (NO scavenger) indicates the role of endogenous NO as a negative modulator of CCD activity. Differences in the spatial distribution of NO in the primary and lateral roots further highlight the involvement of NO in SL-modulated root morphogenesis in sunflower seedlings. Present work provides new report on the negative modulation of SL biosynthesis through modulation of CCD activity by endogenous nitric oxide during SL-modulated LR development.

  4. Endogenous leukotriene formation during anaphylactic shock

    International Nuclear Information System (INIS)

    Keppler, A.; Oerning, L.; Bernstroem, K.; Hammarstroem, S.

    1987-01-01

    Leukotriene (LT)C 4 is a biologically active substance, presumed to play major roles as a mediator of allergic and anaphylactic reactions. It is formed e.g. by basophilic and eosinophilic leukocytes, monocytes, macrophages, and mast cells. In cells having IgE receptors, bridging of these by divalent anti-IgE-receptor antibodies or by interaction between receptor-bound IgE and anti-IgE will induce LTC 4 formation. Leukotriene formation has also been demonstrated in other in vitro models of immediate hypersensivity. The biological actions of LTC 4 , comprise induction of airway obstruction, constriction of coronary arteries, hypotension, and plasma extravasation. Leukotriene formation in vivo may mediate anaphylactic shock symptoms and cause the death of an animal. In order to prove the presumed mediator role of this substance in anaphylactic reactions, it is necessary to demonstrate its endogenous formation during shock. Studies on the metabolism of LTC 4 have revealed rapid catabolism by various transformations of the peptide substituent. Recently, three metabolites were demonstrated to be excreted as end-products in man (LTE 4 ,) and the rat (N-acetyl LTE 4 and N-acetyl 11-trans LTE 4 ). By monitoring biliary N-acetyl LTE 4 levels, endogenous leukotriene formation in the rat was demonstrated in vivo after tissue trauma and endotoxin shock. We now wish to report evidence for endogenous leukotriene C 4 production during anaphylactic shock in guinea pigs. 37 refs. (author)

  5. Chromatin preferences of the perichromosomal layer constituent pKi-67.

    Science.gov (United States)

    Traut, Walther; Endl, Elmar; Garagna, Silvia; Scholzen, Thomas; Schwinger, Eberhard; Gerdes, Johannes; Winking, Heinz

    2002-01-01

    The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the 'perichromosomal layer'. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.

  6. Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation.

    Science.gov (United States)

    Bate, Clive; Nolan, William; Williams, Alun

    2016-01-01

    The prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP(C) in prion formation was examined using a cell painting technique. PrP(Sc) formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP(C). In contrast, PrP(C) containing a GPI anchor from which the sialic acid had been removed (desialylated PrP(C)) was not converted to PrP(Sc). Furthermore, the presence of desialylated PrP(C) inhibited the production of PrP(Sc) within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP(C) contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP(C). Desialylated PrP(C) was less sensitive to cholesterol depletion than PrP(C) and was not released from cells by treatment with glimepiride. The presence of desialylated PrP(C) in neurons caused the dissociation of cytoplasmic phospholipase A2 from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A2. These findings show that the sialic acid moiety of the GPI attached to PrP(C) modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP(Sc) formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Mutation of the Streptococcus gordonii Thiol-Disulfide Oxidoreductase SdbA Leads to Enhanced Biofilm Formation Mediated by the CiaRH Two-Component Signaling System.

    Directory of Open Access Journals (Sweden)

    Lauren Davey

    Full Text Available Streptococcus gordonii is a commensal inhabitant of human oral biofilms. Previously, we identified an enzyme called SdbA that played an important role in biofilm formation by S. gordonii. SdbA is thiol-disulfide oxidoreductase that catalyzes disulfide bonds in secreted proteins. Surprisingly, inactivation of SdbA results in enhanced biofilm formation. In this study we investigated the basis for biofilm formation by the ΔsdbA mutant. The results revealed that biofilm formation was mediated by the interaction between the CiaRH and ComDE two-component signalling systems. Although it did not affect biofilm formation by the S. gordonii parent strain, CiaRH was upregulated in the ΔsdbA mutant and it was essential for the enhanced biofilm phenotype. The biofilm phenotype was reversed by inactivation of CiaRH or by the addition of competence stimulating peptide, the production of which is blocked by CiaRH activity. Competition assays showed that the enhanced biofilm phenotype also corresponded to increased oral colonization in mice. Thus, the interaction between SdbA, CiaRH and ComDE affects biofilm formation both in vitro and in vivo.

  8. Temporal analysis of meiotic DNA double-strand break formation and repair in Drosophila females.

    Science.gov (United States)

    Mehrotra, S; McKim, K S

    2006-11-24

    Using an antibody against the phosphorylated form of His2Av (gamma-His2Av), we have described the time course for the series of events leading from the formation of a double-strand break (DSB) to a crossover in Drosophila female meiotic prophase. MEI-P22 is required for DSB formation and localizes to chromosomes prior to gamma-His2Av foci. Drosophila females, however, are among the group of organisms where synaptonemal complex (SC) formation is not dependent on DSBs. In the absence of two SC proteins, C(3)G and C(2)M, the number of DSBs in oocytes is significantly reduced. This is consistent with the appearance of SC protein staining prior to gamma-His2Av foci. However, SC formation is incomplete or absent in the neighboring nurse cells, and gamma-His2Av foci appear with the same kinetics as in oocytes and do not depend on SC proteins. Thus, competence for DSB formation in nurse cells occurs with a specific timing that is independent of the SC, whereas in the oocytes, some SC proteins may have a regulatory role to counteract the effects of a negative regulator of DSB formation. The SC is not sufficient for DSB formation, however, since DSBs were absent from the heterochromatin even though SC formation occurs in these regions. All gamma-His2Av foci disappear before the end of prophase, presumably as repair is completed and crossovers are formed. However, oocytes in early prophase exhibit a slower response to X-ray-induced DSBs compared to those in the late pachytene stage. Assuming all DSBs appear as gamma-His2Av foci, there is at least a 3:1 ratio of noncrossover to crossover products. From a comparison of the frequency of gamma-His2Av foci and crossovers, it appears that Drosophila females have only a weak mechanism to ensure a crossover in the presence of a low number of DSBs.

  9. Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion

    International Nuclear Information System (INIS)

    Garg, Himanshu; Fuller, Frederick J.; Tompkins, Wayne A.F.

    2004-01-01

    Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU 1 failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation

  10. ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation

    Directory of Open Access Journals (Sweden)

    Yuko Atsumi

    2015-12-01

    Full Text Available In response to DNA double-strand breaks (DSBs, H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.

  11. Biomaterial Substrate-Mediated Multicellular Spheroid Formation and Their Applications in Tissue Engineering.

    Science.gov (United States)

    Tseng, Ting-Chen; Wong, Chui-Wei; Hsieh, Fu-Yu; Hsu, Shan-Hui

    2017-12-01

    Three-dimentional (3D) multicellular aggregates (spheroids), compared to the traditional 2D monolayer cultured cells, are physiologically more similar to the cells in vivo. So far there are various techniques to generate 3D spheroids. Spheroids obtained from different methods have already been applied to regenerative medicine or cancer research. Among the cell spheroids created by different methods, the substrate-derived spheroids and their forming mechanism are unique. This review focuses on the formation of biomaterial substrate-mediated multicellular spheroids and their applications in tissue engineering and tumor models. First, the authors will describe the special chitosan substrate-derived mesenchymal stem cell (MSC) spheroids and their greater regenerative capacities in various tissues. Second, the authors will describe tumor spheroids derived on chitosan and hyaluronan substrates, which serve as a simple in vitro platform to study 3D tumor models or to perform cancer drug screening. Finally, the authors will mention the self-assembly process for substrate-derived multiple cell spheroids (co-spheroids), which may recapitulate the heterotypic cell-cell interaction for co-cultured cells or crosstalk between different types of cells. These unique multicellular mono-spheroids or co-spheroids represent a category of 3D cell culture with advantages of biomimetic cell-cell interaction, better functionalities, and imaging possibilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Immunological Reaction in TNF-α-Mediated Osteoclast Formation and Bone Resorption In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Hideki Kitaura

    2013-01-01

    Full Text Available Tumor necrosis factor-α (TNF-α is a cytokine produced by monocytes, macrophages, and T cells and is induced by pathogens, endotoxins, or related substances. TNF-α may play a key role in bone metabolism and is important in inflammatory bone diseases such as rheumatoid arthritis. Cells directly involved in osteoclastogenesis include macrophages, which are osteoclast precursor cells, osteoblasts, or stromal cells. These cells express receptor activator of NF-κB ligand (RANKL to induce osteoclastogenesis, and T cells, which secrete RANKL, promote osteoclastogenesis during inflammation. Elucidating the detailed effects of TNF-α on bone metabolism may enable the identification of therapeutic targets that can efficiently suppress bone destruction in inflammatory bone diseases. TNF-α is considered to act by directly increasing RANK expression in macrophages and by increasing RANKL in stromal cells. Inflammatory cytokines such as interleukin- (IL- 12, IL-18, and interferon-γ (IFN-γ strongly inhibit osteoclast formation. IL-12, IL-18, and IFN-γ induce apoptosis in bone marrow cells treated with TNF-α  in vitro, and osteoclastogenesis is inhibited by the interactions of TNF-α-induced Fas and Fas ligand induced by IL-12, IL-18, and IFN-γ. This review describes and discusses the role of cells concerned with osteoclast formation and immunological reactions in TNF-α-mediated osteoclastogenesis in vitro and in vivo.

  13. Mediation analysis allowing for exposure-mediator interactions and causal interpretation: theoretical assumptions and implementation with SAS and SPSS macros

    Science.gov (United States)

    Valeri, Linda; VanderWeele, Tyler J.

    2012-01-01

    Mediation analysis is a useful and widely employed approach to studies in the field of psychology and in the social and biomedical sciences. The contributions of this paper are several-fold. First we seek to bring the developments in mediation analysis for non linear models within the counterfactual framework to the psychology audience in an accessible format and compare the sorts of inferences about mediation that are possible in the presence of exposure-mediator interaction when using a counterfactual versus the standard statistical approach. Second, the work by VanderWeele and Vansteelandt (2009, 2010) is extended here to allow for dichotomous mediators and count outcomes. Third, we provide SAS and SPSS macros to implement all of these mediation analysis techniques automatically and we compare the types of inferences about mediation that are allowed by a variety of software macros. PMID:23379553

  14. Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure

    International Nuclear Information System (INIS)

    Falk, Martin; Lukášová, Emilie; Štefančíková, Lenka; Baranová, Elena; Falková, Iva; Ježková, Lucie; Davídková, Marie; Bačíková, Alena; Vachelová, Jana; Michaelidesová, Anna; Kozubek, Stanislav

    2014-01-01

    Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction. - Highlights: ► DSB repair is absent in mature granulocytes with condensed chromatin. ► Repair proteins and γH2AX appear in immature stages but rarely colocalize. ► γH2AX persist long times in these cells and DSB repair is inefficient. ► Even though, γH2AX foci “move” out of the dense chromatin. ► 53BP1 enters HP1β domains only after their decondensation

  15. Aspirin and lipid mediators in the cardiovascular system.

    Science.gov (United States)

    Schrör, Karsten; Rauch, Bernhard H

    2015-09-01

    Aspirin is an unique compound because it bears two active moieties within one and the same molecule: a reactive acetyl group and the salicylate metabolite. Salicylate has some effects similar to aspirin, however only at higher concentrations, usually in the millimolar range, which are not obtained at conventional antiplatelet aspirin doses of 100-300 mg/day. Pharmacological actions of aspirin in the cardiovascular system at these doses are largely if not entirely due to target structure acetylation. Several classes of lipid mediators become affected: Best known is the cyclooxygenase-1 (COX-1) in platelets with subsequent inhibition of thromboxane and, possibly, thrombin formation. By this action, aspirin also inhibits paracrine thromboxane functions on other lipid mediators, such as the platelet storage product sphingosine-1-phosphate (S1P), an inflammatory mediator. Acetylation of COX-2 allows for generation of 15-(R)HETE and subsequent formation of "aspirin-triggered lipoxin" (ATL) by interaction with white cell lipoxygenases. In the cardiovascular system, aspirin also acetylates eNOS with subsequent upregulation of NO formation and enhanced expression of the antioxidans heme-oxygenase-1. This action is possibly also COX-2/ATL mediated. Many more acetylation targets have been identified in live cells by quantitative acid-cleavable activity-based protein profiling and might result in discovery of even more aspirin targets in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Mediation analysis allowing for exposure-mediator interactions and causal interpretation: theoretical assumptions and implementation with SAS and SPSS macros.

    Science.gov (United States)

    Valeri, Linda; Vanderweele, Tyler J

    2013-06-01

    Mediation analysis is a useful and widely employed approach to studies in the field of psychology and in the social and biomedical sciences. The contributions of this article are several-fold. First we seek to bring the developments in mediation analysis for nonlinear models within the counterfactual framework to the psychology audience in an accessible format and compare the sorts of inferences about mediation that are possible in the presence of exposure-mediator interaction when using a counterfactual versus the standard statistical approach. Second, the work by VanderWeele and Vansteelandt (2009, 2010) is extended here to allow for dichotomous mediators and count outcomes. Third, we provide SAS and SPSS macros to implement all of these mediation analysis techniques automatically, and we compare the types of inferences about mediation that are allowed by a variety of software macros. (PsycINFO Database Record (c) 2013 APA, all rights reserved).

  17. Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

    Science.gov (United States)

    Kabulski, Jarod L.

    The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein

  18. Numerical study of Q-ball formation in gravity mediation

    International Nuclear Information System (INIS)

    Hiramatsu, Takashi; Kawasaki, Masahiro; Takahashi, Fuminobu

    2010-01-01

    We study Q-ball formation in the expanding universe on 1D, 2D and 3D lattice simulations. We obtain detailed Q-ball charge distributions, and find that the distribution is peaked at Q 3D peak ≅ 1.9 × 10 −2 (|Φ in |/m) 2 , which is greater than the existing result by about 60%. Based on the numerical simulations, we discuss how the Q-ball formation proceeds. Also we make a comment on possible deviation of the charge distributions from what was conjectured in the past

  19. The question about contemporary state of Russian mediation institute

    Directory of Open Access Journals (Sweden)

    Kurmanbaev M.M.

    2016-11-01

    Full Text Available this article considers the question about introduction and development of Russian mediation institute. According to the authors the adoption of the Law on Mediation testifies that the state gives a new impetus to the process of constitutional state formation and to the development of alternative methods of dispute resolution. The problematic aspects of the mediation development are described in this article. As the results of the study the authors suggest to enhance the effectiveness of media-tion, which is a special form of interposition.

  20. Mediator kinase module and human tumorigenesis.

    Science.gov (United States)

    Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

    2015-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

  1. CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

    Science.gov (United States)

    Tasan, Ipek; Sustackova, Gabriela; Zhang, Liguo; Kim, Jiah; Sivaguru, Mayandi; HamediRad, Mohammad; Wang, Yuchuan; Genova, Justin; Ma, Jian; Belmont, Andrew S; Zhao, Huimin

    2018-06-15

    Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

  2. Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B.

    Science.gov (United States)

    Rao, Minxi; Smith, Brian C; Marletta, Michael A

    2015-05-05

    Nitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis. Bacterial nitric oxide (NO) signaling via heme-nitric oxide/oxygen binding (H-NOX) proteins regulates biofilm formation, playing an important role in protecting bacteria from oxidative stress and other environmental stresses. Biofilms are also an important part of symbiosis, allowing the organism to remain in a

  3. Distinct Neural Mechanisms Mediate Olfactory Memory Formation at Different Timescales

    Science.gov (United States)

    McNamara, Ann Marie; Magidson, Phillip D.; Linster, Christiane; Wilson, Donald A.; Cleland, Thomas A.

    2008-01-01

    Habituation is one of the oldest forms of learning, broadly expressed across sensory systems and taxa. Here, we demonstrate that olfactory habituation induced at different timescales (comprising different odor exposure and intertrial interval durations) is mediated by different neural mechanisms. First, the persistence of habituation memory is…

  4. Dewetting-mediated pattern formation inside the coffee ring

    Science.gov (United States)

    Li, Weibin; Lan, Ding; Wang, Yuren

    2017-04-01

    The rearrangement of particles in the final stage of droplet evaporation has been investigated by utilizing differential interference contrast microscopy and the formation mechanism of a network pattern inside a coffee ring has been revealed. A tailored substrate with a circular hydrophilic domain is prepared to obtain thin liquid film containing monolayer particles. Real-time bottom-view images show that the evolution of a dry patch could be divided into three stages: rupture initiation, dry patch expansion, and drying of the residual liquid. A growing number of dry patches will repeat these stages to form the network patterns inside the ringlike stain. It can be shown that the suction effect promotes the rupture of the liquid film and the formation of the dry patch. The particle-assembling process is totally controlled by the liquid film dewetting and dominated by the surface tension of the liquid film, which eventually determine the ultimate deposition patterns.

  5. Identification of mitochondrial electron transport chain-mediated NADH radical formation by EPR spin-trapping techniques.

    Science.gov (United States)

    Matsuzaki, Satoshi; Kotake, Yashige; Humphries, Kenneth M

    2011-12-20

    The mitochondrial electron transport chain (ETC) is a major source of free radical production. However, due to the highly reactive nature of radical species and their short lifetimes, accurate detection and identification of these molecules in biological systems is challenging. The aim of this investigation was to determine the free radical species produced from the mitochondrial ETC by utilizing EPR spin-trapping techniques and the recently commercialized spin-trap, 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). We demonstrate that this spin-trap has the preferential quality of having minimal mitochondrial toxicity at concentrations required for radical detection. In rat heart mitochondria and submitochondrial particles supplied with NADH, the major species detected under physiological pH was a carbon-centered radical adduct, indicated by markedly large hyperfine coupling constant with hydrogen (a(H) > 2.0 mT). In the presence of the ETC inhibitors, the carbon-centered radical formation was increased and exhibited NADH concentration dependency. The same carbon-centered radical could also be produced with the NAD biosynthesis precursor, nicotinamide mononucleotide, in the presence of a catalytic amount of NADH. The results support the conclusion that the observed species is a complex I derived NADH radical. The formation of the NADH radical could be blocked by hydroxyl radical scavengers but not SOD. In vitro experiments confirmed that an NADH-radical is readily formed by hydroxyl radical but not superoxide anion, further implicating hydroxyl radical as an upstream mediator of NADH radical production. These findings demonstrate the identification of a novel mitochondrial radical species with potential physiological significance and highlight the diverse mechanisms and sites of production within the ETC.

  6. Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

    Science.gov (United States)

    Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

    2011-08-01

    Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

  7. Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

    Science.gov (United States)

    Eyboulet, Fanny; Wydau-Dematteis, Sandra; Eychenne, Thomas; Alibert, Olivier; Neil, Helen; Boschiero, Claire; Nevers, Marie-Claire; Volland, Hervé; Cornu, David; Redeker, Virginie; Werner, Michel; Soutourina, Julie

    2015-10-30

    Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

    Directory of Open Access Journals (Sweden)

    M Cremer

    2009-06-01

    Full Text Available Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has been linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly causally involved in the formation of cell type specific heterochromatin compartments, composed of (pericentromeric regions and chromosomal subregions from neighboring chromosome territories, which contain silent genes.

  9. Autophagy adaptor protein p62/SQSTM1 and autophagy-related gene Atg5 mediate autophagosome formation in response to Mycobacterium tuberculosis infection in dendritic cells.

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    Shintaro Seto

    Full Text Available Mycobacterium tuberculosis is an intracellular pathogen that can survive within phagocytic cells by inhibiting phagolysosome biogenesis. However, host cells can control the intracellular M. tuberculosis burden by the induction of autophagy. The mechanism of autophagosome formation to M. tuberculosis has been well studied in macrophages, but remains unclear in dendritic cells. We therefore characterized autophagosome formation in response to M. tuberculosis infection in dendritic cells. Autophagy marker protein LC3, autophagy adaptor protein p62/SQSTM1 (p62 and ubiquitin co-localized to M. tuberculosis in dendritic cells. Mycobacterial autophagosomes fused with lysosomes during infection, and major histcompatibility complex class II molecules (MHC II also localized to mycobacterial autophagosomes. The proteins p62 and Atg5 function in the initiation and progression of autophagosome formation to M. tuberculosis, respectively; p62 mediates ubiquitination of M. tuberculosis and Atg5 is involved in the trafficking of degradative vesicles and MHC II to mycobacterial autophagosomes. These results imply that the autophagosome formation to M. tuberculosis in dendritic cells promotes the antigen presentation of mycobacterial peptides to CD4(+ T lymphocytes via MHC II.

  10. Leptin regulates bone formation via the sympathetic nervous system

    Science.gov (United States)

    Takeda, Shu; Elefteriou, Florent; Levasseur, Regis; Liu, Xiuyun; Zhao, Liping; Parker, Keith L.; Armstrong, Dawna; Ducy, Patricia; Karsenty, Gerard

    2002-01-01

    We previously showed that leptin inhibits bone formation by an undefined mechanism. Here, we show that hypothalamic leptin-dependent antiosteogenic and anorexigenic networks differ, and that the peripheral mediators of leptin antiosteogenic function appear to be neuronal. Neuropeptides mediating leptin anorexigenic function do not affect bone formation. Leptin deficiency results in low sympathetic tone, and genetic or pharmacological ablation of adrenergic signaling leads to a leptin-resistant high bone mass. beta-adrenergic receptors on osteoblasts regulate their proliferation, and a beta-adrenergic agonist decreases bone mass in leptin-deficient and wild-type mice while a beta-adrenergic antagonist increases bone mass in wild-type and ovariectomized mice. None of these manipulations affects body weight. This study demonstrates a leptin-dependent neuronal regulation of bone formation with potential therapeutic implications for osteoporosis.

  11. CONFLICT MANAGEMENT VIA SYSTEMICALLY PLANNED PEER MEDIATION

    OpenAIRE

    Evangelos C. Papakitsos; Konstantinos Karakiozis

    2016-01-01

    Conflicts and confrontations between students are a key-feature of school life. Especially in recent years, both the scientific and the educational community are particularly sensitive to bullying issues in the school context. Peer mediation (or school mediation) is an alternative way to manage conflicts at school. A critical evaluation of this practice is attempted, as well as its contribution to the formation of a positive attitude in school and to the decreasing of school-bullying incident...

  12. Assay for Human Rad51-Mediated DNA Displacement Loop Formation

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Steven Raynard and Patrick Sung Corresponding author ([]()) ### INTRODUCTION Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded D...

  13. Optimization of Si–C reaction temperature and Ge thickness in C-mediated Ge dot formation

    Energy Technology Data Exchange (ETDEWEB)

    Satoh, Yuhki, E-mail: yu-ki@ecei.tohoku.ac.jp; Itoh, Yuhki; Kawashima, Tomoyuki; Washio, Katsuyoshi

    2016-03-01

    To form Ge dots on a Si substrate, the effect of thermal reaction temperature of sub-monolayer C with Si (100) was investigated and the deposited Ge thickness was optimized. The samples were prepared by solid-source molecular beam epitaxy with an electron-beam gun for C sublimation and a Knudsen cell for Ge evaporation. C of 0.25 ML was deposited on Si (100) at a substrate temperature of 200 °C, followed by a high-temperature treatment at the reaction temperature (T{sub R}) of 650–1000 °C to create Si–C bonds. Ge equivalent to 2 to 5 nm thick was subsequently deposited at 550 °C. Small and dense dots were obtained for T{sub R} = 750 °C but the dot density decreased and the dot diameter varied widely in the case of lower and higher T{sub R}. A dot density of about 2 × 10{sup 10} cm{sup −2} was achieved for Ge deposition equivalent to 3 to 5 nm thick and a standard deviation of dot diameter was the lowest of 10 nm for 5 nm thick Ge. These results mean that C-mediated Ge dot formation was strongly influenced not only by the c(4 × 4) reconstruction condition through the Si–C reaction but also the relationship between the Ge deposition thickness and the exposed Si (100)-(2 × 1) surface area. - Highlights: • The effect of Si–C reaction temperature on Ge dot formation was investigated. • Small and dense dots were obtained for T{sub R} = 750 °C. • The dot density of about 2 × 10{sup 10} cm{sup −2} was achieved for Ge = 3 to 5 nm. • The standard deviation of dot diameter was the lowest of 10 nm at Ge = 5 nm.

  14. BOOKTUBERS MOVEMENT: EMERGING PRACTICES OF READING MEDIATION

    Directory of Open Access Journals (Sweden)

    Claudia Souza Teixeira

    2016-12-01

    Full Text Available This paper intents to investigate reading mediation practices develop by the booktubers, young people that use the YouTube in order to share information about books and to interact with other readers. We begin presenting some concepts related to reading and mediation activities, and then, talk about some aspects of cyberspace, the formation of virtual communities and the Booktubers phenomena. At last, as a result of a documental analysis of 100 videos posted by booktubers, we show the characteristics of these emerging mediation reading practices and the types of books that are more commented by them. We conclude that the conventional practices of reading mediation are the bases for the booktubers’ actuation, but they are adapted to the new kinds of elaboration, creation and organization enabled by the Internet.

  15. Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

    Directory of Open Access Journals (Sweden)

    Huzoor Akbar

    Full Text Available Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation.We utilized the Mx-cre;Cdc42(lox/lox inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+ mice. Platelets isolated from Cdc42(-/-, as compared to Cdc42(+/+, mice exhibited (a diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b inhibition of filopodia formation on immobilized CRP or fibrinogen, (c inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/- mice compared with Cdc42(+/+ mice.Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

  16. LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

    Science.gov (United States)

    Krivega, Ivan; Dean, Ann

    2017-08-21

    Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  17. DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation.

    Science.gov (United States)

    Smets, Martha; Link, Stephanie; Wolf, Patricia; Schneider, Katrin; Solis, Veronica; Ryan, Joel; Meilinger, Daniela; Qin, Weihua; Leonhardt, Heinrich

    2017-04-15

    DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells. © The Author 2017. Published by Oxford University Press.

  18. Inhibition of Hb Binding to GP1bα Abrogates Hb-Mediated Thrombus Formation on Immobilized VWF and Collagen under Physiological Shear Stress.

    Science.gov (United States)

    Annarapu, Gowtham K; Singhal, Rashi; Peng, Yuandong; Guchhait, Prasenjit

    2016-01-01

    Reports including our own describe that intravascular hemolysis increases the risk of thrombosis in hemolytic disorders. Our recent study shows that plasma Hb concentrations correlate directly with platelet activation in patients with paroxysmal nocturnal hemoglobinuria (PNH). The binding of Hb to glycoprotein1bα (GP1bα) increases platelet activation. A peptide AA1-50, designed from N-terminal amino acid sequence of GP1bα significantly inhibits the Hb binding to GP1bα as well as Hb-induced platelet activation. This study further examined if the Hb-mediated platelet activation plays any significant role in thrombus formation on subendothelium matrix under physiological flow shear stresses and the inhibition of Hb-platelet interaction can abrogate the above effects of Hb. Study performed thrombus formation assay in vitro by perfusing whole blood over immobilized VWF or collagen type I in presence of Hb under shear stresses simulating arterial or venous flow. The Hb concentrations ranging from 5 to 10 μM, commonly observed level in plasma of the hemolytic patients including PNH, dose-dependently increased thrombus formation on immobilized VWF under higher shear stress of 25 dyne/cm2, but not at 5 dyne/cm2. The above Hb concentrations also increased thrombus formation on immobilized collagen under both shear stresses of 5 and 25 dyne/cm2. The peptide AA1-50 abrogated invariably the above effects of Hb on thrombus formation. This study therefore indicates that the Hb-induced platelet activation plays a crucial role in thrombus formation on immobilized VWF or collagen under physiological flow shear stresses. Thus suggesting a probable role of this mechanism in facilitating thrombosis under hemolytic conditions.

  19. Iodine-mediated coastal particle formation: an overview of the Reactive Halogens in the Marine Boundary Layer (RHaMBLe Roscoff coastal study

    Directory of Open Access Journals (Sweden)

    G. McFiggans

    2010-03-01

    Full Text Available This paper presents a summary of the measurements made during the heavily-instrumented Reactive Halogens in the Marine Boundary Layer (RHaMBLe coastal study in Roscoff on the North West coast of France throughout September 2006. It was clearly demonstrated that iodine-mediated coastal particle formation occurs, driven by daytime low tide emission of molecular iodine, I2, by macroalgal species fully or partially exposed by the receding waterline. Ultrafine particle concentrations strongly correlate with the rapidly recycled reactive iodine species, IO, produced at high concentrations following photolysis of I2. The heterogeneous macroalgal I2 sources lead to variable relative concentrations of iodine species observed by path-integrated and in situ measurement techniques.

    Apparent particle emission fluxes were associated with an enhanced apparent depositional flux of ozone, consistent with both a direct O3 deposition to macroalgae and involvement of O3 in iodine photochemistry and subsequent particle formation below the measurement height. The magnitude of the particle formation events was observed to be greatest at the lowest tides with the highest concentrations of ultrafine particles growing to the largest sizes, probably by the condensation of anthropogenically-formed condensable material. At such sizes the particles should be able to act as cloud condensation nuclei at reasonable atmospheric supersaturations.

  20. Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture.

    Science.gov (United States)

    Eychenne, Thomas; Novikova, Elizaveta; Barrault, Marie-Bénédicte; Alibert, Olivier; Boschiero, Claire; Peixeiro, Nuno; Cornu, David; Redeker, Virginie; Kuras, Laurent; Nicolas, Pierre; Werner, Michel; Soutourina, Julie

    2016-09-15

    Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts. © 2016 Eychenne et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS.

    Science.gov (United States)

    Bassi, D; Cappa, F; Gazzola, S; Orrù, L; Cocconcelli, P S

    2017-04-15

    In Streptococcus thermophilus , gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the IS Sth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment. IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable

  2. RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

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    Claudinéia Pereira Costa

    Full Text Available Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk, thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs β bursicon genes prevented the complete formation and tanning (melanization/sclerotization of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs β and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs β and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

  3. Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

    Science.gov (United States)

    Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

    Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

  4. Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo

    OpenAIRE

    Eyboulet, Fanny; Wydau-Dematteis, Sandra; Eychenne, Thomas; Alibert, Olivier; Neil, Helen; Boschiero, Claire; Nevers, Marie-Claire; Volland, Herv?; Cornu, David; Redeker, Virginie; Werner, Michel; Soutourina, Julie

    2015-01-01

    Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation i...

  5. Shoot bending promotes flower bud formation by miRNA-mediated regulation in apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Libo; Zhang, Dong; Zhao, Caiping; Li, Youmei; Ma, Juanjuan; An, Na; Han, Mingyu

    2016-02-01

    Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down-regulated and up-regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering-related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  6. 2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

    Science.gov (United States)

    Sethupathy, Sivasamy; Nithya, Chari; Pandian, Shunmugiah Karutha

    2015-01-01

    The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

  7. Characterization of the effect of serum and chelating agents on Staphylococcus aureus biofilm formation; chelating agents augment biofilm formation through clumping factor B

    Science.gov (United States)

    Abraham, Nabil Mathew

    Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We

  8. Computer-Mediated Communication Systems

    Directory of Open Access Journals (Sweden)

    Bin Yu

    2011-10-01

    Full Text Available The essence of communication is to exchange and share information. Computers provide a new medium to human communication. CMC system, composed of human and computers, absorbs and then extends the advantages of all former formats of communication, embracing the instant interaction of oral communication, the abstract logics of printing dissemination, and the vivid images of movie and television. It also creates a series of new communication formats, such as Hyper Text, Multimedia etc. which are the information organizing methods, and cross-space message delivering patterns. Benefiting from the continuous development of technique and mechanism, the computer-mediated communication makes the dream of transmitting information cross space and time become true, which will definitely have a great impact on our social lives.

  9. The roles of the actin cytoskeleton in fear memory formation

    Directory of Open Access Journals (Sweden)

    Raphael eLamprecht

    2011-07-01

    Full Text Available The formation and storage of fear memory is needed to adapt behavior and avoid danger during subsequent fearful events. However, fear memory may also play a significant role in stress and anxiety disorders. When fear becomes disproportionate to that necessary to cope with a given stimulus, or begins to occur in inappropriate situations, a fear or anxiety disorder exists. Thus, the study of cellular and molecular mechanisms underpinning fear memory may shed light on the formation of memory and on anxiety and stress related disorders. Evidence indicates that fear learning leads to changes in neuronal synaptic transmission and morphology in brain areas underlying fear memory formation including the amygdala and hippocampus. The actin cytoskeleton has been shown to participate in these key neuronal processes. Recent findings show that the actin cytoskeleton is needed for fear memory formation and extinction. Moreover, the actin cytoskeleton is involved in synaptic plasticity and in neuronal morphogenesis in brain areas that mediate fear memory. The actin cytoskeleton may therefore mediate between synaptic transmission during fear learning and long-term cellular alterations mandatory for fear memory formation.

  10. Expression of the proteoglycan syndecan-4 and the mechanism by which it mediates stress fiber formation in folliculostellate cells in the rat anterior pituitary gland.

    Science.gov (United States)

    Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Tsukada, Takehiro; Ly, Floren; Kikuchi, Motoshi; Yashiro, Takashi

    2012-08-01

    Folliculostellate (FS) cells in the anterior pituitary gland appear to have multifunctional properties. FS cells connect to each other at gap junctions and thereby form a histological and functional network. We have performed a series of studies on network formation in FS cells and recently reported that FS cells markedly prolong their cytoplasmic processes and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In this study, we investigated the mechanism of this extension of FS cell cytoplasmic processes under the influence of laminin and found that laminin promoted stress fiber formation within FS cells. Next, we noted that formation of stress fibers in FS cells was mediated by syndecan-4, a transmembrane proteoglycan that binds ECM and soluble factors via their extracellular glycosaminoglycan chain. We then observed that expressions of syndecan-4 and α-actinin (a microfilament bundling protein that cross-links actin stress fibers in FS cells) were upregulated by laminin. Using specific siRNA of syndecan-4, actin polymerization of FS cells was inhibited. Our findings suggest that FS cells received a signal from laminin-syndecan-4 interaction, which resulted in morphological changes, and that the formation of a morphological and functional network in FS cells was transduced by a syndecan-4-dependent mechanism in the presence of ECM.

  11. Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

    Directory of Open Access Journals (Sweden)

    Yumiko Kurokawa

    2008-04-01

    Full Text Available In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog-mediated homologous recombination (HR and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA, which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

  12. Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

    Directory of Open Access Journals (Sweden)

    Yvonne Lorat

    Full Text Available DNA double-strand breaks (DSBs generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair.Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent and heterochromatin (electron-dense in cortical neurons of irradiated mouse brain.While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads, occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage.Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing

  13. SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Mickaël Durand-Dubief

    2012-09-01

    Full Text Available Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.

  14. Relations between immune and mediator receptors of mouse lymphocytes

    International Nuclear Information System (INIS)

    Ado, A.D.; Alekseeva, T.A.; Kravchenko, S.A.

    1985-01-01

    This paper examines the action of the specific muscarinic antogonist tritium-quinuclidinyl benzilate (tritium-QNB) on immune rosette formation in mice. It is shown that since the specific muscarini antagonist tritium-QNB inhibits immune rosette formation, this process must be regarded as interconnected with muscarinic receptors of lymphocytes. Interaction of immune (antigen-binding) and mediator receptors, however, is an important factor maintaining immune homeostasis at a certain level

  15. Mediator and Cohesin Connect Gene Expression and Chromatin Architecture

    Science.gov (United States)

    Kagey, Michael H.; Newman, Jamie J.; Bilodeau, Steve; Zhan, Ye; Orlando, David A.; van Berkum, Nynke L.; Ebmeier, Christopher C.; Goossens, Jesse; Rahl, Peter B.; Levine, Stuart S.; Taatjes, Dylan J.; Dekker, Job; Young, Richard A.

    2010-01-01

    Summary Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell. PMID:20720539

  16. The roles of Eph receptors in contextual fear conditioning memory formation.

    Science.gov (United States)

    Dines, Monica; Grinberg, Svetlana; Vassiliev, Maria; Ram, Alon; Tamir, Tal; Lamprecht, Raphael

    2015-10-01

    Eph receptors regulate glutamate receptors functions, neuronal morphology and synaptic plasticity, cellular events believed to be involved in memory formation. In this study we aim to explore the roles of Eph receptors in learning and memory. Toward that end, we examined the roles of EphB2 and EphA4 receptors, key regulators of synaptic functions, in fear conditioning memory formation. We show that mice lacking EphB2 (EphB2(-/-)) are impaired in short- and long-term contextual fear conditioning memory. Mice that express a carboxy-terminally truncated form of EphB2 that lacks forward signaling, instead of the full EphB2, are impaired in long-term, but not short-term, contextual fear conditioning memory. Long-term contextual fear conditioning memory is attenuated in CaMKII-cre;EphA4(lx/-) mice where EphA4 is removed from all pyramidal neurons of the forebrain. Mutant mice with targeted kinase-dead EphA4 (EphA4(KD)) exhibit intact long-term contextual fear conditioning memory showing that EphA4 kinase-mediated forward signaling is not needed for contextual fear memory formation. The ability to form long-term conditioned taste aversion (CTA) memory is not impaired in the EphB2(-/-) and CaMKII-cre;EphA4(lx/-) mice. We conclude that EphB2 forward signaling is required for long-term contextual fear conditioning memory formation. In contrast, EphB2 mediates short-term contextual fear conditioning memory formation in a forward signaling-independent manner. EphA4 mediates long-term contextual fear conditioning memory formation in a kinase-independent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Interference-mediated synaptonemal complex formation with embedded crossover designation

    Science.gov (United States)

    Zhang, Liangran; Espagne, Eric; de Muyt, Arnaud; Zickler, Denise; Kleckner, Nancy E.

    2014-01-01

    Biological systems exhibit complex patterns at length scales ranging from the molecular to the organismic. Along chromosomes, events often occur stochastically at different positions in different nuclei but nonetheless tend to be relatively evenly spaced. Examples include replication origin firings, formation of chromatin loops along chromosome axes and, during meiosis, localization of crossover recombination sites (“crossover interference”). We present evidence in the fungus Sordaria macrospora that crossover interference is part of a broader pattern that includes synaptonemal complex (SC) nucleation. This pattern comprises relatively evenly spaced SC nucleation sites, among which a subset are crossover sites that show a classical interference distribution. This pattern ensures that SC forms regularly along the entire length of the chromosome as required for the maintenance of homolog pairing while concomitantly having crossover interactions locally embedded within the SC structure as required for both DNA recombination and structural events of chiasma formation. This pattern can be explained by a threshold-based designation and spreading interference process. This model can be generalized to give diverse types of related and/or partially overlapping patterns, in two or more dimensions, for any type of object. PMID:25380597

  18. Mediation Analysis with Multiple Mediators.

    Science.gov (United States)

    VanderWeele, T J; Vansteelandt, S

    2014-01-01

    Recent advances in the causal inference literature on mediation have extended traditional approaches to direct and indirect effects to settings that allow for interactions and non-linearities. In this paper, these approaches from causal inference are further extended to settings in which multiple mediators may be of interest. Two analytic approaches, one based on regression and one based on weighting are proposed to estimate the effect mediated through multiple mediators and the effects through other pathways. The approaches proposed here accommodate exposure-mediator interactions and, to a certain extent, mediator-mediator interactions as well. The methods handle binary or continuous mediators and binary, continuous or count outcomes. When the mediators affect one another, the strategy of trying to assess direct and indirect effects one mediator at a time will in general fail; the approach given in this paper can still be used. A characterization is moreover given as to when the sum of the mediated effects for multiple mediators considered separately will be equal to the mediated effect of all of the mediators considered jointly. The approach proposed in this paper is robust to unmeasured common causes of two or more mediators.

  19. Hippocampal and ventral medial prefrontal activation during retrieval-mediated learning supports novel inference.

    Science.gov (United States)

    Zeithamova, Dagmar; Dominick, April L; Preston, Alison R

    2012-07-12

    Memory enables flexible use of past experience to inform new behaviors. Although leading theories hypothesize that this fundamental flexibility results from the formation of integrated memory networks relating multiple experiences, the neural mechanisms that support memory integration are not well understood. Here, we demonstrate that retrieval-mediated learning, whereby prior event details are reinstated during encoding of related experiences, supports participants' ability to infer relationships between distinct events that share content. Furthermore, we show that activation changes in a functionally coupled hippocampal and ventral medial prefrontal cortical circuit track the formation of integrated memories and successful inferential memory performance. These findings characterize the respective roles of these regions in retrieval-mediated learning processes that support relational memory network formation and inferential memory in the human brain. More broadly, these data reveal fundamental mechanisms through which memory representations are constructed into prospectively useful formats. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. High salinity facilitates dolomite precipitation mediated by Haloferax volcanii DS52

    Science.gov (United States)

    Qiu, Xuan; Wang, Hongmei; Yao, Yanchen; Duan, Yong

    2017-08-01

    Although most modern dolomites occur in hypersaline environments, the effects of elevated salinity on the microbial mediation of dolomite precipitation have not been fully evaluated. Here we report results of dolomite precipitation in association with a batch culture of Haloferax volcanii DS52, a halophilic archaeon, under various salinities (from 120‰ to 360‰) and the impact of salinity on microbe-mediated dolomite formation. The mineral phases, morphology and atomic arrangement of the precipitates were analyzed by XRD, SEM and TEM, respectively. The amount of amino acids on the archaeal cell surface was quantified by HPLC/MS. The XRD analysis indicated that disordered dolomite formed successfully with the facilitation of cells harvested from cultures with relatively high salinities (200‰ and 280‰) but was not observed in association with cells harvested from cultures with lower salinity (120‰) or the lysates of cells harvested from extremely high salinity (360‰). The TEM analysis demonstrated that the crystals from cultures with a salinity of 200‰ closely matched that of dolomite. Importantly, we found that more carboxyl groups were presented on the cell surface under high salinity conditions to resist the high osmotic pressure, which may result in the subsequent promotion of dolomite formation. Our finding suggests a link between variations in the hydro-chemical conditions and the formation of dolomite via microbial metabolic activity and enhances our understanding about the mechanism of microbially mediated dolomite formation under high salinity conditions.

  1. Singlet oxygen-mediated formation of protein peroxides within cells

    International Nuclear Information System (INIS)

    Wright, A.; Policarpio, V.

    2003-01-01

    Full text: Singlet oxygen is generated by a number of cellular, enzymatic and chemical reactions as well as by exposure to UV, or visible light in the presence of a sensitizer; as a consequence this oxidant has been proposed as a damaging agent in a number of pathologies including photo-aging and skin cancer. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study it is shown that illumination of viable, sensitizer-loaded, THP-1 (human monocyte-like) cells with visible light gives rise to intra-cellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of the sensitizer, is enhanced in D 2 O, and decreased by azide; these data are consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to ca. 1.5 nmoles peroxide per 10 6 cells using rose bengal as sensitizer, or 10 nmoles per mg cell protein and account for up to ca. 15% of the O 2 consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, the cellular protein peroxide levels decreases with t 1/2 ca. 4 hrs at 37 deg C, and this is associated with increased cell lysis. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions, gives rise to radicals as detected by EPR spin trapping. These protein peroxides, and radicals derived from them, can inactivate key cellular enzymes (including caspases, GAPDH and glutathione reductase) and induce DNA base oxidation, strand breaks and DNA-protein cross-links. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer gives rise to novel long-lived, but reactive, intra-cellular protein peroxides via singlet oxygen-mediated

  2. HAb18G/CD147 regulates vinculin-mediated focal adhesion and cytoskeleton organization in cultured human hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Qiang Liang

    Full Text Available Focal adhesions (FAs, integrin-mediated macromolecular complexes located at the cell membrane extracellular interface, have been shown to regulate cell adhesion and migration. Our previous studies have indicated that HAb18G/CD147 (CD147 is involved in cytoskeleton reorganization and FA formation in human hepatocellular carcinoma (HCC cells. However, the precise mechanisms underlying these processes remain unclear. In the current study, we determined that CD147 was involved in vinculin-mediated FA focal adhesion formation in HCC cells. We also found that deletion of CD147 led to reduced vinculin-mediated FA areas (P<0.0001, length/width ratios (P<0.0001, and mean intensities (P<0.0001. CD147 promoted lamellipodia formation by localizing Arp2/3 to the leading edge of the cell. Deletion of CD147 significantly reduced the fluorescence (t1/2 recovery times (22.7±3.3 s of vinculin-mediated focal adhesions (P<0.0001. In cell-spreading assays, CD147 was found to be essential for dynamic focal adhesion enlargement and disassembly. Furthermore, the current data showed that CD147 reduced tyrosine phosphorylation in vinculin-mediated focal adhesions, and enhanced the accumulation of the acidic phospholipid phosphatidylinositol-4, 5-bisphosphate (PIP2. Together, these results revealed that CD147 is involved in vinculin-mediated focal adhesion formation, which subsequently promotes cytoskeleton reorganization to facilitate invasion and migration of human HCC cells.

  3. Concept Formation Skills in Long-Term Cochlear Implant Users

    OpenAIRE

    Castellanos, Irina; Kronenberger, William G.; Beer, Jessica; Colson, Bethany G.; Henning, Shirley C.; Ditmars, Allison; Pisoni, David B.

    2014-01-01

    This study investigated if a period of auditory sensory deprivation followed by degraded auditory input and related language delays affects visual concept formation skills in long-term prelingually deaf cochlear implant (CI) users. We also examined if concept formation skills are mediated or moderated by other neurocognitive domains (i.e., language, working memory, and executive control). Relative to normally hearing (NH) peers, CI users displayed significantly poorer performance in several s...

  4. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    Directory of Open Access Journals (Sweden)

    Chantelle Ahlenstiel

    2015-01-01

    Full Text Available Transcriptional gene silencing (TGS of mammalian genes can be induced by short interfering RNA (siRNA targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA, which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation.

  5. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Somanath, Sangeeta [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Partridge, Christopher J. [Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Churchill Hospital, University of Oxford, Oxford, OX3 7LJ (United Kingdom); Marshall, Catriona [Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT (United Kingdom); Rowe, Tony [CSL Limited, 45 Poplar Road, Parkville, Victoria 3052 (Australia); Turner, Mark D., E-mail: mark.turner@ntu.ac.uk [Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, NG11 8NS (United Kingdom)

    2016-04-29

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  6. Snapin mediates insulin secretory granule docking, but not trans-SNARE complex formation

    International Nuclear Information System (INIS)

    Somanath, Sangeeta; Partridge, Christopher J.; Marshall, Catriona; Rowe, Tony; Turner, Mark D.

    2016-01-01

    Secretory granule exocytosis is a tightly regulated process requiring granule targeting, tethering, priming, and membrane fusion. At the heart of this process is the SNARE complex, which drives fusion through a coiled-coil zippering effect mediated by the granule v-SNARE protein, VAMP2, and the plasma membrane t-SNAREs, SNAP-25 and syntaxin-1A. Here we demonstrate that in pancreatic β-cells the SNAP-25 accessory protein, snapin, C-terminal H2 domain binds SNAP-25 through its N-terminal Sn-1 domain. Interestingly whilst snapin binds SNAP-25, there is only modest binding of this complex with syntaxin-1A under resting conditions. Instead synataxin-1A appears to be recruited in response to secretory stimulation. These results indicate that snapin plays a role in tethering insulin granules to the plasma membrane through coiled coil interaction of snapin with SNAP-25, with full granule fusion competency only resulting after subsequent syntaxin-1A recruitment triggered by secretory stimulation. - Highlights: • Snapin mediates granule docking. • Snapin binds SNAP-25. • SNARE complex forms downstream.

  7. Soluble lymphocytic mediators

    Science.gov (United States)

    Pick, E.

    1974-01-01

    The effect of a number of drugs on the production of macrophage migration inhibitory factor (MIF) by antigen-stimulated sensitized guinea-pig lymph node cells was studied. The drugs were present during the entire culture period and eliminated from supernatants by dialysis. It was found that MIF secretion is inhibited by exogenous dibutyryl cyclic AMP and by theophylline and chlorphenesin, two agents raising the endogenous level of cyclic AMP. On the other hand, isoproterenol, which stimulates cyclic AMP generation in several tissues, did not block MIF production. The formation of the mediator was also suppressed by the microfilament-affecting drug, cytochalasin B. The microtubular disruptive agents, colchicine and vinblastine sulphate, did not influence MIF production. It is concluded that: (a) endogenous cyclic AMP may act as a regulator of MIF production; (b) the activity of contractile microfilaments is probably required for MIF formation; and (c) microtubules are not involved in the secretory process. PMID:4369184

  8. Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis

    DEFF Research Database (Denmark)

    Hawkins, C L; Brown, B E; Davies, Michael Jonathan

    2001-01-01

    . In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin...... trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane......-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently...

  9. Proinflammatory Adhesion Molecules Facilitate Polychlorinated Biphenyl–Mediated Enhancement of Brain Metastasis Formation

    OpenAIRE

    Sipos, Eszter; Chen, Lei; András, Ibolya E.; Wrobel, Jagoda; Zhang, Bei; Pu, Hong; Park, Minseon; Eum, Sung Yong; Toborek, Michal

    2012-01-01

    Polychlorinated biphenyls (PCBs) are environmental toxicants that cause vascular inflammation and facilitate the development of brain metastases. The crucial event in metastasis formation is adhesion of blood-borne tumor cells to the vascular endothelium, followed by their transcapillary migration. The aim of the present study was to examine the mechanisms of PCB118-induced brain metastasis formation at the blood-brain barrier level with the focus on tumor cell adhesion to the brain endotheli...

  10. PIC Activation through Functional Interplay between Mediator and TFIIH.

    Science.gov (United States)

    Malik, Sohail; Molina, Henrik; Xue, Zhu

    2017-01-06

    The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Polymer-mediated formation of polyoxomolybdate nanomaterials

    Science.gov (United States)

    Wan, Quan

    A polymer-mediated synthetic pathway to a polyoxomolybdate nanomaterial is investigated in this work. Block copolymers or homopolymers containing poly(ethylene oxide) (PEO) are mixed with a MoO2(OH)(OOH) aqueous solution to form a golden gel or viscous solution. As revealed by synchrotron X-ray scattering measurements, electron microscopy, and other characterization techniques, the final dark blue polyoxomolybdate product is a highly ordered simple cubic network similar to certain zeolite structure but with a much larger lattice constant of ˜5.2 nm. The average size of the cube-like single crystals is close to 1 mum. Based on its relatively low density (˜2.2 g/cm3), the nanomaterial can be highly porous if the amount of the residual polymer can be substantially reduced. The valence of molybdenum is ˜5.7 based on cerimetric titration, representing the mixed-valence nature of the polyoxomolybdate structure. The self-assembled structures (if any) of the polymer gel do not have any correlation with the final polyoxomolybdate nanostructure, excluding the possible role of polymers being a structure-directing template. On the other hand, the PEO polymer stabilizes the precursor molybdenum compound through coordination between its ether oxygen atoms and molybdenum atoms, and reduces the molybdenum (VI) precursor compound with its hydroxyl group being a reducing agent. The rare simple cubic ordering necessitates the existence of special affinities among the polyoxomolybdate nanosphere units resulted from the reduction reaction. Our mechanism study shows that the acidified condition is necessary for the synthesis of the mixed-valence polyoxomolybdate clusters, while H2O2 content modulates the rate of the reduction reaction. The polymer degradation is evidenced by the observation of a huge viscosity change, and is likely through a hydrolysis process catalyzed by molybdenum compounds. Cube-like polyoxomolybdate nanocrystals with size of ˜40 nm are obtained by means of

  12. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian; Skjoedt, Mikkel-Ole

    2010-01-01

    Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation...... was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides...

  13. Contribution of T cell-mediated immunity to the resistance to staphlococcal infection

    International Nuclear Information System (INIS)

    Tsuda, S.; Sasai, Y.; Minami, K.; Nomoto, K.

    1978-01-01

    Abscess formation in nude mice after subcutaneous inoculation of Staphylococcus aureus (S. aureus) was more extensive and prolonged as compared with that in phenotypically normal littermates. Abscess formation in nude mice was augmented markedly by whole-body irradiation. Not only T cell-mediated immunity but also radiosensitive, nonimmune phagocytosis appear to contribute to the resistance against staphylococcal infection

  14. Underpotential deposition-mediated layer-by-layer growth of thin films

    Science.gov (United States)

    Wang, Jia Xu; Adzic, Radoslav R.

    2015-05-19

    A method of depositing contiguous, conformal submonolayer-to-multilayer thin films with atomic-level control is described. The process involves the use of underpotential deposition of a first element to mediate the growth of a second material by overpotential deposition. Deposition occurs between a potential positive to the bulk deposition potential for the mediating element where a full monolayer of mediating element forms, and a potential which is less than, or only slightly greater than, the bulk deposition potential of the material to be deposited. By cycling the applied voltage between the bulk deposition potential for the mediating element and the material to be deposited, repeated desorption/adsorption of the mediating element during each potential cycle can be used to precisely control film growth on a layer-by-layer basis. This process is especially suitable for the formation of a catalytically active layer on core-shell particles for use in energy conversion devices such as fuel cells.

  15. Replication-independent chromatin loading of Dnmt1 during G2 and M phases

    Science.gov (United States)

    Easwaran, Hariharan P; Schermelleh, Lothar; Leonhardt, Heinrich; Cardoso, M Cristina

    2004-01-01

    The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation. PMID:15550930

  16. History of the Institute of Mediation in India

    Directory of Open Access Journals (Sweden)

    Ekaterina P. Rusakova

    2016-12-01

    Full Text Available In this article author studies history of the institute of mediation formation and development in India. In the process of research author emphasizes that as a consequence of India's rapid economic development, number of disputes that are associated with them inevitably increases. Mediation is one of the popular methods of resolving disputes these days. In conclusion author emphasizes that the place and role of mediation is explained not only by the modern business culture of this country, but also because from a long time the population preferred to resolve conflicts through negotiations and with the participation of mediators. And the fact that the ancient model of peaceful settlement of disputes has survived to this day is due to the high level of poverty, cultural and religious diversity, and the preservation of traditional and community values and norms. It can be stated that the use of mediation, among other things, including traditional forms of alternative dispute resolution, has had a beneficial effect on the Indian judicial system, both in resolving problems with excessive workload on courts, and in delaying the adjudication of disputes.

  17. Optimisation of Surface-Initiated Photoiniferter-Mediated Polymerisation under Confinement, and the Formation of Block Copolymers in Mesoporous Films

    Directory of Open Access Journals (Sweden)

    Jessica C. Tom

    2017-10-01

    Full Text Available Nature as the ultimate inspiration can direct, gate, and selectively transport species across channels to fulfil a specific targeted function. Harnessing such precision over local structure and functionality at the nanoscale is expected to lead to indispensable developments in synthetic channels for application in catalysis, filtration and sensing, and in drug delivery. By combining mesoporous materials with localised charge-switchable poly(2-(dimethylaminoethyl methacrylate (PDMAEMA brushes, precisely controlling pore filling and exploring the possibility of incorporating two different responsive polymers, we hope to approach the precision control of natural systems in the absence of an external force. Here, we report a simple one-step approach to prepare a mesoporous silica thin film with ~8 nm pores functionalised with a photoiniferter by combining sol–gel chemistry and evaporation-induced self-assembly (EISA. We show that surface-initiated photoiniferter-mediated polymerisation (SI-PIMP allows the incorporation of a high polymer content up to geometrical pore blocking by the simple application of UV light in the presence of a monomer and solvent, proceeding in a controlled manner in pore sizes below 10 nm, with the potential to tune the material properties through the formation of surface-grafted block copolymers.

  18. The Mediator complex: a master coordinator of transcription and cell lineage development.

    Science.gov (United States)

    Yin, Jing-wen; Wang, Gang

    2014-03-01

    Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

  19. Role of the synaptobrevin C terminus in fusion pore formation

    DEFF Research Database (Denmark)

    Ngatchou, Annita N; Kisler, Kassandra; Fang, Qinghua

    2010-01-01

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore...... stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular...

  20. Mechanism of Nitrogenase H 2 Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

    Energy Technology Data Exchange (ETDEWEB)

    Khadka, Nimesh [Department of Chemistry; Milton, Ross D. [Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States; Shaw, Sudipta [Department of Chemistry; Lukoyanov, Dmitriy [Department; Dean, Dennis R. [Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States; Minteer, Shelley D. [Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States; Raugei, Simone [Pacific Northwest National Laboratory, Richland, Washington 99352, United States; Hoffman, Brian M. [Department; Seefeldt, Lance C. [Department of Chemistry

    2017-09-15

    Nitrogenase catalyzes the reduction of dinitrogen (N2) to ammonia (NH3) with obligatory reduction of protons (H+) to dihydrogen (H2) through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides at its active site FeMo-cofactor. The overall rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to MoFe protein without Fe protein and ATP hydrolysis, thereby eliminating the normal rate-limiting step. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, changes linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate limiting step. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the proton from S-H+ moves to the hydride in Fe-H-, predicting the number and magnitude of the observed H/D isotope effect. This study not only reveals the mechanism of H2 formation, but also illustrates a strategy for mechanistic study that can be applied to other enzymes and to biomimetic complexes.

  1. Signaling factors in stem cell-mediated repair of infarcted myocardium

    NARCIS (Netherlands)

    Vandervelde, S; van Luyn, MJA; Tio, RA; Harmsen, MC

    Myocardial infarction leads to scar formation and subsequent reduced cardiac performance. The ultimate therapy after myocardial infarction would pursue stem cell-based regeneration. The aim of stem cell-mediated cardiac repair embodies restoration of cardiac function by regeneration of healthy

  2. Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons

    Science.gov (United States)

    Murphy, Diane D.; Cole, Nelson B.; Segal, Menahem

    1998-01-01

    Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons. PMID:9736750

  3. The impact of vaporized nanoemulsions on ultrasound-mediated ablation.

    Science.gov (United States)

    Zhang, Peng; Kopechek, Jonathan A; Porter, Tyrone M

    2013-01-01

    The clinical feasibility of using high-intensity focused ultrasound (HIFU) for ablation of solid tumors is limited by the high acoustic pressures and long treatment times required. The presence of microbubbles during sonication can increase the absorption of acoustic energy and accelerate heating. However, formation of microbubbles within the tumor tissue remains a challenge. Phase-shift nanoemulsions (PSNE) have been developed as a means for producing microbubbles within tumors. PSNE are emulsions of submicron-sized, lipid-coated, and liquid perfluorocarbon droplets that can be vaporized into microbubbles using short (5 MPa) acoustic pulses. In this study, the impact of vaporized phase-shift nanoemulsions on the time and acoustic power required for HIFU-mediated thermal lesion formation was investigated in vitro. PSNE containing dodecafluoropentane were produced with narrow size distributions and mean diameters below 200 nm using a combination of sonication and extrusion. PSNE was dispersed in albumin-containing polyacrylamide gel phantoms for experimental tests. Albumin denatures and becomes opaque at temperatures above 58°C, enabling visual detection of lesions formed from denatured albumin. PSNE were vaporized using a 30-cycle, 3.2-MHz, at an acoustic power of 6.4 W (free-field intensity of 4,586 W/cm(2)) pulse from a single-element, focused high-power transducer. The vaporization pulse was immediately followed by a 15-s continuous wave, 3.2-MHz signal to induce ultrasound-mediated heating. Control experiments were conducted using an identical procedure without the vaporization pulse. Lesion formation was detected by acquiring video frames during sonication and post-processing the images for analysis. Broadband emissions from inertial cavitation (IC) were passively detected with a focused, 2-MHz transducer. Temperature measurements were acquired using a needle thermocouple. Bubbles formed at the HIFU focus via PSNE vaporization enhanced HIFU-mediated heating

  4. Data Mediation with Enterprise Level Security

    Directory of Open Access Journals (Sweden)

    Kevin E. Foltz

    2017-10-01

    Full Text Available Enterprise Level Security (ELS is an architecture for enabling information sharing with strong security guarantees. It is built upon basic tenets and concepts that shape its component technologies and implementation. One challenge in sharing information is that the source and recipient of the information may use different units, protocols, data formats, or tools to process information. As a result, a transformation of the data is needed before the recipient can use the information. These conversions introduce potential security weaknesses into an ELS system, so an approach for enterprise-wide mediation is required. Methods in common use today, such as a man-in-the-middle (MITM translation and an online mediation service, do not preserve the basic ELS tenets and concepts. This paper examines these existing approaches and compares them with two new approaches designed to preserve ELS security. It looks at the complete picture of security, performance, and ease of implementation, offering a framework for choosing the best mediation approach based on the data sharing context.

  5. Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis.

    Science.gov (United States)

    Erkhembaatar, Munkhsoyol; Choi, Eun-Joo; Lee, Hak-Yong; Lee, Choong Hun; Lee, Young-Rae; Kim, Min Seuk

    2015-07-14

    Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca(2+) mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. Our results demonstrated that POEE suppressed RANKL-induced Ca(2+) oscillations by inhibition of Ca(2+) release from internal Ca(2+) stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.

  6. Collective motion of cells mediates segregation and pattern formation in co-cultures.

    Directory of Open Access Journals (Sweden)

    Elod Méhes

    Full Text Available Pattern formation by segregation of cell types is an important process during embryonic development. We show that an experimentally yet unexplored mechanism based on collective motility of segregating cells enhances the effects of known pattern formation mechanisms such as differential adhesion, mechanochemical interactions or cell migration directed by morphogens. To study in vitro cell segregation we use time-lapse videomicroscopy and quantitative analysis of the main features of the motion of individual cells or groups. Our observations have been extensive, typically involving the investigation of the development of patterns containing up to 200,000 cells. By either comparing keratocyte types with different collective motility characteristics or increasing cells' directional persistence by the inhibition of Rac1 GTP-ase we demonstrate that enhanced collective cell motility results in faster cell segregation leading to the formation of more extensive patterns. The growth of the characteristic scale of patterns generally follows an algebraic scaling law with exponent values up to 0.74 in the presence of collective motion, compared to significantly smaller exponents in case of diffusive motion.

  7. Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation

    DEFF Research Database (Denmark)

    Brochmann, Rikke Prejh; Toft, Anders; Ciofu, Oana

    2014-01-01

    The bactericidal effect of several major types of antibiotics has recently been demonstrated to be dependent on the formation of toxic amounts of hydroxyl radicals (OH·) resulting from oxidative stress in metabolically active cells. Since killing by the antimicrobial peptide colistin does...... not require bacterial metabolic activity, we tested whether the bactericidal effect of colistin depends on the formation of OH·. In Pseudomonas aeruginosa cultures, OH-mediated killing by ciprofloxacin was demonstrated by decreased bacterial survival and induction of 3'-(p-hydroxyphenyl) fluorescein (HPF......) fluorescence. OH·-mediated killing by ciprofloxacin was further confirmed by rescue of cells and reduction of HPF fluorescence due to prevention of OH· accumulation by scavenging with thiourea, by chelating with dipyridyl, by decreasing metabolism as well as by anoxic growth. In contrast, no formation of OH...

  8. Genome-wide identification of GLABRA3 downstream genes for anthocyanin biosynthesis and trichome formation in Arabidopsis.

    Science.gov (United States)

    Gao, Chenhao; Li, Dong; Jin, Changyu; Duan, Shaowei; Qi, Shuanghui; Liu, Kaige; Wang, Hanchen; Ma, Haoli; Hai, Jiangbo; Chen, Mingxun

    2017-04-01

    GLABRA3 (GL3), a bHLH transcription factor, has previously proved to be involved in anthocyanin biosynthesis and trichome formation in Arabidopsis, however, its downstream targeted genes are still largely unknown. Here, we found that GL3 was widely present in Arabidopsis vegetative and reproductive organs. New downstream targeted genes of GL3 for anthocyanin biosynthesis and trichome formation were identified in young shoots and expanding true leaves by RNA sequencing. GL3-mediated gene expression was tissue specific in the two biological processes. This study provides new clues to further understand the GL3-mediated regulatory network of anthocyanin biosynthesis and trichome formation in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Hyperglycemia-Induced Modulation of the Physiognomy and Angiogenic Potential of Fibroblasts Mediated by Matrix Metalloproteinase-2: Implications for Venous Stenosis Formation Associated with Hemodialysis Vascular Access in Diabetic Milieu.

    Science.gov (United States)

    Janardhanan, Rajiv; Kilari, Sreenivasulu; Leof, Edward B; Misra, Sanjay

    2015-01-01

    It is hypothesized that venous stenosis formation associated with hemodialysis vascular-access failure is caused by hypoxia-mediated fibroblast-to-myofibroblast differentiation accompanied by proliferation and migration, and that diabetic patients have worse clinical outcomes. The aim of this study was to determine the functional and gene expression outcomes of matrix metalloproteinase-2 (Mmp-2) silencing in fibroblasts cultured under hyperglycemia and euglycemia with hypoxic and normoxic stimuli. AKR-2B fibroblasts were stably transduced using lentivirus-mediated shRNA-Mmp-2 or scrambled controls and subjected to hypoxia or normoxia under hyperglycemic or euglycemic conditions for 24 and 72 h. Gene expression of vascular endothelial growth factor-A (Vegf-A), Vegfr-1, Mmp-2, Mmp-9 and tissue inhibitors of matrix metalloproteinases (Timps) were determined by RT-PCR. Collagen I and IV secretion and cellular proliferation and migration were determined. Under hyperglycemic conditions, there is a significant reduction in the average gene expression of Vegf-A and Mmp-9, with an increase in Timp-1 at 24 h of hypoxia (p < 0.05) in Mmp-2-silenced fibroblasts when compared to controls. In addition, there is a decrease in collagen I and IV secretion and cellular migration. The euglycemic cells were able to reverse these findings. These findings demonstrate the rationale for using anti-Mmp-2 therapy in dialysis patients with hemodialysis vascular access in helping to reduce stenosis formation. © 2016 The Author(s) Published by S. Karger AG, Basel.

  10. Mediator subunit18 controls flowering time and floral organ identity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Zhengui Zheng

    Full Text Available Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18 affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC and AGAMOUS (AG. A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.

  11. DNA-mediated bacterial aggregation is dictated by acid-base interactions

    NARCIS (Netherlands)

    Das, Theerthankar; Krom, Bastiaan P.; van der Mei, Henny C.; Busscher, Henk J.; Sharma, Prashant K.

    2011-01-01

    Extracellular DNA (eDNA) plays a significant role in bacterial biofilm formation and aggregation. Here, for the first time, we present a physico-chemical analysis of the DNA-mediated aggregation for three bacterial strains (Streptococcus mutans LT11, Pseudomonas aeruginosa PAO1 and Staphylococcus

  12. Non-Identity-Mediated CRISPR-Bacteriophage Interaction Mediated via the Csy and Cas3 Proteins ▿#

    Science.gov (United States)

    Cady, Kyle C.; O'Toole, George A.

    2011-01-01

    Studies of the Escherichia, Neisseria, Thermotoga, and Mycobacteria clustered regularly interspaced short palindromic repeat (CRISPR) subtypes have resulted in a model whereby CRISPRs function as a defense system against bacteriophage infection and conjugative plasmid transfer. In contrast, we previously showed that the Yersinia-subtype CRISPR region of Pseudomonas aeruginosa strain UCBPP-PA14 plays no detectable role in viral immunity but instead is required for bacteriophage DMS3-dependent inhibition of biofilm formation by P. aeruginosa. The goal of this study is to define the components of the Yersinia-subtype CRISPR region required to mediate this bacteriophage-host interaction. We show that the Yersinia-subtype-specific CRISPR-associated (Cas) proteins Csy4 and Csy2 are essential for small CRISPR RNA (crRNA) production in vivo, while the Csy1 and Csy3 proteins are not absolutely required for production of these small RNAs. Further, we present evidence that the core Cas protein Cas3 functions downstream of small crRNA production and that this protein requires functional HD (predicted phosphohydrolase) and DEXD/H (predicted helicase) domains to suppress biofilm formation in DMS3 lysogens. We also determined that only spacer 1, which is not identical to any region of the DMS3 genome, mediates the CRISPR-dependent loss of biofilm formation. Our evidence suggests that gene 42 of phage DMS3 (DMS3-42) is targeted by CRISPR2 spacer 1 and that this targeting tolerates multiple point mutations between the spacer and DMS3-42 target sequence. This work demonstrates how the interaction between P. aeruginosa strain UCBPP-PA14 and bacteriophage DMS3 can be used to further our understanding of the diverse roles of CRISPR system function in bacteria. PMID:21398535

  13. Inhibition of neointima formation by local delivery of estrogen receptor alpha and beta specific agonists

    NARCIS (Netherlands)

    Krom, Y.D.; Pires, N.M.M.; Jukema, J.W.; Vries, M.R. de; Frants, R.R.; Havekes, L.M.; Dijk, K.W. van; Quax, P.H.A.

    2007-01-01

    Objective: Neointima formation is the underlying mechanism of (in-stent) restenosis. 17β-Estradiol (E2) is known to inhibit injury-induced neointima formation and post-angioplasty restenosis. Estrogen receptor alpha (ERα) has been demonstrated to mediate E2 anti-restenotic properties. However, the

  14. Nanoscale E-Cadherin ligand patterns show threshold size for cellular adhesion and adherence junction formation

    DEFF Research Database (Denmark)

    Kristensen, Stine H; Pedersen, Gitte Albinus; Nejsum, Lene Niemann

    2012-01-01

    The role of ligand spatial distribution on the formation of cadherin mediated cell-cell contacts is studied utilizing nanopatterns of E-cadherin ligands. Protein patches ranging in size from 100 nm to 800 nm prepared by colloidal lithography critically influence adhesion, spreading and formation ...

  15. Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

    Science.gov (United States)

    Rolloos, Martijn; Hooykaas, Paul J J; van der Zaal, Bert J

    2015-02-09

    Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

  16. A critical role of platelet TGF-β release in podoplanin-mediated tumour invasion and metastasis.

    Science.gov (United States)

    Takemoto, Ai; Okitaka, Mina; Takagi, Satoshi; Takami, Miho; Sato, Shigeo; Nishio, Makoto; Okumura, Sakae; Fujita, Naoya

    2017-02-08

    The tumour microenvironment is critical for various characteristics of tumour malignancies. Platelets, as part of the tumour microenvironment, are associated with metastasis formation via increasing the rate of tumour embolus formation in microvasculature. However, the mechanisms underlying the ability of tumour cells to acquire invasiveness and extravasate into target organs at the site of embolization remain unclear. In this study, we reported that platelet aggregation-inducing factor podoplanin expressed on tumour cell surfaces were found to not only promote the formation of tumour-platelet aggregates via interaction with platelets, but also induced the epithelial-mesenchymal transition (EMT) of tumour cells by enhancing transforming growth factor-β (TGF-β) release from platelets. In vitro and in vivo analyses revealed that podoplanin-mediated EMT resulted in increased invasiveness and extravasation of tumour cells. Treatment of mice with a TGF-β-neutralizing antibody statistically suppressed podoplanin-mediated distant metastasis in vivo, suggesting that podoplanin promoted haematogenous metastasis in part by releasing TGF-β from platelets that was essential for EMT of tumour cells. Therefore, our findings suggested that blocking the TGF-β signalling pathway might be a promising strategy for suppressing podoplanin-mediated haematogenous metastasis in vivo.

  17. Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

    Science.gov (United States)

    Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

    2015-01-01

    Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

  18. Grounding formative assessment in high-school chemistry classrooms: Connections between professional development and teacher practice

    Science.gov (United States)

    Cisterna Alburquerque, Dante Igor

    This study describes and analyzes the experiences of two high-school chemistry teachers who participated in a team-based professional development program to learn about and enact formative assessment in their classrooms. The overall purpose of this study is to explain how participation in this professional development influenced both teachers' classroom enactment of formative assessment practices. This study focuses on 1) teachers' participation in the professional development program, 2) teachers' enactment of formative assessment, and 3) factors that enabled or hindered enactment of formative assessment. Drawing on cultural-historical activity theory (CHAT) and using evidence from teacher lessons, teacher interviews, professional development meetings as data sources, this single embedded case study analyzes how these two teachers who participated in the same learning team and have similar characteristics (i.e., teaching in the same school, teaching the same courses and population of students, and using the same materials) differentially used the professional development learning about formative assessment as mediating tools to improve their classroom instruction. The learning team experience contributed to both teachers' development of a better understanding of formative assessment---especially in recognizing that their current grading and assessment practices were not appropriate to promote student learning---and the co-creation of artifacts to gather evidence of students' ideas. Although both teachers demonstrated understanding about how formative assessment may serve to promote student learning and had a set of tools available to utilize for formative assessment use, they did not enact these tools in the same way. One teacher appropriated formative assessment as mediating tool to verify if the students were following her explanations, and to check if the students were able to provide the correct response. The other teacher used the mediating tool to promote

  19. Mediation Analysis with Multiple Mediators

    OpenAIRE

    VanderWeele, T.J.; Vansteelandt, S.

    2014-01-01

    Recent advances in the causal inference literature on mediation have extended traditional approaches to direct and indirect effects to settings that allow for interactions and non-linearities. In this paper, these approaches from causal inference are further extended to settings in which multiple mediators may be of interest. Two analytic approaches, one based on regression and one based on weighting are proposed to estimate the effect mediated through multiple mediators and the effects throu...

  20. Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin

    OpenAIRE

    Have, ten, R.

    2000-01-01

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich medium. This procedure resulted in a successful LiP production of 600 U/L. Peptone in the culture medium was shown to interfere with the standard LiP assay in which the formation of veratraldehyde (V...

  1. Therapeutic Applicability of Anti-Inflammatory and Proresolving Polyunsaturated Fatty Acid-Derived Lipid Mediators

    Directory of Open Access Journals (Sweden)

    Gerard L. Bannenberg

    2010-01-01

    Full Text Available The enzymatic oxygenation of polyunsaturated fatty acids by lipoxygenases and cyclo-oxygenases is a resourceful mode of formation of specific autacoids that regulate the extent and pace of the inflammatory response. Arachidonate-derived eicosanoids, such as lipoxin A4, prostaglandin (PGD2, PGF2α, PGE2, and PGD2-derived cyclopentenones exert specific roles in counter-regulating inflammation and turning on resolution. Recently recognized classes of autacoids derived from long-chain ω-3 polyunsaturated fatty acids, the E- and D-series resolvins, protectin D1, and maresin 1, act as specialized mediators to dampen inflammation actively, afford tissue protection, stimulate host defense, and activate resolution. It is held that counter-regulatory lipid mediators and the specific molecular pathways activated by such endogenous agonists may be suitable for pharmacological use in the treatment of inflammatory disease. The anti-inflammatory drug aspirin is a striking example of a drug that is able to act in such a manner, namely through triggering the formation of 15-epi-lipoxin A4 and aspirin-triggered resolvins. Different aspects of the therapeutic applicability of lipid mediators have been addressed here, and indicate that the development of innovative pharmacotherapy based on anti-inflammatory and proresolution lipid mediators presents novel prospects for the treatment of inflammatory disease.

  2. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  3. Cylindromatosis mediates neuronal cell death in vitro and in vivo.

    Science.gov (United States)

    Ganjam, Goutham K; Terpolilli, Nicole Angela; Diemert, Sebastian; Eisenbach, Ina; Hoffmann, Lena; Reuther, Christina; Herden, Christiane; Roth, Joachim; Plesnila, Nikolaus; Culmsee, Carsten

    2018-01-19

    The tumor-suppressor cylindromatosis (CYLD) is a deubiquitinating enzyme and key regulator of cell proliferation and inflammation. A genome-wide siRNA screen linked CYLD to receptor interacting protein-1 (RIP1) kinase-mediated necroptosis; however, the exact mechanisms of CYLD-mediated cell death remain unknown. Therefore, we investigated the precise role of CYLD in models of neuronal cell death in vitro and evaluated whether CYLD deletion affects brain injury in vivo. In vitro, downregulation of CYLD increased RIP1 ubiquitination, prevented RIP1/RIP3 complex formation, and protected neuronal cells from oxidative death. Similar protective effects were achieved by siRNA silencing of RIP1 or RIP3 or by pharmacological inhibition of RIP1 with necrostatin-1. In vivo, CYLD knockout mice were protected from trauma-induced brain damage compared to wild-type littermate controls. These findings unravel the mechanisms of CYLD-mediated cell death signaling in damaged neurons in vitro and suggest a cell death-mediating role of CYLD in vivo.

  4. Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin

    NARCIS (Netherlands)

    Have, ten R.

    2000-01-01

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich

  5. Characterization of novel methyltransferases METTL22 and FAM86A.1

    OpenAIRE

    Ali, Qamar

    2012-01-01

    Proteins are subjected to various post-translational modifications (PTMs) that affect their activity, interaction and localization. Methylation is one such PTM that is well known to play a regulatory role in heterochromatin and euchromatin formation through methyl marks on histone tails, and it has recently been shown that regulation through methylation is also applicable to non-histone proteins. A recent characterization of protein methyltransferase (MTase) METTL21D led to the discovery of a...

  6. The mediator complex in genomic and non-genomic signaling in cancer.

    Science.gov (United States)

    Weber, Hannah; Garabedian, Michael J

    2018-05-01

    Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

    Science.gov (United States)

    Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

    2015-07-01

    Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  8. ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

    International Nuclear Information System (INIS)

    Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

    2013-01-01

    Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

  9. Waste treatment in NUCEF facility with silver mediated electrochemical oxidation technique

    International Nuclear Information System (INIS)

    Umeda, M.; Sugikawa, S.

    2000-01-01

    Silver mediated electrochemical oxidation technique has been considered one of promising candidates for alpha-bearing waste treatment. Destruction tests of organic compounds, such as insoluble tannin, TBP and dodecane, were carried out by this technique and the experimental data such as destruction rates, current efficiencies and intermediates were obtained. These compounds could be completely mineralized without the formation of reactive organic nitrate associated to safety hazards. On the basis of these results, the applicability of silver mediated electrochemical oxidation technique to waste treatment in NUCEF was evaluated. (authors)

  10. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis

    2013-10-01

    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

  11. Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity.

    Science.gov (United States)

    Hu, Ping; Huang, Ping; Chen, Min Wei

    2013-10-01

    Sortase A is an enzyme responsible for the covalent attachment of Pac proteins to the cell wall in Streptococcus mutans. It has been shown to play a role in modulating the surface properties and the biofilm formation and influence the cariogenicity of S. mutans. Curcumin, an active ingredient of turmeric, was reported to be an inhibitor for Staphylococcus aureus sortase A. The aim of this study was to investigate the inhibitory ability of curcumin against S. mutans sortase A and the effect of curcumin for biofilm formation. The antimicrobial activity of the curcumin to the S. mutans and inhibitory ability of the curcumin against the purified sortase A in vitro were detected. Western-blot and real-time PCR were used to analysis the sortase A mediated Pac protein changes when the S. mutans was cultured with curcumin. The curcumin on the S. mutans biofilm formation was determined by biofilm formation analysis. Curcumin can inhibit purified S. mutans sortase A with a half-maximal inhibitory concentration (IC50) of (10.2±0.7)μmol/l, which is lower than minimum inhibitory concentration (MIC) of 175μmol/l. Curcumin (15μmol/l) was found to release the Pac protein to the supernatant and reduce S. mutans biofilm formation. These results indicated that curcumin is an S. mutans sortase A inhibitor and has promising anti-caries characteristics through an anti-adhesion-mediated mechanism. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  12. Mediation analysis with time varying exposures and mediators.

    Science.gov (United States)

    VanderWeele, Tyler J; Tchetgen Tchetgen, Eric J

    2017-06-01

    In this paper we consider causal mediation analysis when exposures and mediators vary over time. We give non-parametric identification results, discuss parametric implementation, and also provide a weighting approach to direct and indirect effects based on combining the results of two marginal structural models. We also discuss how our results give rise to a causal interpretation of the effect estimates produced from longitudinal structural equation models. When there are time-varying confounders affected by prior exposure and mediator, natural direct and indirect effects are not identified. However, we define a randomized interventional analogue of natural direct and indirect effects that are identified in this setting. The formula that identifies these effects we refer to as the "mediational g-formula." When there is no mediation, the mediational g-formula reduces to Robins' regular g-formula for longitudinal data. When there are no time-varying confounders affected by prior exposure and mediator values, then the mediational g-formula reduces to a longitudinal version of Pearl's mediation formula. However, the mediational g-formula itself can accommodate both mediation and time-varying confounders and constitutes a general approach to mediation analysis with time-varying exposures and mediators.

  13. Rab5 induces Rac-independent lamellipodia formation and cell migration

    NARCIS (Netherlands)

    Spaargaren, M.; Bos, J. L.

    1999-01-01

    Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative

  14. RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

    Science.gov (United States)

    Ouyang, Jie-Xiu; Luo, Tao; Sun, Hui-Yun; Huang, Jian; Tang, Dan-Feng; Wu, Lei; Zheng, Yue-Hui; Zheng, Li-Ping

    2013-08-01

    Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

  15. Exploring the Mediating Effect of E-social Capital Between Community Members Interaction and Consumer Engagement

    OpenAIRE

    Yan Bingsheng; Li Lihua; Sun Hongtao

    2017-01-01

    This article explored the effect of instrumental interaction and relational interaction on consumer engagement (community engagement and brand engagement) among community members. The mediating effect of E-social capital was investigated as well. The research results showed that: both instrumental interaction and interpersonal interaction promote the formation of E-social capital (online trust and online reciprocity); online trust plays a partial mediating role between community interaction (...

  16. Role of β-h elimination in rhodium-mediated carbene insertion polymerization

    NARCIS (Netherlands)

    Finger, M.; Reek, J.N.H.; de Bruin, B.

    2011-01-01

    The importance of β-H elimination as a possible mechanism to induce chain termination/transfer and/or the formation of stereodefects in the Rh(diene)-mediated oligomerization and polymerization of carbenes has been studied by means of different approaches. As a remarkable feature, this reaction is

  17. Visible-light-mediated selective arylation of cysteine in batch and flow

    NARCIS (Netherlands)

    Bottecchia, C.; Rubens, M.; Gunnoo, S.B.; Hessel, V.; Madder, A.

    2017-01-01

    A mild visible-light-mediated strategy for cysteine arylation is presented. The method relies on the use of eosin Y as a metal-free photocatalyst and aryldiazonium salts as arylating agents. The reaction can be significantly accelerated in a microflow reactor, whilst allowing the in situ formation

  18. Identification of the elementary structural units of the DNA damage response.

    Science.gov (United States)

    Natale, Francesco; Rapp, Alexander; Yu, Wei; Maiser, Andreas; Harz, Hartmann; Scholl, Annina; Grulich, Stephan; Anton, Tobias; Hörl, David; Chen, Wei; Durante, Marco; Taucher-Scholz, Gisela; Leonhardt, Heinrich; Cardoso, M Cristina

    2017-06-12

    Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

  19. Interventional Effects for Mediation Analysis with Multiple Mediators.

    Science.gov (United States)

    Vansteelandt, Stijn; Daniel, Rhian M

    2017-03-01

    The mediation formula for the identification of natural (in)direct effects has facilitated mediation analyses that better respect the nature of the data, with greater consideration of the need for confounding control. The default assumptions on which it relies are strong, however. In particular, they are known to be violated when confounders of the mediator-outcome association are affected by the exposure. This complicates extensions of counterfactual-based mediation analysis to settings that involve repeatedly measured mediators, or multiple correlated mediators. VanderWeele, Vansteelandt, and Robins introduced so-called interventional (in)direct effects. These can be identified under much weaker conditions than natural (in)direct effects, but have the drawback of not adding up to the total effect. In this article, we adapt their proposal to achieve an exact decomposition of the total effect, and extend it to the multiple mediator setting. Interestingly, the proposed effects capture the path-specific effects of an exposure on an outcome that are mediated by distinct mediators, even when-as often-the structural dependence between the multiple mediators is unknown, for instance, when the direction of the causal effects between the mediators is unknown, or there may be unmeasured common causes of the mediators.

  20. Pyoverdine and PQS Mediated Subpopulation Interactions Involved in Pseudomonas aeruginosa Biofilm Formation

    DEFF Research Database (Denmark)

    Yang, Liang; Nilsson, Martin; Gjermansen, Morten

    2009-01-01

    Using flow chamber-grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that formation of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron...

  1. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    International Nuclear Information System (INIS)

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-01-01

    Carbachol and guanine nucleotides stimulate formation of the ( 3 H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with ( 3 H) inositol. Carbachol alone has little effect on formation of the ( 3 H) inositol phosphates (IPs), but GTPγS causes synergistic accumulation of ( 3 H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTPγS all support formation of the ( 3 H)IPs, with or without hormone, but GTPγS is the most effective. In the presence of GTPγS, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the ( 3 H)IPs occur at ∼ 5 μM and ∼ 100 μM carbachol, respectively; values close to those seen in intact cells. GTPγS alone stimulates formation of the ( 3 H)IPs after a brief lag time. The combination of GTPγS and carbachol both increases the rate of, and decreases the lag in, formation of the ( 3 H)IPs. LiCl increases ( 3 H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of ( 3 H)IP3. GTPγS and carbachol cause formation of ( 3 H)IPs in the absence of Ca ++ , but formation induced by GTPγS with or without carbachol is Ca ++ -sensitive over a range of physiological concentrations. Although carbachol, Ca ++ , and GTPγS all have effects on formation of ( 3 H)IPs, GTPγS appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell

  2. The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs.

    Science.gov (United States)

    Hecker, Andreas; Brand, Luise H; Peter, Sébastien; Simoncello, Nathalie; Kilian, Joachim; Harter, Klaus; Gaudin, Valérie; Wanke, Dierk

    2015-07-01

    Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein basic pentacysteine6 (BPC6) interacts with like heterochromatin protein1 (LHP1), a PRC1 component, and associates with vernalization2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution. © 2015 American Society of Plant Biologists. All Rights Reserved.

  3. Estimation of causal mediation effects for a dichotomous outcome in multiple-mediator models using the mediation formula.

    Science.gov (United States)

    Wang, Wei; Nelson, Suchitra; Albert, Jeffrey M

    2013-10-30

    Mediators are intermediate variables in the causal pathway between an exposure and an outcome. Mediation analysis investigates the extent to which exposure effects occur through these variables, thus revealing causal mechanisms. In this paper, we consider the estimation of the mediation effect when the outcome is binary and multiple mediators of different types exist. We give a precise definition of the total mediation effect as well as decomposed mediation effects through individual or sets of mediators using the potential outcomes framework. We formulate a model of joint distribution (probit-normal) using continuous latent variables for any binary mediators to account for correlations among multiple mediators. A mediation formula approach is proposed to estimate the total mediation effect and decomposed mediation effects based on this parametric model. Estimation of mediation effects through individual or subsets of mediators requires an assumption involving the joint distribution of multiple counterfactuals. We conduct a simulation study that demonstrates low bias of mediation effect estimators for two-mediator models with various combinations of mediator types. The results also show that the power to detect a nonzero total mediation effect increases as the correlation coefficient between two mediators increases, whereas power for individual mediation effects reaches a maximum when the mediators are uncorrelated. We illustrate our approach by applying it to a retrospective cohort study of dental caries in adolescents with low and high socioeconomic status. Sensitivity analysis is performed to assess the robustness of conclusions regarding mediation effects when the assumption of no unmeasured mediator-outcome confounders is violated. Copyright © 2013 John Wiley & Sons, Ltd.

  4. Estimation of Causal Mediation Effects for a Dichotomous Outcome in Multiple-Mediator Models using the Mediation Formula

    Science.gov (United States)

    Nelson, Suchitra; Albert, Jeffrey M.

    2013-01-01

    Mediators are intermediate variables in the causal pathway between an exposure and an outcome. Mediation analysis investigates the extent to which exposure effects occur through these variables, thus revealing causal mechanisms. In this paper, we consider the estimation of the mediation effect when the outcome is binary and multiple mediators of different types exist. We give a precise definition of the total mediation effect as well as decomposed mediation effects through individual or sets of mediators using the potential outcomes framework. We formulate a model of joint distribution (probit-normal) using continuous latent variables for any binary mediators to account for correlations among multiple mediators. A mediation formula approach is proposed to estimate the total mediation effect and decomposed mediation effects based on this parametric model. Estimation of mediation effects through individual or subsets of mediators requires an assumption involving the joint distribution of multiple counterfactuals. We conduct a simulation study that demonstrates low bias of mediation effect estimators for two-mediator models with various combinations of mediator types. The results also show that the power to detect a non-zero total mediation effect increases as the correlation coefficient between two mediators increases, while power for individual mediation effects reaches a maximum when the mediators are uncorrelated. We illustrate our approach by applying it to a retrospective cohort study of dental caries in adolescents with low and high socioeconomic status. Sensitivity analysis is performed to assess the robustness of conclusions regarding mediation effects when the assumption of no unmeasured mediator-outcome confounders is violated. PMID:23650048

  5. Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta

    International Nuclear Information System (INIS)

    Hirai, Yohei; Radisky, Derek; Boudreau, Rosanne; Simian, Marina; Stevens, Mary E.; Oka, Yumiko; Takebe, Kyoko; Niwa, Shinichiro; Bissell, Mina J.

    2002-01-01

    We have previously shown that epimorphin, a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which epimorphin mediates luminal morphogenesis. Treatment of cells with epimorphin to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, as constitutive expression of LIP was sufficient to produce lumen formation, while constitutive expression of LAP blocked epimorphin-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which epimorphin expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for epimorphin in luminal morphogenesis through control of C/EBPbeta expression

  6. Human CYP2E1 mediates the formation of glycidamide from acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Settels, Eva; Appel, Klaus E. [Federal Institute for Risk Assessment, Center for Experimental Toxicology, Berlin (Germany); Bernauer, Ulrike; Gundert-Remy, Ursula [Federal Institute for Risk Assessment, Department of Safety of Substances and Preparations, Berlin (Germany); Palavinskas, Richard; Klaffke, Horst S. [Federal Institute for Risk Assessment, Center for Analytical Chemistry, Berlin (Germany)

    2008-10-15

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes trademark, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes trademark, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes trademark; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species. (orig.)

  7. Causal mediation analysis with multiple causally non-ordered mediators.

    Science.gov (United States)

    Taguri, Masataka; Featherstone, John; Cheng, Jing

    2018-01-01

    In many health studies, researchers are interested in estimating the treatment effects on the outcome around and through an intermediate variable. Such causal mediation analyses aim to understand the mechanisms that explain the treatment effect. Although multiple mediators are often involved in real studies, most of the literature considered mediation analyses with one mediator at a time. In this article, we consider mediation analyses when there are causally non-ordered multiple mediators. Even if the mediators do not affect each other, the sum of two indirect effects through the two mediators considered separately may diverge from the joint natural indirect effect when there are additive interactions between the effects of the two mediators on the outcome. Therefore, we derive an equation for the joint natural indirect effect based on the individual mediation effects and their interactive effect, which helps us understand how the mediation effect works through the two mediators and relative contributions of the mediators and their interaction. We also discuss an extension for three mediators. The proposed method is illustrated using data from a randomized trial on the prevention of dental caries.

  8. The Influence of Receptor-Mediated Interactions on Reaction-Diffusion Mechanisms of Cellular Self-organisation

    KAUST Repository

    Klika, Vá clav; Baker, Ruth E.; Headon, Denis; Gaffney, Eamonn A.

    2011-01-01

    formation, motivating numerous theoretical and experimental studies, though its verification at the molecular level in biological systems has remained elusive. In this work, we consider the influence of receptor-mediated dynamics within the framework

  9. Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    International Nuclear Information System (INIS)

    Fu, Wen; Gorodeski, George I; McCormick, Tom; Qi, Xiaoping; Luo, Liping; Zhou, Lingyin; Li, Xin; Wang, Bing-Cheng; Gibbons, Heidi E; Abdul-Karim, Fadi W

    2009-01-01

    The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X 7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X 7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X 7 action. Skin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X 7 -null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X 7 pore formation) were determined by confocal laser microscopy. (a) Co-application on the skin of the P2X 7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X 7 -receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X 7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the

  10. Ribosome-catalyzed formation of an abnormal peptide analogue

    International Nuclear Information System (INIS)

    Roesser, J.R.; Chorghade, M.S.; Hecht, S.M.

    1986-01-01

    The peptidyl-tRNA analogue N-(chloracetyl) phenylalanyl-tRNA/sup Phe/ was prepared by chemical aminoacylation and prebound to the P site of Escherichia coli ribosomes in response to poly(uridylic acid). Admixture of phenylalanyl-tRNA/sup Phe/ to the A site resulted in the formation of two dipeptides, one of which was found by displacement of chloride ion from the peptidyl-tRNA. This constitutes the first example of ribosome-mediated formation of a peptide of altered connectivity and suggests a need for revision of the current model of peptide bond formation. Also suggested by the present finding is the feasibility of utilizing tRNAs to prepare polypeptides of altered connectivity in an in vitro protein biosynthesizing system. [ 32 P]-oligo(rA), [ 3 H]- and [ 14 C] phenylalanines were used in the assay of the peptidye-tRNA analogue

  11. Does animal-mediated seed dispersal facilitate the formation of Pinus armandii-Quercus aliena var. acuteserrata forests?

    Science.gov (United States)

    Yu, Fei; Wang, Dexiang; Yi, Xianfeng; Shi, Xiaoxiao; Huang, Yakun; Zhang, Hongwu; Zhang, XinPing

    2014-01-01

    The Pinus armandii and Quercus aliena var. acuteserrata mixed forest is one of the major forest types in the Qinling Mountains, China. P. armandii is considered to be a pioneer species during succession and it is usually invaded by late successional Q. aliena var. acuteserrata. However, the mechanism that underlies its invasion remains unclear. In the present study, we tracked seed dispersal of P. armandii and Q. aliena var. acuteserrata using coded plastic tags in the western, middle and eastern Qinling Mountains to elucidate the invasion process in the mixed forests. Our results indicated that the seeds of both P. armandii and Q. aliena var. acuteserrata were removed rapidly in the Qinling Mountains, and there were no differences in the seed removal rates between the two species. There were significant differences in rodent seed-eating and caching strategies between the two tree species. For P. armandii, seeds were more likely to be eaten in situ than those of Q. aliena var. acuteserrata in all plots. By contrast, the acorns of Q. aliena var. acuteserrata were less frequently eaten in situ, but more likely to be removed and cached. Q. aliena var. acuteserrata acorns had significantly longer dispersal distances than P. armandii seeds in all plots. Although P. armandii seeds were less likely to be dispersed into the Q. aliena var. acuteserrata stands, over 30% of the released acorns were transported into the P. armandii stands where they established five seedlings. Based on the coupled recruitment patterns of P. armandii and Q. aliena var. acuteserrata, we suggest that the animal-mediated seed dispersal contributes to the formation of Pinus armandii-Quercus aliena var. acuteserrata forests.

  12. Endocytic pathways mediating oligomeric Aβ42 neurotoxicity

    Directory of Open Access Journals (Sweden)

    Laxton Kevin

    2010-05-01

    Full Text Available Abstract Background One pathological hallmark of Alzheimer's disease (AD is amyloid plaques, composed primarily of amyloid-β peptide (Aβ. Over-production or diminished clearance of the 42 amino acid form of Aβ (Aβ42 in the brain leads to accumulation of soluble Aβ and plaque formation. Soluble oligomeric Aβ (oAβ has recently emerged to be as a likely proximal cause of AD. Results Here we demonstrate that endocytosis is critical in mediating oAβ42-induced neurotoxicity and intraneuronal accumulation of Aβ. Inhibition of clathrin function either with a pharmacological inhibitor, knock-down of clathrin heavy chain expression, or expression of the dominant-negative mutant of clathrin-assembly protein AP180 did not block oAβ42-induced neurotoxicity or intraneuronal accumulation of Aβ. However, inhibition of dynamin and RhoA by expression of dominant negative mutants reduced neurotoxicity and intraneuronal Aβ accumulation. Pharmacologic inhibition of the dynamin-mediated endocytic pathway by genistein also reduced neurotoxicity. Conclusions These data suggest that dynamin-mediated and RhoA-regulated endocytosis are integral steps for oligomeric Aβ42-induced neurotoxicity and intraneuronal Aβ accumulation.

  13. [SINEs in mammalian genomes can serve as additional signals in formation of facultative heterochromatin].

    Science.gov (United States)

    Usmanova, N M; Kazakov, V I; Tomilin, N V

    2008-01-01

    Using computer-based methods we determined the global distribution of short interspersed nuclear elements (SINEs) in the human and mouse X chromosomes. It has been shown that this distributions is similar to the distributions of CpG islands and genes but is different from the distribution of LINE1 elements. Since SINEs (human Alu and mouse B2) may have binding sites for Polycomb protein YY1, we suggest that these repeats can serve as additional signals ("boosters") in Polycomb-dependent silencing of gene rich segments during X inactivation.

  14. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization...... research is concerned with long-term structural changes involving media, culture, and society, i.e. the influences of the media are understood in relation to how media are implicated in social and cultural changes and how these processes come to create new conditions for human communication and interaction...

  15. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    International Nuclear Information System (INIS)

    Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

    2013-01-01

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis

  16. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ha Young, E-mail: hayoung@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Sang Doo [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Baek, Suk-Hwan [Department of Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Choi, Joon Hyuk [Department of Pathology, College of Medicine, Yeungnam University, Daegu 705-717 (Korea, Republic of); Cho, Kyung-Hyun [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Zabel, Brian A. [Palo Alto Institute for Research and Education, Veterans Affairs Hospital, Palo Alto, CA 94304 (United States); Bae, Yoe-Sik, E-mail: yoesik@skku.edu [Department of Biological Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710 (Korea, Republic of)

    2013-03-29

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

  17. Mammalian mediator 19 mediates H1299 lung adenocarcinoma cell clone conformation, growth, and metastasis.

    Science.gov (United States)

    Xu, Lu-Lu; Guo, Shu-Liang; Ma, Su-Ren; Luo, Yong-Ai

    2012-01-01

    Mammalian mediator (MED) is a multi-protein coactivator that has been identified by several research groups. The involvement of the MED complex subunit 19 (MED 19) in the metastasis of lung adenocarcinoma cell line (H1299), which expresses the MED 19 subunit, was here investigated. When MED 19 expression was decreased by RNA interference H1299 cells demonstrated reduced clone formation, arrest in the S phase of the cell cycle, and lowered metastatic capacity. Thus, MED 19 appears to play important roles in the biological behavior of non-small cell lung carcinoma cells. These findings may be important for the development of novel lung carcinoma treatments.

  18. Analysis of HDAC6 and BAG3-Aggresome Pathways in African Swine Fever Viral Factory Formation

    Directory of Open Access Journals (Sweden)

    Raquel Muñoz-Moreno

    2015-04-01

    Full Text Available African swine fever virus (ASFV is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes.

  19. What the Logs Can Tell You: Mediation to Implement Feedback in Training

    Science.gov (United States)

    Maluf, David; Wiederhold, Gio; Abou-Khalil, Ali; Norvig, Peter (Technical Monitor)

    2000-01-01

    The problem addressed by Mediation to Implement Feedback in Training (MIFT) is to customize the feedback from training exercizes by exploiting knowledge about the training scenario, training objectives, and specific student/teacher needs. We achieve this by inserting an intelligent mediation layer into the information flow from observations collected during training exercises to the display and user interface. Knowledge about training objectives, scenarios, and tasks is maintained in the mediating layer. A designer constraint is that domain experts must be able to extend mediators by adding domain-specific knowledge that supports additional aggregations, abstractions, and views of the results of training exercises. The MIFT mediation concept is intended to be integrated with existing military training exercise management tools and reduce the cost of developing and maintaining separate feedback and evaluation tools for every training simulator and every set of customer needs. The MIFT Architecture is designed as a set of independently reusable components which interact with each other through standardized formalisms such as the Knowledge Interchange Format (KIF) and Knowledge Query and Manipulation Language (KQML).

  20. Characterization of the cellular response triggered by gold nanoparticle-mediated laser manipulation.

    Science.gov (United States)

    Kalies, Stefan; Keil, Sebastian; Sender, Sina; Hammer, Susanne C; Antonopoulos, Georgios C; Schomaker, Markus; Ripken, Tammo; Murua Escobar, Hugo; Meyer, Heiko; Heinemann, Dag

    2015-11-01

    Laser-based transfection techniques have proven high applicability in several cell biologic applications. The delivery of different molecules using these techniques has been extensively investigated. In particular, new high-throughput approaches such as gold nanoparticle–mediated laser transfection allow efficient delivery of antisense molecules or proteins into cells preserving high cell viabilities. However, the cellular response to the perforation procedure is not well understood. We herein analyzed the perforation kinetics of single cells during resonant gold nanoparticle–mediated laser manipulation with an 850-ps laser system at a wavelength of 532 nm. Inflow velocity of propidium iodide into manipulated cells reached a maximum within a few seconds. Experiments based on the inflow of FM4-64 indicated that the membrane remains permeable for a few minutes for small molecules. To further characterize the cellular response postmanipulation, we analyzed levels of oxidative heat or general stress. Although we observed an increased formation of reactive oxygen species by an increase of dichlorofluorescein fluorescence, heat shock protein 70 was not upregulated in laser-treated cells. Additionally, no evidence of stress granule formation was visible by immunofluorescence staining. The data provided in this study help to identify the cellular reactions to gold nanoparticle–mediated laser manipulation.

  1. Stability and dynamic of strain mediated adatom superlattices on Cu

    Science.gov (United States)

    Kappus, Wolfgang

    2013-03-01

    Substrate strain mediated adatom equilibrium density distributions have been calculated for Cu surfaces using two complementing methods. A hexagonal adatom superlattice in a coverage range up to 0.045 ML is derived for repulsive short range interactions. For zero short range interactions a hexagonal superstructure of adatom clusters is derived in a coverage range about 0.08 ML. Conditions for the stability of the superlattice against formation of dimers or clusters and degradation are analyzed using simple neighborhood models. Such models are also used to investigate the dynamic of adatoms within their superlattice neighborhood. Collective modes of adatom diffusion are proposed from the analogy with bulk lattice dynamics and methods for measurement are suggested. The recently put forward explanation of surface state mediated interactions for superstructures found in scanning tunneling microscopy experiments is put in question and strain mediated interactions are proposed as an alternative.

  2. A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

    DEFF Research Database (Denmark)

    Lappann, M.; Claus, H.; van Alen, T.

    2010-01-01

    formation, whereas biofilm formation of cc with low point prevalence (ST-8 cc and ST-11 cc) was eDNA-independent. For initial biofilm formation, a ST-32 cc type strain, but not a ST-11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N......-acetylmuramyl-l-alanine amidase genes. In late biofilms, outer membrane phospholipase A-dependent autolysis, which was observed in most cc, but not in ST-8 and ST-11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an e....... On the contrary, spreaders (ST-11 and ST-8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates....

  3. Intercultural Mediation

    OpenAIRE

    Dragos Marian Radulescu; Denisa Mitrut

    2012-01-01

    The Intercultural Mediator facilitates exchanges between people of different socio-cultural backgrounds and acts as a bridge between immigrants and national and local associations, health organizations, services and offices in order to foster integration of every single individual. As the use mediation increases, mediators are more likely to be involved in cross-cultural mediation, but only the best mediators have the opportunity to mediate cross border business disputes or international poli...

  4. Maintaining Pedagogical Integrity of a Computer Mediated Course Delivery in Social Foundations

    Science.gov (United States)

    Stewart, Shelley; Cobb-Roberts, Deirdre; Shircliffe, Barbara J.

    2013-01-01

    Transforming a face to face course to a computer mediated format in social foundations (interdisciplinary field in education), while maintaining pedagogical integrity, involves strategic collaboration between instructional technologists and content area experts. This type of planned partnership requires open dialogue and a mutual respect for prior…

  5. Factor H C-Terminal Domains Are Critical for Regulation of Platelet/Granulocyte Aggregate Formation

    Directory of Open Access Journals (Sweden)

    Adam Z. Blatt

    2017-11-01

    Full Text Available Platelet/granulocyte aggregates (PGAs increase thromboinflammation in the vasculature, and PGA formation is tightly controlled by the complement alternative pathway (AP negative regulator, Factor H (FH. Mutations in FH are associated with the prothrombotic disease atypical hemolytic uremic syndrome (aHUS, yet it is unknown whether increased PGA formation contributes to the thrombosis seen in patients with aHUS. Here, flow cytometry assays were used to evaluate the effects of aHUS-related mutations on FH regulation of PGA formation and characterize the mechanism. Utilizing recombinant fragments of FH spanning the entire length of the protein, we mapped the regions of FH most critical for limiting AP activity on the surface of isolated human platelets and neutrophils, as well as the regions most critical for regulating PGA formation in human whole blood stimulated with thrombin receptor-activating peptide (TRAP. FH domains 19–20 were the most critical for limiting AP activity on platelets, neutrophils, and at the platelet/granulocyte interface. The role of FH in PGA formation was attributed to its ability to regulate AP-mediated C5a generation. AHUS-related mutations in domains 19–20 caused differential effects on control of PGA formation and AP activity on platelets and neutrophils. Our data indicate FH C-terminal domains are key for regulating PGA formation, thus increased FH protection may have a beneficial impact on diseases characterized by increased PGA formation, such as cardiovascular disease. Additionally, aHUS-related mutations in domains 19–20 have varying effects on control of TRAP-mediated PGA formation, suggesting that some, but not all, aHUS-related mutations may cause increased PGA formation that contributes to excessive thrombosis in patients with aHUS.

  6. Mediation analysis with multiple versions of the mediator.

    Science.gov (United States)

    Vanderweele, Tyler J

    2012-05-01

    The causal inference literature has provided definitions of direct and indirect effects based on counterfactuals that generalize the approach found in the social science literature. However, these definitions presuppose well-defined hypothetical interventions on the mediator. In many settings, there may be multiple ways to fix the mediator to a particular value, and these various hypothetical interventions may have very different implications for the outcome of interest. In this paper, we consider mediation analysis when multiple versions of the mediator are present. Specifically, we consider the problem of attempting to decompose a total effect of an exposure on an outcome into the portion through the intermediate and the portion through other pathways. We consider the setting in which there are multiple versions of the mediator but the investigator has access only to data on the particular measurement, not information on which version of the mediator may have brought that value about. We show that the quantity that is estimated as a natural indirect effect using only the available data does indeed have an interpretation as a particular type of mediated effect; however, the quantity estimated as a natural direct effect, in fact, captures both a true direct effect and an effect of the exposure on the outcome mediated through the effect of the version of the mediator that is not captured by the mediator measurement. The results are illustrated using 2 examples from the literature, one in which the versions of the mediator are unknown and another in which the mediator itself has been dichotomized.

  7. Morphology and Ultrastructure of Brain Tissue and Fat Body from the Flesh Fly, Sarcophaga bullata Parker (Diptera: Sarcophagidae, Envenomated by the Ectoparasitic Wasp Nasonia vitripennis (Walker (Hymenoptera: Pteromalidae

    Directory of Open Access Journals (Sweden)

    David B. Rivers

    2011-01-01

    Full Text Available This study tested the hypothesis that venom from the ectoparasitic wasp Nasonia vitripennis targets brain tissue and fat body from its flesh fly host, Sarcophaga bullata. By 1 h postenvenomation, some brain neurons began to show irregularities in nuclear shape, and though they were predominately euchromatic, there was evidence of heterochromatin formation. Irregularity in the nuclear envelope became more prominent by 3 h after envenomation, as did the condensation of heterochromatin. The severity of ultrastructural changes continued to increase until at least 24 h after parasitoid attack. At this point, cellular swelling and extensive heterochromatic inclusions were evident, multivesicular bodies occurred in the cytoplasm of some cells, and the rough endoplasmic reticulum was dilated in many of the cells. Immunohistochemical staining revealed significant apoptosis in neurons located in brain tissues. By contrast, there was no evidence of any morphological or ultrastructural disturbances in fat body tissues up to 24 h after envenomation, nor did any of the cells display signs of cell death.

  8. Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

    Directory of Open Access Journals (Sweden)

    Jonathan H. Foley

    2016-03-01

    Full Text Available Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC. In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

  9. Silent decision: HP1 protein escorts heterochromatic RNAs to their destiny

    Science.gov (United States)

    Ren, Jie; Martienssen, Robert A

    2012-01-01

    EMBO J advance online publication 06072012; doi:10.1016/j.molcel.2012.05.009 Heterochromatin is classically perceived to be refractory to transcription because of its compact structure. However, Keller et al (2012) now demonstrated that heterochromatic transcripts can accumulate even when heterochromatin is normally packaged. By tracking down the fate of these heterochromatic RNAs, they revealed a new post-transcriptional mechanism of silencing in heterochromatin that involves the dynamic turnover of HP1Swi6 between its free, chromatin-bound and RNA-bound forms. The latter form escorts heterochromatic RNA to degradation. PMID:22705945

  10. Digital Identity Formation: Socially Being Real and Present on Digital Networks

    Science.gov (United States)

    Bozkurt, Aras; Tu, Chih-Hsiung

    2016-01-01

    Social networks have become popular communication and interaction environments recently. As digital environments, so as ecosystems, they have potential in terms of networked learning as they fulfill some roles such as mediating an environment for digital identity formation and providing social and emotional presence. Based on this phenomenon, the…

  11. Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation

    NARCIS (Netherlands)

    Croes, M.; Kruyt, M. C.; Groen, W. M.; Van Dorenmalen, K. M.A.; Dhert, W. J.A.; Öner, F. C.; Alblas, J.

    2018-01-01

    Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is

  12. mediation: R Package for Causal Mediation Analysis

    Directory of Open Access Journals (Sweden)

    Dustin Tingley

    2014-09-01

    Full Text Available In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a treatment but also understanding the process in which the treatment causally affects the outcome. Causal mediation analysis is frequently used to assess potential causal mechanisms. The mediation package implements a comprehensive suite of statistical tools for conducting such an analysis. The package is organized into two distinct approaches. Using the model-based approach, researchers can estimate causal mediation effects and conduct sensitivity analysis under the standard research design. Furthermore, the design-based approach provides several analysis tools that are applicable under different experimental designs. This approach requires weaker assumptions than the model-based approach. We also implement a statistical method for dealing with multiple (causally dependent mediators, which are often encountered in practice. Finally, the package also offers a methodology for assessing causal mediation in the presence of treatment noncompliance, a common problem in randomized trials.

  13. Flagellar motility is critical for Listeria monocytogenes biofilm formation.

    Science.gov (United States)

    Lemon, Katherine P; Higgins, Darren E; Kolter, Roberto

    2007-06-01

    The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.

  14. mediation: R package for causal mediation analysis

    OpenAIRE

    Tingley, Dustin; Yamamoto, Teppei; Hirose, Kentaro; Keele, Luke; Imai, Kosuke

    2012-01-01

    In this paper, we describe the R package mediation for conducting causal mediation analysis in applied empirical research. In many scientific disciplines, the goal of researchers is not only estimating causal effects of a treatment but also understanding the process in which the treatment causally affects the outcome. Causal mediation analysis is frequently used to assess potential causal mechanisms. The mediation package implements a comprehensive suite of statistical tools for conducting su...

  15. Nitric oxide-mediated bystander signal transduction induced by heavy-ion microbeam irradiation

    Science.gov (United States)

    Tomita, Masanori; Matsumoto, Hideki; Funayama, Tomoo; Yokota, Yuichiro; Otsuka, Kensuke; Maeda, Munetoshi; Kobayashi, Yasuhiko

    2015-07-01

    In general, a radiation-induced bystander response is known to be a cellular response induced in non-irradiated cells after receiving bystander signaling factors released from directly irradiated cells within a cell population. Bystander responses induced by high-linear energy transfer (LET) heavy ions at low fluence are an important health problem for astronauts in space. Bystander responses are mediated via physical cell-cell contact, such as gap-junction intercellular communication (GJIC) and/or diffusive factors released into the medium in cell culture conditions. Nitric oxide (NO) is a well-known major initiator/mediator of intercellular signaling within culture medium during bystander responses. In this study, we investigated the NO-mediated bystander signal transduction induced by high-LET argon (Ar)-ion microbeam irradiation of normal human fibroblasts. Foci formation by DNA double-strand break repair proteins was induced in non-irradiated cells, which were co-cultured with those irradiated by high-LET Ar-ion microbeams in the same culture plate. Foci formation was suppressed significantly by pretreatment with an NO scavenger. Furthermore, NO-mediated reproductive cell death was also induced in bystander cells. Phosphorylation of NF-κB and Akt were induced during NO-mediated bystander signaling in the irradiated and bystander cells. However, the activation of these proteins depended on the incubation time after irradiation. The accumulation of cyclooxygenase-2 (COX-2), a downstream target of NO and NF-κB, was observed in the bystander cells 6 h after irradiation but not in the directly irradiated cells. Our findings suggest that Akt- and NF-κB-dependent signaling pathways involving COX-2 play important roles in NO-mediated high-LET heavy-ion-induced bystander responses. In addition, COX-2 may be used as a molecular marker of high-LET heavy-ion-induced bystander cells to distinguish them from directly irradiated cells, although this may depend on the time

  16. Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

    Science.gov (United States)

    Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

    2016-01-01

    ABSTRACT Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

  17. The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

    Science.gov (United States)

    Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

    2016-02-01

    Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

  18. SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

    Science.gov (United States)

    Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W

    2017-05-01

    SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre -driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.

  19. Formation of potential antigens from radiographic contrast media

    International Nuclear Information System (INIS)

    Nilsson, R.; Ehrenberg, L.; Fedorcsak, I.

    1987-01-01

    The use of radiographic contrast media is occasionally accompanied by more or less serious adverse effects, evidently of complex etiology, following intravascular administration. Some of these reactions are suspected of having an allergic basis. The in vitro and in vivo formation of iodinated serum proteins following gamma irradiation in the presence of two commonly used radiographic contrast media is demonstrated. Non-toxic concentrations of ascorbate present during the irradiation is shown to prevent the formation of such iodo-proteins in vitro as well as in vivo. The amounts of potentially antigenic iodoprotein formed during radiographic procedures will certainly be very small, but this quantity may be sufficient to elicit a hypersensitivity reaction in cases when an individual has been previously sensitized to immunologically similar iodo-proteins, a mechanism that could account for certain rare and unpredictable reations. The radiation induced formation of iodo-proteins may also serve as a model for the formation of iodine containing antigens mediated by a free radical mechanism, i.e. in the metabolism of iodinated compounds like erythrosine, a widely used colouring agent for certain foods. (orig.)

  20. Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release

    International Nuclear Information System (INIS)

    Lees, R.K.; MacDonald, H.R.; Sinclair, N.R.; University of Western Ontario London

    1977-01-01

    The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes generated in vitro in mixed leukocyte cultures were incubated for various periods of time at 37degC with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37degC, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51 Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51 Cr-release assay involving EDTA addition gave comparable result to the cloning inhibition assay. These results raise the possibility that the events leading to 51 Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability

  1. Liarozole inhibits transforming growth factor-β3–mediated extracellular matrix formation in human three-dimensional leiomyoma cultures

    Science.gov (United States)

    Levy, Gary; Malik, Minnie; Britten, Joy; Gilden, Melissa; Segars, James; Catherino, William H.

    2014-01-01

    Objective To investigate the impact of liarozole on transforming growth factor-β3 (TGF-β3) expression, TGF-β3 controlled profibrotic cytokines, and extracellular matrix formation in a three-dimensional (3D) leiomyoma model system. Design Molecular and immunohistochemical analysis in a cell line evaluated in a three-dimensional culture. Setting Laboratory study. Patient(s) None. Intervention(s) Treatment of leiomyoma and myometrial cells with liarozole and TGF-β3 in a three-dimensional culture system. Main Outcome Measure(s) Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blotting to assess fold gene and protein expression of TGF-β3 and TGF-β3 regulated fibrotic cytokines: collagen 1A1 (COL1A1), fibronectin, and versican before and after treatment with liarozole, and confirmatory immunohistochemical stains of treated three-dimensional cultures. Result(s) Both TGF-β3 gene and protein expression were elevated in leiomyoma cells compared with myometrium in two-dimensional and 3D cultures. Treatment with liarozole decreased TGF-β3 gene and protein expression. Extracellular matrix components versican, COL1A1, and fibronectin were also decreased by liarozole treatment in 3D cultures. Treatment of 3D cultures with TGF-β3 increased gene expression and protein production of COL1A1, fibronectin, and versican. Conclusion(s) Liarozole decreased TGF-β3 and TGF-β3–mediated extracellular matrix expression in a 3D uterine leiomyoma culture system. PMID:24825427

  2. Towards the synthesis of batrachotoxin-formation of alkynyl stannanes

    International Nuclear Information System (INIS)

    Sultan, A.; Raza, A.

    2013-01-01

    Batrachotoxin, isolated from frogs belonging to the genus Phyllobates, is a very potent neurotoxin and a steroidal alkaloid that has been found to block the Na+ channels in nerves and muscles resulting in arrhythmias or cardiac arrest leading to death. Research on this toxin is limited due to difficult isolation but many attempts have been made towards its total and partial synthesis. The present work indicates our efforts towards the formation of steroidal skeleton of this neurotoxin in high/improved yield by employing radical mediated cyclization which led to many interesting results. The successful model study towards the formation of the steroidal skeleton is also reported here. (author)

  3. Simultaneous coastal measurements of ozone deposition fluxes and iodine-mediated particle emission fluxes with subsequent CCN formation

    Directory of Open Access Journals (Sweden)

    J. D. Whitehead

    2010-01-01

    Full Text Available Here we present the first observations of simultaneous ozone deposition fluxes and ultrafine particle emission fluxes over an extensive infra-littoral zone. Fluxes were measured by the eddy covariance technique at the Station Biologique de Roscoff, on the coast of Brittany, north-west France. This site overlooks a very wide (3 km littoral zone controlled by very deep tides (9.6 m exposing extensive macroalgae beds available for significant iodine mediated photochemical production of ultrafine particles. The aspect at the Station Biologique de Roscoff provides an extensive and relatively flat, uniform fetch within which micrometeorological techniques may be utilized to study links between ozone deposition to macroalgae (and sea water and ultrafine particle production.

    Ozone deposition to seawater at high tide was significantly slower (vd[O3]=0.302±0.095 mm s−1 than low tidal deposition. A statistically significant difference in the deposition velocities to macroalgae at low tide was observed between night time (vd[O3]=1.00±0.10 mm s−1 and daytime (vd[O3]=2.05±0.16 mm s−1 when ultrafine particle formation results in apparent particle emission. Very high emission fluxes of ultrafine particles were observed during daytime periods at low tides ranging from 50 000 particles cm−2 s−1 to greater than 200 000 particles cm−2 s−1 during some of the lowest tides. These emission fluxes exhibited a significant relationship with particle number concentrations comparable with previous observations at another location. Apparent particle growth rates were estimated to be in the range 17–150 nm h−1 for particles in the size range 3–10 nm. Under certain conditions, particle growth may be inferred to continue to greater than 120 nm over tens

  4. Acute Social Stress Engages Synergistic Activity of Stress Mediators in the VTA to Promote Pavlovian Reward Learning

    OpenAIRE

    Kan, Russell; Pomrenze, Matthew; Tovar-Diaz, Jorge; Morikawa, Hitoshi; Drew, Michael; Pahlavan, Bahram

    2017-01-01

    Stressful events rapidly trigger activity-dependent synaptic plasticity in certain brain areas, driving the formation of aversive memories. However, it remains unclear how stressful experience affects plasticity mechanisms to regulate learning of appetitive events, such as intake of addictive drugs or palatable foods. Using rats, we show that two acute stress mediators, corticotropin-releasing factor (CRF) and norepinephrine (NE), enhance plasticity of NMDA receptor-mediated glutamatergic tra...

  5. What carries a mediation process? Configural analysis of mediation.

    Science.gov (United States)

    von Eye, Alexander; Mun, Eun Young; Mair, Patrick

    2009-09-01

    Mediation is a process that links a predictor and a criterion via a mediator variable. Mediation can be full or partial. This well-established definition operates at the level of variables even if they are categorical. In this article, two new approaches to the analysis of mediation are proposed. Both of these approaches focus on the analysis of categorical variables. The first involves mediation analysis at the level of configurations instead of variables. Thus, mediation can be incorporated into the arsenal of methods of analysis for person-oriented research. Second, it is proposed that Configural Frequency Analysis (CFA) can be used for both exploration and confirmation of mediation relationships among categorical variables. The implications of using CFA are first that mediation hypotheses can be tested at the level of individual configurations instead of variables. Second, this approach leaves the door open for different types of mediation processes to exist within the same set. Using a data example, it is illustrated that aggregate-level analysis can overlook mediation processes that operate at the level of individual configurations.

  6. ADN ribosomique 5S chez Arabidopsis thaliana : dynamique chromatinienne et ARN polymérase IV

    OpenAIRE

    Douet , Julien

    2008-01-01

    In Arabidopsis thaliana, 5S rRNA genes are found clustered at pericentromeric heterochromatin of chromosomes 3, 4 and 5. 5S rRNA genes transcription is epigenetically regulated through the formation of specific chromatin structure. In order to determine the events that lead to the establishment of such structures, a study during the first steps of post-germinative plant development was done. Unexpectedly, we observed a decondensation followed by a rapid "re"condensation of 5S rDNA chromatin. ...

  7. Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    Directory of Open Access Journals (Sweden)

    Fu Wen

    2009-04-01

    BzATP. (g Pore formation and the augmented calcium influx were depended on the expression of the P2X7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8. Conclusion (a P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b The P2X7-dependent apoptosis is mediated by calcium influx via P2X7 pores, and involves the caspase-9 (mitochondrial pathway. (c The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7 receptor. (d Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.

  8. Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

    Science.gov (United States)

    Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

    2017-09-29

    Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Profession of mediator as the professional provider of the mediation process

    Directory of Open Access Journals (Sweden)

    Jernej Šoštar

    2017-03-01

    Full Text Available The civil mediation programme, which is a court-connected programme, established as a form of alternative dispute resolution, is increasingly gaining ground as a field with its own theoretical and practical knowledge, principles and basic rules. Mediation has already set up its own body of knowledge, based on studies, classification of cases and the analyses of the results. In this article, we examine whether in the context of the development of mediation in Slovenia we might already talk about the profession of the mediator, defined as a provider of the mediation process. We examine the court-connected civil mediation and mediators who mediate at the court-connected civil mediation, and define them theoretically. By interviewing the mediation experts and mediators we examine their opinions about mediators and the court mediation. We examine the legal basis for the court-connected mediation programmes in Slovenia as well as in the European Union. Proceeding from our findings we conclude that the legal regulation of the court mediation in Slovenia is well established, and that the mediators of the court-connected civil mediation programmes can be accepted as the professional providers of the mediation process.

  10. Violence in gender culture: problem of typology formation (part 2

    Directory of Open Access Journals (Sweden)

    I. O. Svyatnenko

    2017-07-01

    Psychic level includes the creation of conditions for the emergence of psycho-emotional disorders against the backdrop of comprehensive demoralization (emotional manipulation and the imposition of a complex of guilt and low self-esteem; formatting of male micro communications (creation of conditions for limitation of the time of communication with other men and female mediation of such communication.

  11. Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization

    OpenAIRE

    Case, Lindsay B.; Waterman, Clare M.

    2011-01-01

    At the leading lamellipodium of migrating cells, protrusion of an Arp2/3-nucleated actin network is coupled to formation of integrin-based adhesions, suggesting that Arp2/3-mediated actin polymerization and integrin-dependent adhesion may be mechanistically linked. Arp2/3 also mediates actin polymerization in structures distinct from the lamellipodium, in "ventral F-actin waves" that propagate as spots and wavefronts along the ventral plasma membrane. Here we show that integrins engage the ex...

  12. Efficient inhibition of the formation of joint adhesions by ERK2 small interfering RNAs

    International Nuclear Information System (INIS)

    Li, Fengfeng; Ruan, Hongjiang; Fan, Cunyi; Zeng, Bingfang; Wang, Chunyang; Wang, Xiang

    2010-01-01

    Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which extracellular signal-regulated kinase (ERK)2 is considered to be crucial. Based on these theories, we examined the effects of a lentivirus-mediated small interfering RNA (siRNA) targeting ERK2 on the suppression of joint adhesion formation in vivo. The effects were assessed in vivo from different aspects including the adhesion score, histology and joint contracture angle. We found that the adhesions in the ERK2 siRNA group became soft and weak, and were easily stretched. Accordingly, the flexion contracture angles in the ERK2 siRNA group were also reduced (P < 0.05 compared with the control group). The animals appeared healthy, with no signs of impaired wound healing. In conclusion, local delivery of a lentivirus-mediated siRNA targeting ERK2 can ameliorate joint adhesion formation effectively and safely.

  13. Crack formation and prevention in colloidal drops

    Science.gov (United States)

    Kim, Jin Young; Cho, Kun; Ryu, Seul-A.; Kim, So Youn; Weon, Byung Mook

    2015-08-01

    Crack formation is a frequent result of residual stress release from colloidal films made by the evaporation of colloidal droplets containing nanoparticles. Crack prevention is a significant task in industrial applications such as painting and inkjet printing with colloidal nanoparticles. Here, we illustrate how colloidal drops evaporate and how crack generation is dependent on the particle size and initial volume fraction, through direct visualization of the individual colloids with confocal laser microscopy. To prevent crack formation, we suggest use of a versatile method to control the colloid-polymer interactions by mixing a nonadsorbing polymer with the colloidal suspension, which is known to drive gelation of the particles with short-range attraction. Gelation-driven crack prevention is a feasible and simple method to obtain crack-free, uniform coatings through drying-mediated assembly of colloidal nanoparticles.

  14. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Permanent link: https://www.ias.ac.in/article/fulltext/jbsc/041/04/0759-0786. Keywords. Epigenetics; genomic imprinting; heterochromatin; HP1; KRAB-ZFP. Abstract. Twenty five years ago it was proposed that conserved components of constitutive heterochromatin assemble heterochromatinlikecomplexes in euchromatin ...

  15. Defining an EPOR- regulated transcriptome for primary progenitors, including Tnfr-sf13c as a novel mediator of EPO- dependent erythroblast formation.

    Directory of Open Access Journals (Sweden)

    Seema Singh

    Full Text Available Certain concepts concerning EPO/EPOR action modes have been challenged by in vivo studies: Bcl-x levels are elevated in maturing erythroblasts, but not in their progenitors; truncated EPOR alleles that lack a major p85/PI3K recruitment site nonetheless promote polycythemia; and Erk1 disruption unexpectedly bolsters erythropoiesis. To discover novel EPO/EPOR action routes, global transcriptome analyses presently are applied to interrogate EPO/EPOR effects on primary bone marrow-derived CFUe-like progenitors. Overall, 160 EPO/EPOR target transcripts were significantly modulated 2-to 21.8-fold. A unique set of EPO-regulated survival factors included Lyl1, Gas5, Pim3, Pim1, Bim, Trib3 and Serpina 3g. EPO/EPOR-modulated cell cycle mediators included Cdc25a, Btg3, Cyclin-d2, p27-kip1, Cyclin-g2 and CyclinB1-IP-1. EPO regulation of signal transduction factors was also interestingly complex. For example, not only Socs3 plus Socs2 but also Spred2, Spred1 and Eaf1 were EPO-induced as negative-feedback components. Socs2, plus five additional targets, further proved to comprise new EPOR/Jak2/Stat5 response genes (which are important for erythropoiesis during anemia. Among receptors, an atypical TNF-receptor Tnfr-sf13c was up-modulated >5-fold by EPO. Functionally, Tnfr-sf13c ligation proved to both promote proerythroblast survival, and substantially enhance erythroblast formation. The EPOR therefore engages a sophisticated set of transcriptome response circuits, with Tnfr-sf13c deployed as one novel positive regulator of proerythroblast formation.

  16. Microbial exopolysaccharide-mediated synthesis and stabilization of metal nanoparticles.

    Science.gov (United States)

    Sathiyanarayanan, Ganesan; Dineshkumar, Krishnamoorthy; Yang, Yung-Hun

    2017-11-01

    Exopolysaccharides (EPSs) are structurally and functionally valuable biopolymer secreted by different prokaryotic and eukaryotic microorganisms in response to biotic/abiotic stresses and to survive in extreme environments. Microbial EPSs are fascinating in various industrial sectors due to their excellent material properties and less toxic, highly biodegradable, and biocompatible nature. Recently, microbial EPSs have been used as a potential template for the rapid synthesis of metallic nanoparticles and EPS-mediated metal reduction processes are emerging as simple, harmless, and environmentally benign green chemistry approaches. EPS-mediated synthesis of metal nanoparticles is a distinctive metabolism-independent bio-reduction process due to the formation of interfaces between metal cations and the polyanionic functional groups (i.e. hydroxyl, carboxyl and amino groups) of the EPS. In addition, the range of physicochemical features which facilitates the EPS as an efficient stabilizing or capping agents to protect the primary structure of the metal nanoparticles with an encapsulation film in order to separate the nanoparticle core from the mixture of composites. The EPS-capping also enables the further modification of metal nanoparticles with expected material properties for multifarious applications. The present review discusses the microbial EPS-mediated green synthesis/stabilization of metal nanoparticles, possible mechanisms involved in EPS-mediated metal reduction, and application prospects of EPS-based metal nanoparticles.

  17. Pro-Resolving Mediators in Regulating and Conferring Macrophage Function

    Directory of Open Access Journals (Sweden)

    Jesmond Dalli

    2017-11-01

    Full Text Available Macrophages are central in coordinating the host response to both sterile and infective insults. Clearance of apoptotic cells and cellular debris is a key biological action preformed by macrophages that paves the way to the resolution of local inflammation, repair and regeneration of damaged tissues, and re-establishment of function. The essential fatty acid-derived autacoids termed specialized pro-resolving mediators (SPM play central roles in promoting these processes. In the present article, we will review the role of microvesicles in controlling macrophage efferocytosis and SPM production. We will also discuss the role of both apoptotic cells and microvesicles in providing substrate for transcellular biosynthesis of several SPM families during efferocyotsis. In addition, this article will discuss the biological actions of the recently uncovered macrophage-derived SPM termed maresins. These mediators are produced via 14-lipoxygenation of docosahexaenoic acid that is either enzymatically converted to mediators carrying two hydroxyl groups or to autacoids that are peptide-lipid conjugates, coined maresin conjugates in tissue regeneration. The formation of these mediators is temporally regulated during acute self-limited infectious-inflammation where they promote the uptake and clearance of apoptotic cells, regulate several aspects of the tissue repair and regeneration, and display potent anti-nociceptive actions.

  18. Analysis on the Word-formation of English Netspeak Neologism

    Directory of Open Access Journals (Sweden)

    Wei Liu

    2014-12-01

    Full Text Available The emergence of computer-mediated communication provides a resourceful database for language researchers as well as learners. This study focuses on the Internet neologisms, a derivative of new media age, which in many ways affects the netizens in terms of communication. The collected data are examined empirically to figure out the characteristics of netspeak neologisms and their patterns of formation. It suggests that the most frequently occurring word-formation process of netspeak neologisms is compounding, subsequently, blending, affixation, old words with new meaning, acronyms, conversion, and clipping. Through probing into each process, the examples are illustrated and sub-categories are listed in terms of blending for further understanding. This study has proven that the diversity of word-formation processes of English netspeak neologism and may shed light on the creativity of language in the online context.

  19. Synthesis of Monodispersed Tantalum(V) oxide Nanospheres by an Ethylene Glycol Mediated Route

    Science.gov (United States)

    Tantalum(V) oxide (Ta2O5) nanospheres have been synthesized by a very simple ethylene glycol mediated route. The two-step process involves the formation of glycolate nanoparticles and their subsequent hydrolysis and calcination to generate the final Ta2O5 nanospheres. The synthes...

  20. ON THE ANATOMY OF MEDIATIZED NEWS BROADCASTING: METAMORPHOSES AND MULTIPLE NARRATIVES

    Directory of Open Access Journals (Sweden)

    Demétrio de Azeredo Soster

    2013-06-01

    Full Text Available This article discusses mediatization in news journalism, which is characterized by the institution, in current times, of a new “place” for the media. From this standpoint, they are no longer regarded as mere media, but as structuring elements of a new communicational bios. In the course of society’s transition from the media towards a mediatized society, news production practices become complex and process practices, as well as narrative and content distribution forms emerge, that are distinct from those seen until then. Television news broadcast emerges, therefore, in a new format, made up of the cluster of diverse media that refer to such programs, demanding new identification and recognition protocols. These changes will be examined as they relate to the news telecasts TV Folha, of TV Cultura, based on four movements resulting from the mediatization process in journalism: self-referentiality, co-referentiality, decentralization and dialogy.

  1. On the anatomy of mediatized news broadcasting: metamorphoses and multiple narratives

    Directory of Open Access Journals (Sweden)

    Fabiana Piccinin

    2013-12-01

    Full Text Available This article discusses mediatization in news journalism, which is characterized by the institution, in current times, of a new “place” for the media. From this standpoint, they are no longer regarded as mere media, but as structuring elements of a new communicational bios. In the course of society’s transition from the media towards a mediatized society, news production practices become complex and process practices, as well as narrative and content distribution forms emerge, that are distinct from those seen until then. Television news broadcast emerges, therefore, in a new format, made up of the cluster of diverse media that refer to such programs, demanding new identification and recognition protocols. These changes will be examined as they relate to the news telecasts TV Folha, of TV Cultura, based on four movements resulting from the mediatization process in journalism: self-referentiality, co-referentiality, decentralization and dialogy.

  2. The Anticancer Effects of Radachlorin-mediated Photodynamic Therapy in the Human Endometrial Adenocarcinoma Cell Line HEC-1-A.

    Science.gov (United States)

    Kim, Su-Mi; Rhee, Yun-Hee; Kim, Jong-Soo

    2017-11-01

    We investigated the effect of photodynamic therapy (PDT) using radachlorin on invasion, vascular formation and apoptosis by targeting epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathways in the HEC-1-A endometrial adenocarcinoma cell line. To investigate the apoptotic pathway, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and western blot analysis. We also evaluated the effects of PDT on tubular capillary formation in and invasion by HEC-1-A cells with a tube formation assay, invasion assay, prostaglandin E2 (PGE2) assay, and western blot analysis. PDT had anticancer effects on HEC-1-A through activation of the intrinsic pathway of apoptosis via caspase-9 and poly-(ADP-ribose) polymerase (PARP). PDT also inhibited tubular capillary formation in and invasion by HEC-1-A under VEGF pretreatment, that resulted from down-regulation of VEGFR2, EGFR, Ras homolog gene family/ member A (RhoA) and PGE2. These results are indicative of the specificity of radachlorin-mediated PDT to VEGF. The major advantage of radachlorin-mediated PDT is its selectivity for cancer tissue while maintaining adjacent normal endometrial tissue. Therefore, radachlorin-mediated PDT might offer high anticancer efficacy for endometrial adenocarcinoma and an especially useful modality for preserving fertility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  3. Computer-mediated interdisciplinary teams: theory and reality.

    Science.gov (United States)

    Vroman, Kerryellen; Kovacich, Joann

    2002-05-01

    The benefit of experience, tempered with the wisdom of hindsight and 5 years of text-based, asynchronous, computer-mediated, interdisciplinary team communications, provides the energy, insights and data shared in this article. Through the theoretical lens of group dynamics and the epistemology of interdisciplinary teaming, we analyze the interactions of a virtual interdisciplinary team to provide an understanding and appreciation of collaborative interdisciplinary communication in the context of interactive technologies. Whilst interactive technologies may require new patterns of language similar to that of learning a foreign language, what is communicated in the interdisciplinary team process does not change. Most important is the recognition that virtual teams, similar to their face-to-face counterparts, undergo the same challenges of interdisciplinary teaming and group developmental processes of formation: forming, storming, norming, performing, and transforming. After examining these dynamics of communication and collaboration in the context of the virtual team, the article concludes with guidelines facilitating interdisciplinary team computer-mediated communication.

  4. Laccase/mediator assisted degradation of triarylmethane dyes in a continuous membrane reactor.

    Science.gov (United States)

    Chhabra, Meenu; Mishra, Saroj; Sreekrishnan, Trichur Ramaswamy

    2009-08-10

    Laccase/mediator systems are important bioremediation agents as the rates of reactions can be enhanced in the presence of the mediators. The decolorization mechanism of two triarylmethane dyes, namely, Basic Green 4 and Acid Violet 17 is reported using Cyathus bulleri laccase. Basic Green 4 was decolorized through N-demethylation by laccase alone, while in mediator assisted reactions, dye breakdown was initiated from oxidation of carbinol form of the dye. Benzaldehyde and N,N-dimethyl aniline were the major end products. With Acid Violet 17, laccase carried out N-deethylation and in mediator assisted reactions, oxidation of the carbinol form of the dye occurred resulting in formation of formyl benzene sulfonic acid, carboxy benzene sulfonic acid and benzene sulfonic acid. Toxicity analysis revealed that Basic Green 4 was toxic and treatment with laccase/mediators resulted in 80-100% detoxification. The treatment of the textile dye solution using laccase and 2,2'-azino-di-(-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was demonstrated in an enzyme membrane reactor. At a hydraulic retention time of 6h, the process was operated for a period of 15 days with nearly 95% decolorization, 10% reduction in flux and 70% recovery of active ABTS.

  5. Crosslinking of fibrinogen and fibronectin by free radicals : A possible initial step in adhesion formation in osteoarthritis of the temporomandibular joint

    NARCIS (Netherlands)

    Dijkgraaf, LC; Zardeneta, G; Cordewener, FW; Liem, RSB; Schmitz, JP; de Bont, LGM; Milam, SB

    Purpose: Adhesion formation in osteoarthritis (OA) of the temporomandibular joint (TMJ) typically results in a sustained limitation of joint movement. We propose the hypothesis that free-radical-mediated crosslinking of proteins underlies this adhesion formation in affected joints. Free radicals may

  6. Estimation of Causal Mediation Effects for a Dichotomous Outcome in Multiple-Mediator Models using the Mediation Formula

    OpenAIRE

    Wang, Wei; Nelson, Suchitra; Albert, Jeffrey M.

    2013-01-01

    Mediators are intermediate variables in the causal pathway between an exposure and an outcome. Mediation analysis investigates the extent to which exposure effects occur through these variables, thus revealing causal mechanisms. In this paper, we consider the estimation of the mediation effect when the outcome is binary and multiple mediators of different types exist. We give a precise definition of the total mediation effect as well as decomposed mediation effects through individual or sets ...

  7. Extracellular Trap Formation in Response to Trypanosoma cruzi Infection in Granulocytes Isolated From Dogs and Common Opossums, Natural Reservoir Hosts

    Directory of Open Access Journals (Sweden)

    Nicole de Buhr

    2018-05-01

    Full Text Available Granulocytes mediate the first line of defense against infectious diseases in humans as well as animals and they are well known as multitasking cells. They can mediate antimicrobial activity by different strategies depending on the pathogen they encounter. Besides phagocytosis, a key strategy against extracellular pathogens is the formation of extracellular traps (ETs. Those ETs mainly consist of DNA decorated with antimicrobial components and mediate entrapment of various pathogens. In the last years, various studies described ET formation as response to bacteria, viruses and parasites e.g., Trypanosma (T. cruzi. Nevertheless, it is not fully understood, if ET formation helps the immune system to eliminate intracellular parasites. The goal of this study was to analyze ET formation in response to the intracellular parasite Trypanosma (T. cruzi by granulocytes derived from animals that serve as natural reservoir. Thus, we investigated the ET formation in two T. cruzi reservoirs, namely dogs as domestic animal and common opossums (Didelphis marsupialis as wild animal. Granulocytes were harvested from fresh blood by density gradient centrifugation and afterwards incubated with T. cruzi. We conducted the analysis by determination of free DNA and immunofluorescence microscopy. Using both methods, we show that T. cruzi efficiently induces ET formation in granulocytes derived from common opossum as well as dog blood. Most ETs from both animal species as response to T. cruzi are decorated with the protease neutrophil elastase. Since T. cruzi is well known to circulate over years in both analyzed animals as reservoirs, it may be assumed that T. cruzi efficiently evades ET-mediated killing in those animals. Therefore, ETs may not play a major role in efficient elimination of the pathogen from the blood of dogs or common opossums as T. cruzi survives in niches of their body. The characterization of granulocytes in various animals and humans may be helpful

  8. Elevated AKR1C3 expression promotes prostate cancer cell survival and prostate cell-mediated endothelial cell tube formation: implications for prostate cancer progressioan

    International Nuclear Information System (INIS)

    Dozmorov, Mikhail G; Lin, Hsueh-Kung; Azzarello, Joseph T; Wren, Jonathan D; Fung, Kar-Ming; Yang, Qing; Davis, Jeffrey S; Hurst, Robert E; Culkin, Daniel J; Penning, Trevor M

    2010-01-01

    Aldo-keto reductase (AKR) 1C family member 3 (AKR1C3), one of four identified human AKR1C enzymes, catalyzes steroid, prostaglandin, and xenobiotic metabolism. In the prostate, AKR1C3 is up-regulated in localized and advanced prostate adenocarcinoma, and is associated with prostate cancer (PCa) aggressiveness. Here we propose a novel pathological function of AKR1C3 in tumor angiogenesis and its potential role in promoting PCa progression. To recapitulate elevated AKR1C3 expression in cancerous prostate, the human PCa PC-3 cell line was stably transfected with an AKR1C3 expression construct to establish PC3-AKR1C3 transfectants. Microarray and bioinformatics analysis were performed to identify AKR1C3-mediated pathways of activation and their potential biological consequences in PC-3 cells. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and an in vitro Matrigel angiogenesis assays were applied to validate the pro-angiogenic activity of PC3-AKR1C3 transfectants identified by bioinformatics analysis. Microarray and bioinformatics analysis suggested that overexpression of AKR1C3 in PC-3 cells modulates estrogen and androgen metabolism, activates insulin-like growth factor (IGF)-1 and Akt signaling pathways, as well as promotes tumor angiogenesis and aggressiveness. Levels of IGF-1 receptor (IGF-1R) and Akt activation as well as vascular endothelial growth factor (VEGF) expression and secretion were significantly elevated in PC3-AKR1C3 transfectants in comparison to PC3-mock transfectants. PC3-AKR1C3 transfectants also promoted endothelial cell (EC) tube formation on Matrigel as compared to the AKR1C3-negative parental PC-3 cells and PC3-mock transfectants. Pre-treatment of PC3-AKR1C3 transfectants with a selective IGF-1R kinase inhibitor (AG1024) or a non-selective phosphoinositide 3-kinases (PI3K) inhibitor (LY294002) abolished ability of the cells to promote EC tube formation. Bioinformatics

  9. HOIP Deficiency Causes Embryonic Lethality by Aberrant TNFR1-Mediated Endothelial Cell Death

    Directory of Open Access Journals (Sweden)

    Nieves Peltzer

    2014-10-01

    Full Text Available Summary: Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC, consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1 prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF and lymphotoxin-α (LT-α, and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP’s catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death. : HOIP is the main catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC, a crucial regulator of TNF and other immune signaling pathways. Peltzer et al. find that HOIP deficiency results in embryonic lethality at midgestation due to endothelial cell death mediated by TNFR1. Aberrant formation of a TNF-mediated cell-death-inducing complex in HOIP-deficient (but not -proficient cells underlies the phenotype, with the catalytic activity of HOIP required for the control of cell death in response to TNF.

  10. WANDERINGS: RESEARCH AND FORMATION AS A SUBJECTIFYING PROCESSUALITY

    Directory of Open Access Journals (Sweden)

    Juliano dos Santos

    2016-12-01

    Full Text Available This work seeks to explore the relationship between body, subjectivity, and formation of some aspects that involve the “making of research,” whose implementation took place between 2013 and 2015. The cartography (DELEUZE; GUATTARI, 2011; PASSOS et all, 2009; ROLNIK, 1989 elected by us as a method strived to accompany encounters with bodies, dance, contact improvisation, bio-dance, and lives in formation: “Dance and body expression.” This activity was integrated, in the second semester of 2013, the set of activities of the pedagogical axis “Interações Culturais e Humanísticas” at the Universidade Federal do Paraná, Setor Litoral. There were some contributions to the field of education through the intensities mobilized during the research’s production. The process of formation of a researcher presenting smaller elements, minor elements, not teaching itself, the representation or the discovery of the world, but research, movement, doubt, and the invention of worlds, mediation of desire. In this sense, the text that we present proposes a point out some encounters that resonated with the body of the researcher and b explore the effects that, in intensity, mobilized and continue to mobilize a process of formation. Keywords: Formation. Experimentation. Body. Subjectivity.

  11. Concept Formation Skills in Long-Term Cochlear Implant Users

    Science.gov (United States)

    Castellanos, Irina; Kronenberger, William G.; Beer, Jessica; Colson, Bethany G.; Henning, Shirley C.; Ditmars, Allison; Pisoni, David B.

    2015-01-01

    This study investigated if a period of auditory sensory deprivation followed by degraded auditory input and related language delays affects visual concept formation skills in long-term prelingually deaf cochlear implant (CI) users. We also examined if concept formation skills are mediated or moderated by other neurocognitive domains (i.e., language, working memory, and executive control). Relative to normally hearing (NH) peers, CI users displayed significantly poorer performance in several specific areas of concept formation, especially when multiple comparisons and relational concepts were components of the task. Differences in concept formation between CI users and NH peers were fully explained by differences in language and inhibition–concentration skills. Language skills were also found to be more strongly related to concept formation in CI users than in NH peers. The present findings suggest that complex relational concepts may be adversely affected by a period of early prelingual deafness followed by access to underspecified and degraded sound patterns and spoken language transmitted by a CI. Investigating a unique clinical population such as early-implanted prelingually deaf children with CIs can provide new insights into foundational brain–behavior relations and developmental processes. PMID:25583706

  12. Causal mediation analysis with multiple mediators.

    Science.gov (United States)

    Daniel, R M; De Stavola, B L; Cousens, S N; Vansteelandt, S

    2015-03-01

    In diverse fields of empirical research-including many in the biological sciences-attempts are made to decompose the effect of an exposure on an outcome into its effects via a number of different pathways. For example, we may wish to separate the effect of heavy alcohol consumption on systolic blood pressure (SBP) into effects via body mass index (BMI), via gamma-glutamyl transpeptidase (GGT), and via other pathways. Much progress has been made, mainly due to contributions from the field of causal inference, in understanding the precise nature of statistical estimands that capture such intuitive effects, the assumptions under which they can be identified, and statistical methods for doing so. These contributions have focused almost entirely on settings with a single mediator, or a set of mediators considered en bloc; in many applications, however, researchers attempt a much more ambitious decomposition into numerous path-specific effects through many mediators. In this article, we give counterfactual definitions of such path-specific estimands in settings with multiple mediators, when earlier mediators may affect later ones, showing that there are many ways in which decomposition can be done. We discuss the strong assumptions under which the effects are identified, suggesting a sensitivity analysis approach when a particular subset of the assumptions cannot be justified. These ideas are illustrated using data on alcohol consumption, SBP, BMI, and GGT from the Izhevsk Family Study. We aim to bridge the gap from "single mediator theory" to "multiple mediator practice," highlighting the ambitious nature of this endeavor and giving practical suggestions on how to proceed. © 2014 The Authors Biometrics published by Wiley Periodicals, Inc. on behalf of International Biometric Society.

  13. mma: An R Package for Mediation Analysis with Multiple Mediators

    Directory of Open Access Journals (Sweden)

    Qingzhao Yu

    2017-04-01

    Full Text Available Mediation refers to the effect transmitted by mediators that intervene in the relationship between an exposure and a response variable. Mediation analysis has been broadly studied in many fields. However, it remains a challenge for researchers to consider complicated associations among variables and to differentiate individual effects from multiple mediators. [1] proposed general definitions of mediation effects that were adaptable to all different types of response (categorical or continuous, exposure, or mediation variables. With these definitions, multiple mediators of different types can be considered simultaneously, and the indirect effects carried by individual mediators can be separated from the total effect. Moreover, the derived mediation analysis can be performed with general predictive models. That is, the relationships among variables can be modeled using not only generalized linear models but also nonparametric models such as the Multiple Additive Regression Trees. Therefore, more complicated variable transformations and interactions can be considered in analyzing the mediation effects. The proposed method is realized by the R package 'mma'. We illustrate in this paper the proposed method and how to use 'mma' to estimate mediation effects and make inferences.

  14. Nucleolar aggresomes mediate release of pericentric heterochromatin and nuclear destruction of genotoxically treated cancer cells

    Czech Academy of Sciences Publication Activity Database

    Salmina, K.; Huna, A.; Inashkina, I.; Belyayev, Alexander; Krigerts, J.; Paštová, Ladislava; Vazquez-Martin, A.; Erenpreisa, J.

    2017-01-01

    Roč. 8, č. 2 (2017), s. 205-221 ISSN 1949-1034 Institutional support: RVO:67985939 Keywords : aggresome * ALU retrotransposition * autophagy Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 2.387, year: 2016

  15. Proportionate Dwarfism in Mice Lacking Heterochromatin Protein 1 Binding Protein 3 (HP1BP3) Is Associated With Alterations in the Endocrine IGF-1 Pathway.

    Science.gov (United States)

    Garfinkel, Benjamin P; Arad, Shiri; Le, Phuong T; Bustin, Michael; Rosen, Clifford J; Gabet, Yankel; Orly, Joseph

    2015-12-01

    Heterochromatin protein 1 binding protein 3 (HP1BP3) is a recently described histone H1-related protein with roles in chromatin structure and transcriptional regulation. To explore the potential physiological role of HP1BP3, we have previously described an Hp1bp3(-/-) mouse model with reduced postnatal viability and growth. We now find that these mice are proportionate dwarfs, with reduction in body weight, body length, and organ weight. In addition to their small size, microcomputed tomography analysis showed that Hp1bp3(-/-) mice present a dramatic impairment of their bone development and structure. By 3 weeks of age, mice of both sexes have severely impaired cortical and trabecular bone, and these defects persist into adulthood and beyond. Primary cultures of both osteoblasts and osteoclasts from Hp1bp3(-/-) bone marrow and splenocytes, respectively, showed normal differentiation and function, strongly suggesting that the impaired bone accrual is due to noncell autonomous systemic cues in vivo. One major endocrine pathway regulating both body growth and bone acquisition is the IGF regulatory system, composed of IGF-1, the IGF receptors, and the IGF-binding proteins (IGFBPs). At 3 weeks of age, Hp1bp3(-/-) mice exhibited a 60% reduction in circulating IGF-1 and a 4-fold increase in the levels of IGFBP-1 and IGFBP-2. These alterations were reflected in similar changes in the hepatic transcripts of the Igf1, Igfbp1, and Igfbp2 genes. Collectively, these results suggest that HP1BP3 plays a key role in normal growth and bone development by regulating transcription of endocrine IGF-1 components.

  16. Causal Mediation Analysis of Survival Outcome with Multiple Mediators.

    Science.gov (United States)

    Huang, Yen-Tsung; Yang, Hwai-I

    2017-05-01

    Mediation analyses have been a popular approach to investigate the effect of an exposure on an outcome through a mediator. Mediation models with multiple mediators have been proposed for continuous and dichotomous outcomes. However, development of multimediator models for survival outcomes is still limited. We present methods for multimediator analyses using three survival models: Aalen additive hazard models, Cox proportional hazard models, and semiparametric probit models. Effects through mediators can be characterized by path-specific effects, for which definitions and identifiability assumptions are provided. We derive closed-form expressions for path-specific effects for the three models, which are intuitively interpreted using a causal diagram. Mediation analyses using Cox models under the rare-outcome assumption and Aalen additive hazard models consider effects on log hazard ratio and hazard difference, respectively; analyses using semiparametric probit models consider effects on difference in transformed survival time and survival probability. The three models were applied to a hepatitis study where we investigated effects of hepatitis C on liver cancer incidence mediated through baseline and/or follow-up hepatitis B viral load. The three methods show consistent results on respective effect scales, which suggest an adverse estimated effect of hepatitis C on liver cancer not mediated through hepatitis B, and a protective estimated effect mediated through the baseline (and possibly follow-up) of hepatitis B viral load. Causal mediation analyses of survival outcome with multiple mediators are developed for additive hazard and proportional hazard and probit models with utility demonstrated in a hepatitis study.

  17. Activation of PAD4 in NET formation

    Directory of Open Access Journals (Sweden)

    Amanda eRohrbach

    2012-11-01

    Full Text Available Peptidyl arginine deiminases, or PADs, convert arginine residues to the non-ribosomally encoded amino acid citrulline in a variety of protein substrates. PAD4 is expressed in granulocytes and is essential for the formation of neutrophil extracellular traps (NETs via PAD4-mediated histone citrullination. Citrullination of histones is thought to promote NET formation by inducing chromatin decondensation and facilitating the expulsion of chromosomal DNA that is coated with antimicrobial molecules. Numerous stimuli have been reported to lead to PAD4 activation and NET formation. However, how this signaling process proceeds and how PAD4 becomes activated in cells is largely unknown. Herein, we describe the various stimuli and signaling pathways that have been implicated in PAD4 activation and NET formation, including the role of reactive oxygen species generation. To provide a foundation for the above discussion, we first describe PAD4 structure and function, and how these studies led to the development of PAD-specific inhibitors. A comprehensive survey of the receptors and signaling pathways that regulate PAD4 activation will be important for our understanding of innate immunity, and the identification of signaling intermediates in PAD4 activation may also lead to the generation of pharmaceuticals to target NET-related pathogenesis.

  18. Enrofloxacin enhances the formation of neutrophil extracellular traps in bovine granulocytes

    Science.gov (United States)

    Jerjomiceva, Natalja; Seri, Hisham; Völlger, Lena; Wang, Yanming; Zeitouni, Nathalie; Naim, Hassan Y.; von Köckritz-Blickwede, Maren

    2014-01-01

    Several antibiotics are known for their ability to accumulate in neutrophils and thereby modulate the antimicrobial functions of those cells. This manuscript demonstrates for the first time that an antibiotic, namely the fluoroquinolone enrofloxacin, enhances the formation of bovine neutrophil extracellular traps (NETs). When pharmacologically inactivating NADPH oxidase or peptidylarginine deiminase-4, enrofloxacin-induced NET-formation was distinctly reduced. Additionally, when treating the cells with cytochalasin D or nocodazole, the enrofloxacin-mediated NET-induction was abolished, indicating that besides oxidative burst and histone citrullination also the actin and microtubule polymerization are involved in this process. PMID:24642685

  19. Total synthesis of fluoxetine and duloxetine through an in situ imine formation/borylation/transimination and reduction approach.

    Science.gov (United States)

    Calow, Adam D J; Fernández, Elena; Whiting, Andrew

    2014-08-28

    We report efficient, catalytic, asymmetric total syntheses of both (R)-fluoxetine and (S)-duloxetine from α,β-unsaturated aldehydes conducting five sequential one-pot steps (imine formation/copper mediated β-borylation/transimination/reduction/oxidation) followed by the specific ether group formation which deliver the desired products (R)-fluoxetine in 45% yield (96% ee) and (S)-duloxetine in 47% yield (94% ee).

  20. Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation

    Science.gov (United States)

    Huebsch, Nathaniel; Lippens, Evi; Lee, Kangwon; Mehta, Manav; Koshy, Sandeep T.; Darnell, Max C.; Desai, Rajiv M.; Madl, Christopher M.; Xu, Maria; Zhao, Xuanhe; Chaudhuri, Ovijit; Verbeke, Catia; Kim, Woo Seob; Alim, Karen; Mammoto, Akiko; Ingber, Donald E.; Duda, Georg N.; Mooney, David J.

    2015-12-01

    The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel’s elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel’s elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

  1. Polyol-mediated synthesis of copper indium sulphide by solvothermal process

    International Nuclear Information System (INIS)

    Gorai, S.; Chaudhuri, S.

    2005-01-01

    A simple polyol-mediated solvothermal method has been proposed to synthesize copper indium sulphide. XRD studies reveal that the products are well crystallized. SEM indicates rod-like (with different aspect ratio) and star-shaped flake-like morphology of the products. The products are also characterized by optical studies and compositional analysis (XRF). XRF results show the formation of stoichiometric and non-stoichiometric copper indium sulphides depending on the reaction conditions

  2. Analysis of the human HP1 interactome reveals novel binding partners

    DEFF Research Database (Denmark)

    Rosnoblet, Claire; Vandamme, Julien; Völkel, Pamela

    2011-01-01

    Heterochromatin protein 1 (HP1) has first been described in Drosophila as an essential component of constitutive heterochromatin required for stable epigenetic gene silencing. Less is known about the three mammalian HP1 isotypes CBX1, CBX3 and CBX5. Here, we applied a tandem affinity purification...

  3. Reversible assembly of protein-DNA nanostructures triggered by mediated electron transfer

    International Nuclear Information System (INIS)

    Vogt, Stephan; Wenderhold-Reeb, Sabine; Nöll, Gilbert

    2017-01-01

    Stable protein-DNA nanostructures have been assembled by reconstitution of the multi-ligand binding flavoprotein dodecin on top of flavin-terminated dsDNA monolayers on gold electrodes. These structures could be disassembled by electrochemical flavin reduction via mediated electron transfer. For this purpose a negative potential was applied at the Au working electrode in the presence of the redox mediator bis-(ammoniumethyl)-4,4′-bipyridinium tetrabromide. The stepwise formation of the flavin-terminated dsDNA monolayers as well as the binding and electrochemically triggered release of apododecin were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. The assembly and disassembly of the protein-DNA nanostructures were fully reversible processes, which could be carried out multiple times at the same flavin-dsDNA modified surface. When a negative potential was applied in the absence of a redox mediator apododecin could not be released, i.e. direct electron transfer was not possible. As alternative redox mediators also methylene blue and phenosafranine were studied, but in the presence of these molecules apododecin was released without applying a potential, probably because the tricyclic aromatic compounds are able to replace the flavins at the binding sites.

  4. Diffusion mediated agglomeration of CdS nanoparticles via Langmuir–Blodgett technique

    International Nuclear Information System (INIS)

    Das, Nayan Mani; Roy, Dhrubojyoti; Gupta, P.S.

    2013-01-01

    Graphical abstract: - Highlights: • Diffusion mediated agglomeration of CdS nanoparticles are discussed. • Formation of CdS nanoparticles are confirmed by the change of chain length in XRD. • AFM shows the agglomeration of particles with a film swelling of about 5 Å. • UV–vis absorbance suggests that the grown particles show quantum confinement. • Hexagonal form of particle was confirmed by UV–vis reflectivity. - Abstract: We have reported a diffusion mediated agglomeration of cadmium sulphide (CdS) nanoparticles within cadmium arachidate (CdA 2 ) film matrix. The structural morphology and formation of CdS nanoparticles are characterized by X-ray diffraction (XRD), X-ray reflectivity (XRR), atomic force microscopy (AFM) and ultraviolet-visible spectroscopy techniques. X-ray diffraction (XRD) results show a change in bilayer difference from 53.04 Å to 43 Å after the sulphidation. An epitaxial growth of the films by ∼5 Å after sulphidation is confirmed from atomic force microscopy studies. The particle size calculated form UV–vis absorption edges are found to be varying from 2.6 nm to 3.3 nm for the different layers. A lateral dimension of 72–80 nm from AFM measurements and a size of 2.6–3.3 nm have confirmed one side flat pseudo two-dimensional disk-like nanoparticles. UV–vis reflectivity peak at E 1 (A) confirms the formation of hexagonal CdS nanoparticles along the c-axis

  5. Bioelectrochemical ethanol production through mediated acetate reduction by mixed cultures.

    Science.gov (United States)

    Steinbusch, Kirsten J J; Hamelers, Hubertus V M; Schaap, Joris D; Kampman, Christel; Buisman, Cees J N

    2010-01-01

    Biological acetate reduction with hydrogen is a potential method to convert wet biomass waste into ethanol. Since the ethanol concentration and reaction rates are low, this research studies the feasibility of using an electrode, in stead of hydrogen, as an electron donor for biological acetate reduction in conjunction of an electron mediator. Initially, the effect of three selected mediators on metabolic flows during acetate reduction with hydrogen was explored; subsequently, the best performing mediator was used in a bioelectrochemical system to stimulate acetate reduction at the cathode with mixed cultures at an applied cathode potential of -550 mV. In the batch test, methyl viologen (MV) was found to accelerate ethanol production 6-fold and increased ethanol concentration 2-fold to 13.5 +/- 0.7 mM compared to the control. Additionally, MV inhibited n-butyrate and methane formation, resulting in high ethanol production efficiency (74.6 +/- 6%). In the bioelectrochemical system, MV addition to an inoculated cathode led directly to ethanol production (1.82 mM). Hydrogen was coproduced at the cathode (0.0035 Nm(3) hydrogen m(-2) d(-1)), so it remained unclear whether acetate was reduced to ethanol by electrons supplied by the mediator or by hydrogen. As MV reacted irreversibly at the cathode, ethanol production stopped after 5 days.

  6. Singlet oxygen-mediated damage to proteins and its consequences

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2003-01-01

    by the transfer of energy to ground state (triplet) molecular oxygen by either protein-bound, or other, chromophores. Singlet oxygen can also be generated by a range of other enzymatic and non-enzymatic reactions including processes mediated by heme proteins, lipoxygenases, and activated leukocytes, as well...... the absorption of UV radiation by the protein, or bound chromophore groups, thereby generating excited states (singlet or triplets) or radicals via photo-ionisation. The second major process involves indirect oxidation of the protein via the formation and subsequent reactions of singlet oxygen generated...... as radical termination reactions. This paper reviews the data available on singlet oxygen-mediated protein oxidation and concentrates primarily on the mechanisms by which this excited state species brings about changes to both the side-chains and backbone of amino acids, peptides, and proteins. Recent work...

  7. Sensitivity of Electron Transfer Mediated Decay to Ion Pairing.

    Science.gov (United States)

    Pohl, Marvin N; Richter, Clemens; Lugovoy, Evgeny; Seidel, Robert; Slavíček, Petr; Aziz, Emad F; Abel, Bernd; Winter, Bernd; Hergenhahn, Uwe

    2017-08-17

    Ion pairing in electrolyte solutions remains a topic of discussion despite a long history of research. Very recently, nearest-neighbor mediated electronic de-excitation processes of core hole vacancies (electron transfer mediated decay, ETMD) were proposed to carry a spectral fingerprint of local solvation structure and in particular of contact ion pairs. Here, for the first time, we apply electron-electron coincidence detection to a liquid microjet, and record ETMD spectra of Li 1s vacancies in aqueous solutions of lithium chloride (LiCl) in direct comparison to lithium acetate (LiOAc). A change in the ETMD spectrum dependent on the electrolyte anion identity is observed for 4.5 M salt concentration. We discuss these findings within the framework of the formation and presence of contact ion pairs and the unique sensitivity of ETMD spectroscopy to ion pairing.

  8. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation

    NARCIS (Netherlands)

    Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

    2014-01-01

    There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute

  9. Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

    International Nuclear Information System (INIS)

    Lin, Ping; Mobasher, Maral E.; Alawi, Faizan

    2014-01-01

    Highlights: • Dyskerin depletion triggers cellular senescence in U2OS osteosarcoma cells. • Dyskerin-depleted cells are resistant to apoptosis induced by genotoxic stress. • Chromatin relaxation sensitizes dyskerin-depleted cells to apoptosis. - Abstract: Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening

  10. Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52

    DEFF Research Database (Denmark)

    Seong, C.; Sehorn, M.G.; Plate, Iben

    2008-01-01

    A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52...... with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function....... Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad...

  11. ResDE Two-Component Regulatory System Mediates Oxygen Limitation-Induced Biofilm Formation by Bacillus amyloliquefaciens SQR9.

    Science.gov (United States)

    Zhou, Xuan; Zhang, Nan; Xia, Liming; Li, Qing; Shao, Jiahui; Shen, Qirong; Zhang, Ruifu

    2018-04-15

    Efficient biofilm formation and root colonization capabilities facilitate the ability of beneficial plant rhizobacteria to promote plant growth and antagonize soilborne pathogens. Biofilm formation by plant-beneficial Bacillus strains is triggered by environmental cues, including oxygen deficiency, but the pathways that sense these environmental signals and regulate biofilm formation have not been thoroughly elucidated. In this study, we showed that the ResDE two-component regulatory system in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain SQR9 senses the oxygen deficiency signal and regulates biofilm formation. ResE is activated by sensing the oxygen limitation-induced reduction of the NAD + /NADH pool through its PAS domain, stimulating its kinase activity, and resulting in the transfer of a phosphoryl group to ResD. The phosphorylated ResD directly binds to the promoter regions of the qoxABCD and ctaCDEF operons to improve the biosynthesis of terminal oxidases, which can interact with KinB to activate biofilm formation. These results not only revealed the novel regulatory function of the ResDE two-component system but also contributed to the understanding of the complicated regulatory network governing Bacillus biofilm formation. This research may help to enhance the root colonization and the plant-beneficial efficiency of SQR9 and other Bacillus rhizobacteria used in agriculture. IMPORTANCE Bacillus spp. are widely used as bioinoculants for plant growth promotion and disease suppression. The exertion of their plant-beneficial functions is largely dependent on their root colonization, which is closely related to their biofilm formation capabilities. On the other hand, Bacillus is the model bacterium for biofilm study, and the process and molecular network of biofilm formation are well characterized (B. Mielich-Süss and D. Lopez, Environ Microbiol 17:555-565, 2015, https://doi.org/10.1111/1462-2920.12527; L. S. Cairns, L. Hobley, and

  12. mma: An R Package for Mediation Analysis with Multiple Mediators

    OpenAIRE

    Qingzhao Yu; Bin Li

    2017-01-01

    Mediation refers to the effect transmitted by mediators that intervene in the relationship between an exposure and a response variable. Mediation analysis has been broadly studied in many fields. However, it remains a challenge for researchers to consider complicated associations among variables and to differentiate individual effects from multiple mediators. [1] proposed general definitions of mediation effects that were adaptable to all different types of response (categorical or continuous...

  13. Descondensación de la heterocromatina en bovinos criollos portadores de la translocación Robertsoniana (rob1; 29. Acción del inductor 5-azacitidina-C Heterochromatin decondesation in Creole cattle carrier of Robertsonian franslocation (rob 1; 29. Action of 5-azacytidine-C

    Directory of Open Access Journals (Sweden)

    Rody Artigas

    2008-01-01

    Full Text Available La translocación Robertsoniana (rob1; 29 está ampliamente distribuida en razas comerciales y en reservas genéticas de bovinos Criollos americanos. Se ha descrito un enlentecimiento en el desarrollo de embriones portadores de esta aneuploidía, frente a embriones normales. La acción de la 5-aza-C, como agente desmetilante, permitiría descondensar la heterocromatina constitutiva o facultativa. En este trabajo se realiza inducción con el análogo de base 5-aza-C(10mM, 2 hrs en cultivos linfocitarios de una hembra y un macho portadores de la rob1; 29, frente a bovinos Criollos normales. Se controla la acción desmetilante del inductor al identificar la despiralización del cromosoma X de replicación tardía en hembras y permitir el análisis de la despiralización de la cromatina en múltiples regiones de los autosomas (grandes, medianos, pequeños; de la rob1; 29 y del BTA1. Se discute la correlación existente entre regiones desmetiladas con la descondensación de la heterocromatina facultativa (condicional, relacionándola con la inestabilidad genómica, y la reprogramación epigenética.The Robertsonian translocation (rob1; 29 is widely spread in commercial breeds and specially in genetic reserve of American Creole cattle. It is also described a delay on embryo development in front of normal ones. The action of 5-aza-C, as an hypomethylated agent, could permitted to decondensate the constitutive or facultative heterochromatin. In this work we made induction with the 5-aza-C(10mM, 2 hrs analogs, in lymphocyte cultures of female and male carriers and normal Creole cattle. The DNA hypometilation is found in the inactive X chromosome of late replication as it is incorporated during the last hours of cell culture. The decondensing effects of 5-aza-C analogs is observed in multiple regions of the autosomes chromatin, the rob1; 29 and the BTA1. A correlation between hypometilated regions and decondensed of facultative (conditional

  14. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S

    Science.gov (United States)

    Haka, Abigail S.; Barbosa-Lorenzi, Valéria C.; Lee, Hyuek Jong; Falcone, Domenick J.; Hudis, Clifford A.; Dannenberg, Andrew J.

    2016-01-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  15. Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

    Science.gov (United States)

    Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

    2016-01-01

    DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

  16. Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL

    Science.gov (United States)

    Jenkins, Misty R; Stinchcombe, Jane C; Au-Yeung, Byron B; Asano, Yukako; Ritter, Alex T; Weiss, Arthur; Griffiths, Gillian M

    2014-01-01

    T cell receptor (TCR) activation leads to a dramatic reorganisation of both membranes and receptors as the immunological synapse forms. Using a genetic model to rapidly inhibit Zap70 catalytic activity we examined synapse formation between cytotoxic T lymphocytes and their targets. In the absence of Zap70 catalytic activity Vav-1 activation occurs and synapse formation is arrested at a stage with actin and integrin rich interdigitations forming the interface between the two cells. The membranes at the synapse are unable to flatten to provide extended contact, and Lck does not cluster to form the central supramolecular activation cluster (cSMAC). Centrosome polarisation is initiated but aborts before reaching the synapse and the granules do not polarise. Our findings reveal distinct roles for Zap70 as a structural protein regulating integrin-mediated control of actin vs its catalytic activity that regulates TCR-mediated control of actin and membrane remodelling during formation of the immunological synapse. DOI: http://dx.doi.org/10.7554/eLife.01310.001 PMID:24596147

  17. microRNA-150 inhibits the formation of macrophage foam cells through targeting adiponectin receptor 2

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing [Department of Geratory, Linzi District People’s Hospital of Zibo City, Zibo, Shandong (China); Zhang, Suhua, E-mail: drsuhuangzhang@qq.com [Department of HealthCare, Qilu Hospital of Shandong University (Qingdao), Qingdao City, Qingdao (China)

    2016-08-05

    Transformation of macrophages into foam cells plays a critical role in the pathogenesis of atherosclerosis. The aim of this study was to determine the expression and biological roles of microRNA (miR)-150 in the formation of macrophage foam cells and to identify its functional target(s). Exposure to 50 μg/ml oxidized low-density lipoprotein (oxLDL) led to a significant upregulation of miR-150 in THP-1 macrophages. Overexpression of miR-150 inhibited oxLDL-induced lipid accumulation in THP-1 macrophages, while knockdown of miR-150 enhanced lipid accumulation. apoA-I- and HDL-mediated cholesterol efflux was increased by 66% and 43%, respectively, in miR-150-overexpressing macrophages relative to control cells. In contrast, downregulation of miR-150 significantly reduced cholesterol efflux from oxLDL-laden macrophages. Bioinformatic analysis and luciferase reporter assay revealed adiponectin receptor 2 (AdipoR2) as a direct target of miR-150. Small interfering RNA-mediated downregulation of AdipoR2 phenocopied the effects of miR-150 overexpression, reducing lipid accumulation and facilitating cholesterol efflux in oxLDL-treated THP-1 macrophages. Knockdown of AdipoR2 induced the expression of proliferator-activated receptor gamma (PPARγ), liver X receptor alpha (LXRα), ABCA1, and ABCG1. Moreover, pharmacological inhibition of PPARγ or LXRα impaired AdipoR2 silencing-induced upregulation of ABCA1 and ABCG1. Taken together, our results indicate that miR-150 can attenuate oxLDL-induced lipid accumulation in macrophages via promotion of cholesterol efflux. The suppressive effects of miR-150 on macrophage foam cell formation are mediated through targeting of AdipoR2. Delivery of miR-150 may represent a potential approach to prevent macrophage foam cell formation in atherosclerosis. -- Highlights: •miR-150 inhibits macrophage foam cell formation. •miR-150 accelerates cholesterol efflux from oxLDL-laden macrophages. •miR-150 suppresses macrophage foam cell

  18. Src binds cortactin through an SH2 domain cystine-mediated linkage

    Science.gov (United States)

    Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

    2012-01-01

    Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

  19. Src binds cortactin through an SH2 domain cystine-mediated linkage.

    Science.gov (United States)

    Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

    2012-12-15

    Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

  20. Effect of gamma irradiation on sex chromatin body appearance and the sex chromosome aberrations in the potato tuber moth, phthorimaea operculella (Lepidoptera: Gelechiidae)

    International Nuclear Information System (INIS)

    Makee, H.

    2007-01-01

    Genetic sexing technique based on the construction of a Balanced Lethal Strain (BLS) has been proposed for Phthorimaea operculella (Zeller). The isolation of female with T(W. Z) translocation is a fundamental step to develop such strain. Gamma irradiation was used to induce the requested translocations. The availability of sex-linked morphological marker is required to facilitate the detection of such mutations. Since a visible sex-linked marker has not been found in P. operculella, therefore main aim of our study was to determine the possibility of using sex heterochromatin body as a marker to identify the required translocated females. The appearance of sex heterochromatin body and the analysis of sex chromosomes in F1 females of irradiated P. operculella females were investigated. The percentage of abnormality in sex heterochromatin body in highly polyploid Malpighian tubule nuclei was increased by increasing the applied dose. Based on the appearance of this body, 3 mutant lines were isolated: elongated, small, fragmented lines. W chromosome was easily distinguished from Z chromosome when the analysis of pachytene sex chromosome bivalents of P. operculella females was carried out. The aberrations involved W chromosome directly influenced the appearance of sex heterochromatin body in highly polyploid somatic cells of the isolated mutant lines. The results showed that sex heterochromatin could be used as sex determination and cytogenetic marker in P. operculella. (Author)

  1. Effect of gamma irradiation on sex chromatin body appearance and the sex chromosome aberrations in the potato tuber moth, phthorimaea operculella (Lepidoptera: Gelechiidae)

    International Nuclear Information System (INIS)

    Makee, H.

    2006-05-01

    Genetic sexing technique based on the construction of a Balanced Lethal Strain (BLS) has been proposed for Phthorimaea operculella (Zeller). The isolation of female with T(W; Z) translocation is a fundamental step to develop such strain. Gamma irradiation was used to induce the requested translocations. The availability of sex-linked morphological marker is required to facilitate the detection of such mutations. Since a visible sex-linked marker has not been found in P. operculella, therefore main aim of our study was to determine the possibility of using sex heterochromatin body as a marker to identify the required translocated females. The appearance of sex heterochromatin body and the analysis of sex chromosomes in F1 females of irradiated P. operculella females were investigated. The percentage of abnormality in sex heterochromatin body in highly polyploid Malpighian tubule nuclei was increased by increasing the applied dose. Based on the appearance of this body, 3 mutant lines were isolated: elongated, small, fragmented lines. W chromosome was easily distinguished from Z chromosome when the analysis of pachytene sex chromosome bivalents of P. operculella females was carried out. The aberrations involved W chromosome directly influenced the appearance of sex heterochromatin body in highly polyploid somatic cells of the isolated mutant lines. The results showed that sex heterochromatin could be used as sex determination and cytogenetic marker in P. operculella. (Author)

  2. Inflammation induced mTORC2-Akt-mTORC1 signaling promotes macrophage foam cell formation.

    Science.gov (United States)

    Banerjee, Dipanjan; Sinha, Archana; Saikia, Sudeshna; Gogoi, Bhaskarjyoti; Rathore, Arvind K; Das, Anindhya Sundar; Pal, Durba; Buragohain, Alak K; Dasgupta, Suman

    2018-06-05

    The transformation of macrophages into lipid loaded foam cells is a critical and early event in the pathogenesis of atherosclerosis. Several recent reports highlighted that induction of TLR4 signaling promotes macrophage foam cell formation; however, the underlying molecular mechanisms have not been clearly elucidated. Here, we found that the TLR4 mediated inflammatory signaling communicated with mTORC2-Akt-mTORC1 metabolic cascade in macrophage and thereby promoting lipid uptake and foam cell formation. Mechanistically, LPS treatment markedly upregulates TLR4 mediated inflammatory pathway which by activating mTORC2 induces Akt phosphorylation at serine 473 and that aggravate mTORC1 dependent scavenger receptors expression and consequent lipid accumulation in THP-1 macrophages. Inhibition of mTORC2 either by silencing Rictor expression or inhibiting its association with mTOR notably prevents LPS induced Akt activation, scavenger receptors expression and macrophage lipid accumulation. Although suppression of mTORC1 expression by genetic knockdown of Raptor did not produce any significant change in Akt S473 phosphorylation, however, incubation with Akt activator in Rictor silenced cells failed to promote scavenger receptors expression and macrophage foam cell formation. Thus, present research explored the signaling pathway involved in inflammation induced macrophage foam cells formation and therefore, targeting this pathway might be useful for preventing macrophage foam cell formation. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. RNA-binding IMPs promote cell adhesion and invadopodia formation

    DEFF Research Database (Denmark)

    Vikesaa, Jonas; Hansen, Thomas V O; Jønson, Lars

    2006-01-01

    Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and inva......Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading...... and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular......-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation....

  4. Recent advances in engineering topography mediated antibacterial surfaces

    Science.gov (United States)

    Hasan, Jafar; Chatterjee, Kaushik

    2015-09-01

    The tendency of bacterial cells to adhere and colonize a material surface leading to biofilm formation is a fundamental challenge underlying many different applications including microbial infections associated with biomedical devices and products. Although, bacterial attachment to surfaces has been extensively studied in the past, the effect of surface topography on bacteria-material interactions has received little attention until more recently. We review the recent progress in surface topography based approaches for engineering antibacterial surfaces. Biomimicry of antibacterial surfaces in nature is a popular strategy. Whereas earlier endeavors in the field aimed at minimizing cell attachment, more recent efforts have focused on developing bactericidal surfaces. However, not all such topography mediated bactericidal surfaces are necessarily cytocompatible thus underscoring the need for continued efforts for research in this area for developing antibacterial and yet cytocompatible surfaces for use in implantable biomedical applications. This mini-review provides a brief overview of the current strategies and challenges in the emerging field of topography mediated antibacterial surfaces.

  5. The human hippocampal formation mediates short-term memory of colour-location associations.

    Science.gov (United States)

    Finke, Carsten; Braun, Mischa; Ostendorf, Florian; Lehmann, Thomas-Nicolas; Hoffmann, Karl-Titus; Kopp, Ute; Ploner, Christoph J

    2008-01-31

    The medial temporal lobe (MTL) has long been considered essential for declarative long-term memory, whereas the fronto-parietal cortex is generally seen as the anatomical substrate of short-term memory. This traditional dichotomy is questioned by recent studies suggesting a possible role of the MTL for short-term memory. In addition, there is no consensus on a possible specialization of MTL sub-regions for memory of associative information. Here, we investigated short-term memory for single features and feature associations in three humans with post-surgical lesions affecting the right hippocampal formation and in 10 healthy controls. We used three delayed-match-to-sample tasks with two delays (900/5000 ms) and three set sizes (2/4/6 items). Subjects were instructed to remember either colours, locations or colour-location associations. In colour-only and location-only conditions, performance of patients did not differ from controls. By contrast, a significant group difference was found in the association condition at 5000 ms delay. This difference was largely independent of set size, thus suggesting that it cannot be explained by the increased complexity of the association condition. These findings show that the hippocampal formation plays a significant role for short-term memory of simple visuo-spatial associations, and suggest a specialization of MTL sub-regions for associative memory.

  6. Ferroxidase-Mediated Iron Oxide Biomineralization

    DEFF Research Database (Denmark)

    Zeth, Kornelius; Hoiczyk, Egbert; Okuda, Mitsuhiro

    2016-01-01

    Iron oxide biomineralization occurs in all living organisms and typically involves protein compartments ranging from 5 to 100nm in size. The smallest iron-oxo particles are formed inside dodecameric Dps protein cages, while the structurally related ferritin compartments consist of twice as many......, translocation, oxidation, nucleation, and storage, that are mediated by ferroxidase centers. Thus, compartmentalized iron oxide biomineralization yields uniform nanoparticles strictly determined by the sizes of the compartments, allowing customization for highly diverse nanotechnological applications....... identical protein subunits. The largest known compartments are encapsulins, icosahedra made of up to 180 protein subunits that harbor additional ferritin-like proteins in their interior. The formation of iron-oxo particles in all these compartments requires a series of steps including recruitment of iron...

  7. Concept formation skills in long-term cochlear implant users.

    Science.gov (United States)

    Castellanos, Irina; Kronenberger, William G; Beer, Jessica; Colson, Bethany G; Henning, Shirley C; Ditmars, Allison; Pisoni, David B

    2015-01-01

    This study investigated if a period of auditory sensory deprivation followed by degraded auditory input and related language delays affects visual concept formation skills in long-term prelingually deaf cochlear implant (CI) users. We also examined if concept formation skills are mediated or moderated by other neurocognitive domains (i.e., language, working memory, and executive control). Relative to normally hearing (NH) peers, CI users displayed significantly poorer performance in several specific areas of concept formation, especially when multiple comparisons and relational concepts were components of the task. Differences in concept formation between CI users and NH peers were fully explained by differences in language and inhibition-concentration skills. Language skills were also found to be more strongly related to concept formation in CI users than in NH peers. The present findings suggest that complex relational concepts may be adversely affected by a period of early prelingual deafness followed by access to underspecified and degraded sound patterns and spoken language transmitted by a CI. Investigating a unique clinical population such as early-implanted prelingually deaf children with CIs can provide new insights into foundational brain-behavior relations and developmental processes. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Chemically mediated group formation in soil-dwelling larvae and pupae of the beetle Trypoxylus dichotomus

    Science.gov (United States)

    Kojima, Wataru; Ishikawa, Yukio; Takanashi, Takuma

    2014-09-01

    Many insects form groups through interactions among individuals, and these are often mediated by chemical, acoustic, or visual cues and signals. In spite of the diversity of soil-dwelling insects, their aggregation behaviour has not been examined as extensively as that of aboveground species. We investigated the aggregation mechanisms of larvae of the Japanese rhinoceros beetle Trypoxylus dichotomus, which live in groups in humus soil. In two-choice laboratory tests, 2nd- and 3rd-instar larvae gathered at conspecific larvae irrespective of the kinship. The ablation of maxillae, which bear chemosensilla, abolished aggregation behaviour. Intact larvae also exhibited aggregation behaviour towards a larval homogenate. These results suggest that larval aggregation is mediated by chemical cues. We also demonstrated that the mature larvae of T. dichotomus built their pupal cells close to a mesh bag containing a conspecific pupal cell, which indicated that larvae utilize chemical cues emanating from these cells to select the pupation site. Thus, the larvae of T. dichotomus may use chemical cues from the conspecifics in two different contexts, i.e. larval aggregation and pupation site selection. Using conspecific cues, larvae may be able to choose suitable locations for foraging or building pupal cells. The results of the present study highlight the importance of chemical information in belowground ecology.

  9. E-selectin mediates stem cell adhesion and formation of blood vessels in a murine model of infantile hemangioma.

    Science.gov (United States)

    Smadja, David M; Mulliken, John B; Bischoff, Joyce

    2012-12-01

    Hemangioma stem cells (HemSCs) are multipotent cells isolated from infantile hemangioma (IH), which form hemangioma-like lesions when injected subcutaneously into immune-deficient mice. In this murine model, HemSCs are the primary target of corticosteroid, a mainstay therapy for problematic IH. The relationship between HemSCs and endothelial cells that reside in IH is not clearly understood. Adhesive interactions might be critical for the preferential accumulation of HemSCs and/or endothelial cells in the tumor. Therefore, we studied the interactions between HemSCs and endothelial cells (HemECs) isolated from IH surgical specimens. We found that HemECs isolated from proliferating phase IH, but not involuting phase, constitutively express E-selectin, a cell adhesion molecule not present in quiescent endothelial cells. E-selectin was further increased when HemECs were exposed to vascular endothelial growth factor-A or tumor necrosis factor-α. In vitro, HemSC migration and adhesion was enhanced by recombinant E-selectin but not P-selectin; both processes were neutralized by E-selectin-blocking antibodies. E-selectin-positive HemECs also stimulated migration and adhesion of HemSCs. In vivo, neutralizing antibodies to E-selectin strongly inhibited formation of blood vessels when HemSCs and HemECs were co-implanted in Matrigel. These data suggest that endothelial E-selectin could be a major ligand for HemSCs and thereby promote cellular interactions and vasculogenesis in IH. We propose that constitutively expressed E-selectin on endothelial cells in the proliferating phase is one mediator of the stem cell tropism in IH. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. Non-canonical WOX11-mediated root branching contributes to plasticity in Arabidopsis root system architecture.

    Science.gov (United States)

    Sheng, Lihong; Hu, Xiaomei; Du, Yujuan; Zhang, Guifang; Huang, Hai; Scheres, Ben; Xu, Lin

    2017-09-01

    Lateral roots (LRs), which originate from the growing root, and adventitious roots (ARs), which are formed from non-root organs, are the main contributors to the post-embryonic root system in Arabidopsis However, our knowledge of how formation of the root system is altered in response to diverse inductive cues is limited. Here, we show that WOX11 contributes to root system plasticity. When seedlings are grown vertically on medium, WOX11 is not expressed in LR founder cells. During AR initiation, WOX11 is expressed in AR founder cells and activates LBD16 LBD16 also functions in LR formation and is activated in that context by ARF7 / 19 and not by WOX11 This indicates that divergent initial processes that lead to ARs and LRs may converge on a similar mechanism for primordium development. Furthermore, we demonstrated that when plants are grown in soil or upon wounding on medium, the primary root is able to produce both WOX11 -mediated and non- WOX11 -mediated roots. The discovery of WOX11 -mediated root-derived roots reveals a previously uncharacterized pathway that confers plasticity during the generation of root system architecture in response to different inductive cues. © 2017. Published by The Company of Biologists Ltd.

  11. Complex Mediation

    DEFF Research Database (Denmark)

    Bødker, Susanne; Andersen, Peter Bøgh

    2005-01-01

    This article has its starting point in a large number of empirical findings regarding computer-mediated work. These empirical findings have challenged our understanding of the role of mediation in such work; on the one hand as an aspect of communication and cooperation at work and on the other hand...... as an aspect of human engagement with instruments of work. On the basis of previous work in activity-theoretical and semiotic human—computer interaction, we propose a model to encompass both of these aspects. In a dialogue with our empirical findings we move on to propose a number of types of mediation...... that have helped to enrich our understanding of mediated work and the design of computer mediation for such work....

  12. Evaluation of the human relevance of the constitutive androstane receptor-mediated mode of action for rat hepatocellular tumor formation by the synthetic pyrethroid momfluorothrin.

    Science.gov (United States)

    Okuda, Yu; Kushida, Masahiko; Kikumoto, Hiroko; Nakamura, Yoshimasa; Higuchi, Hashihiro; Kawamura, Satoshi; Cohen, Samuel M; Lake, Brian G; Yamada, Tomoya

    2017-01-01

    High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

  13. Working the Senate from the Outside In: The Mediated Construction of a Feminist Political Campaign.

    Science.gov (United States)

    Vavrus, Mary

    1998-01-01

    Contributes to scholarship on rhetorical criticism, political science, and feminism by examining one aspect of the 1992 campaign year known as the "Year of the Woman." Discusses how the mass-mediated discursive formation positioned five female Senate candidates outside of perceived mainstream cultural beliefs. Investigates ways the…

  14. Formation of Monocrystalline 1D and 2D Architectures via Epitaxial Attachment: Bottom-Up Routes through Surfactant-Mediated Arrays of Oriented Nanocrystals.

    Science.gov (United States)

    Nakagawa, Yoshitaka; Kageyama, Hiroyuki; Oaki, Yuya; Imai, Hiroaki

    2015-06-09

    Monocrystalline architectures with well-defined shapes were achieved by bottom-up routes through epitaxial attachment of Mn3O4 nanocrystals. The crystallographically continuous 1D chains elongated in the a axis and 2D panels having large a or c faces were obtained by removal of the organic mediator from surfactant-mediated 1D and 2D arrays of Mn3O4 nanocrystals, respectively. Our basal approach indicates that the epitaxial attachment through the surfactant-mediated arrays is utilized for fabrication of a wide variety of micrometric architectures from nanometric crystalline units.

  15. Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation.

    Directory of Open Access Journals (Sweden)

    Caitlin K Wotanis

    Full Text Available The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.

  16. Hemoglobin as a nitrite anhydrase: modeling methemoglobin-mediated N2O3 formation.

    Science.gov (United States)

    Hopmann, Kathrin H; Cardey, Bruno; Gladwin, Mark T; Kim-Shapiro, Daniel B; Ghosh, Abhik

    2011-05-27

    Nitrite has recently been recognized as a storage form of NO in blood and as playing a key role in hypoxic vasodilation. The nitrite ion is readily reduced to NO by hemoglobin in red blood cells, which, as it happens, also presents a conundrum. Given NO's enormous affinity for ferrous heme, a key question concerns how it escapes capture by hemoglobin as it diffuses out of the red cells and to the endothelium, where vasodilation takes place. Dinitrogen trioxide (N(2)O(3)) has been proposed as a vehicle that transports NO to the endothelium, where it dissociates to NO and NO(2). Although N(2)O(3) formation might be readily explained by the reaction Hb-Fe(3+)+NO(2)(-)+NO⇌Hb-Fe(2+)+N(2)O(3), the exact manner in which methemoglobin (Hb-Fe(3+)), nitrite and NO interact with one another is unclear. Both an "Hb-Fe(3+)-NO(2)(-)+NO" pathway and an "Hb-Fe(3+)-NO+NO(2)(-) " pathway have been proposed. Neither pathway has been established experimentally. Nor has there been any attempt until now to theoretically model N(2)O(3) formation, the so-called nitrite anhydrase reaction. Both pathways have been examined here in a detailed density functional theory (DFT, B3LYP/TZP) study and both have been found to be feasible based on energetics criteria. Modeling the "Hb-Fe(3+)-NO(2)(-)+NO" pathway proved complex. Not only are multiple linkage-isomeric (N- and O-coordinated) structures conceivable for methemoglobin-nitrite, multiple isomeric forms are also possible for N(2)O(3) (the lowest-energy state has an N-N-bonded nitronitrosyl structure, O(2)N-NO). We considered multiple spin states of methemoglobin-nitrite as well as ferromagnetic and antiferromagnetic coupling of the Fe(3+) and NO spins. Together, the isomerism and spin variables result in a diabolically complex combinatorial space of reaction pathways. Fortunately, transition states could be successfully calculated for the vast majority of these reaction channels, both M(S)=0 and M(S)=1. For a six-coordinate Fe(3+)-O

  17. Supplementation with linoleic acid-rich soybean oil stimulates macrophage foam cell formation via increased oxidative stress and diacylglycerol acyltransferase1-mediated triglyceride biosynthesis.

    Science.gov (United States)

    Rom, Oren; Jeries, Helana; Hayek, Tony; Aviram, Michael

    2017-01-02

    During the last decades there has been a staggering rise in human consumption of soybean oil (SO) and its major polyunsaturated fatty acid linoleic acid (LA). The role of SO or LA in cardiovascular diseases is highly controversial, and their impact on macrophage foam cell formation, the hallmark of early atherogenesis, is unclear. To investigate the effects of high SO or LA intake on macrophage lipid metabolism and the related mechanisms of action, C57BL/6 mice were orally supplemented with increasing levels of SO-based emulsion or equivalent levels of purified LA for 1 month, followed by analyses of lipid accumulation and peroxidation in aortas, serum and in peritoneal macrophages (MPM) of the mice. Lipid peroxidation and triglyceride mass in aortas from SO or LA supplemented mice were dose-dependently and significantly increased. In MPM from SO or LA supplemented mice, lipid peroxides were significantly increased and a marked accumulation of cellular triglycerides was found in accordance with enhanced triglyceride biosynthesis rate and overexpression of diacylglycerol acyltransferase1 (DGAT1), the key enzyme in triglyceride biosynthesis. In cultured J774A.1 macrophages treated with SO or LA, triglyceride accumulated via increased oxidative stress and a p38 mitogen-activated protein kinase (MAPK)-mediated overexpression of DGAT1. Accordingly, anti-oxidants (pomegranate polyphenols), inhibition of p38 MAPK (by SB202190) or DGAT1 (by oleanolic acid), all significantly attenuated SO or LA-induced macrophage triglyceride accumulation. These findings reveal novel mechanisms by which supplementation with SO or LA stimulate macrophage foam cell formation, suggesting a pro-atherogenic role for overconsumption of SO or LA. © 2016 BioFactors, 43(1):100-116, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  18. Sexual Biofilm Formation in Candida tropicalis Opaque Cells

    Science.gov (United States)

    Jones, Stephen K.; Hirakawa, Matthew P.; Bennett, Richard J.

    2014-01-01

    Summary Candida albicans and Candida tropicalis are opportunistic fungal pathogens that can transition between white and opaque phenotypic states. White and opaque cells differ both morphologically and in their responses to environmental signals. In C. albicans, opaque cells respond to sexual pheromones by undergoing conjugation, while white cells are induced by pheromones to form sexual biofilms. Here, we show that sexual biofilm formation also occurs in C. tropicalis but, unlike C. albicans, biofilms are formed exclusively by opaque cells. C. tropicalis biofilm formation was dependent on the pheromone receptors Ste2 and Ste3, confirming the role of pheromone signaling in sexual biofilm development. Structural analysis of C. tropicalis sexual biofilms revealed stratified communities consisting of a basal layer of yeast cells and an upper layer of filamentous cells, together with an extracellular matrix. Transcriptional profiling showed that genes involved in pheromone signaling and conjugation were upregulated in sexual biofilms. Furthermore, FGR23, which encodes an agglutinin-like protein, was found to enhance both mating and sexual biofilm formation. Together, these studies reveal that C. tropicalis opaque cells form sexual biofilms with a complex architecture, and suggest a conserved role for sexual agglutinins in mediating mating, cell cohesion and biofilm formation. PMID:24612417

  19. Photochemical Formation of Aerosol in Planetary Atmospheres: Photon and Water Mediated Chemistry of SO_2

    Science.gov (United States)

    Kroll, Jay A.; Donaldson, D. J.; Vaida, Veronica

    2016-06-01

    Sulfur compounds have been observed in a number of planetary atmospheres throughout our solar system. Our current understanding of sulfur chemistry explains much of what we observe in Earth's atmosphere. However, several discrepancies between modeling and observations of the Venusian atmosphere show there are still problems in our fundamental understanding of sulfur chemistry. This is of particular concern due to the important role sulfur compounds play in the formation of aerosols, which have a direct impact on planetary climates, including Earth's. We investigate the role of water complexes in the hydration of sulfur oxides and dehydration of sulfur acids and will present spectroscopic studies to document such effects. I will present recent work investigating mixtures of SO_2 and water that generate large quantities of aerosol when irradiated with solar UV light, even in the absence of traditional OH chemistry. I will discuss a proposed mechanism for the formation of sulfurous acid (H_2SO_3) and present recent experimental work that supports this proposed mechanism. Additionally, the implications that photon-induced hydration of SO_2 has for aerosol formation in the atmosphere of earth as well as other planetary atmospheres will be discussed.

  20. Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

    Directory of Open Access Journals (Sweden)

    Ohgi Takahashi

    2015-01-01

    Full Text Available Succinimide formation from aspartic acid (Asp residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe as a model compound, we propose the possibility that acetic acid (AA, which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

  1. Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study.

    Science.gov (United States)

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

    2015-01-12

    Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

  2. Characterization of the integrated filamentous phage Pf5 and its involvement in small-colony formation

    NARCIS (Netherlands)

    Mooij, Marlies J.; Drenkard, Eliana; Llamas, María A.; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.; Ausubel, Frederick M.; Bitter, Wilbert

    2007-01-01

    Bacteriophages play an important role in bacterial virulence and phenotypic variation. It has been shown that filamentous bacteriophage Pf4 of Pseudomonas aeruginosa strain PAO1 mediates the formation of small-colony variants (SCVs) in biofilms. This morphology type is associated with parameters of

  3. Steps in the design, development and formative evaluation of obesity prevention-related behavior change trials.

    Science.gov (United States)

    Baranowski, Tom; Cerin, Ester; Baranowski, Janice

    2009-01-21

    Obesity prevention interventions through dietary and physical activity change have generally not been effective. Limitations on possible program effectiveness are herein identified at every step in the mediating variable model, a generic conceptual framework for understanding how interventions may promote behavior change. To minimize these problems, and thereby enhance likely intervention effectiveness, four sequential types of formative studies are proposed: targeted behavior validation, targeted mediator validation, intervention procedure validation, and pilot feasibility intervention. Implementing these studies would establish the relationships at each step in the mediating variable model, thereby maximizing the likelihood that an intervention would work and its effects would be detected. Building consensus among researchers, funding agencies, and journal editors on distinct intervention development studies should avoid identified limitations and move the field forward.

  4. Digital technologies and their impact on Higher Education. The new mediations

    Directory of Open Access Journals (Sweden)

    Juan de Pablos Pons

    2018-03-01

    Full Text Available This work proposes a reflection on the evolution of media in the digital age, requires us to advance in knowledge about mediation and remediation processes, favored by technologies, differentiating them from those generated so far by traditional analogue media (printed book, film, television. Digitization has given rise to new realities and proposals, such as video games, social networks or augmented reality applications. The next step in our media culture is the multiplication and continuous diversification of formats, but also new forms of mediated interaction. The mediation processes generated contribute to the modification of communication models and teaching methods. And this has an impact on educational institutions, and also on higher education. As a contribution to its analysis, the concept of technological remediation is provided and defined. The new information and communication technologies (ICT represent, under the considerations formulated here, an opportunity for change some forms and procedures of main areas of the higher education: teaching, research and management. Being proactive in this new reality is relevant, since the adaptation effort is linked to the survival of the university itself.

  5. Asymmetric Formation of Bridged Benzoxazocines through an Organocatalytic Multicomponent Dienamine-Mediated One-Pot Cascade

    DEFF Research Database (Denmark)

    Ransborg, Lars Krogager; Overgaard, Mette Kiilerich; Hejmanowska, Joanna

    2014-01-01

    An organocatalytic one-pot cascade leading to the stereoselective formation of novel bridged benzoxazocines is presented. The developed methodology is based on the first example of a γ-selective-Mannich-initiated cascade reaction and allows for direct annulation of the bridged benzoxazocines...

  6. Flexible Mediation Analysis With Multiple Mediators.

    Science.gov (United States)

    Steen, Johan; Loeys, Tom; Moerkerke, Beatrijs; Vansteelandt, Stijn

    2017-07-15

    The advent of counterfactual-based mediation analysis has triggered enormous progress on how, and under what assumptions, one may disentangle path-specific effects upon combining arbitrary (possibly nonlinear) models for mediator and outcome. However, current developments have largely focused on single mediators because required identification assumptions prohibit simple extensions to settings with multiple mediators that may depend on one another. In this article, we propose a procedure for obtaining fine-grained decompositions that may still be recovered from observed data in such complex settings. We first show that existing analytical approaches target specific instances of a more general set of decompositions and may therefore fail to provide a comprehensive assessment of the processes that underpin cause-effect relationships between exposure and outcome. We then outline conditions for obtaining the remaining set of decompositions. Because the number of targeted decompositions increases rapidly with the number of mediators, we introduce natural effects models along with estimation methods that allow for flexible and parsimonious modeling. Our procedure can easily be implemented using off-the-shelf software and is illustrated using a reanalysis of the World Health Organization's Large Analysis and Review of European Housing and Health Status (WHO-LARES) study on the effect of mold exposure on mental health (2002-2003). © The Author(s) 2017. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Callous-Unemotional Traits and Effortful Control Mediate the Effect of Parenting Intervention on Preschool Conduct Problems.

    Science.gov (United States)

    Elizur, Yoel; Somech, Lior Y

    2018-02-24

    Parenting intervention (PI) is an effective treatment for children's conduct problems (CP) that has been shown to be mediated by improved parenting practices and parenting self-efficacy. Recently, Hitkashrut's randomized controlled trial demonstrated that ineffective parenting (IP) mediated effects on callous-unemotional (CU) traits and effortful control (EC), while controlling for more general treatment effects on CP. These temperament and personality-based features predict the formation of early-onset antisocial trajectories with poor long-term prognosis. The objective of this study was to use Hitkashrut's 3-wave dataset to test posttreatment EC and CU mediation of treatment effect on 1-year follow-up CP, and to determine whether mediation by each child-level potential mediator remains significant when tested concurrently with the parenting mediator. Parents of 209 3-5 year-old preschoolers (163 boys; 46 girls), with subclinical-clinical range CP were assigned to 14-session co-parent training groups (n = 140 couples), or to minimal intervention control groups (n = 69 couples). Assessments were based on both parents' questionnaires. An intent-to-treat analysis showed that EC and CU traits simultaneously mediated treatment effects on CP in one EC/CU mediational model. The concurrent testing of child- and parent-level mediators showed mediation by IP and CU traits in the CU/IP model, and IP mediation in the EC/IP model. Similar results were obtained in mediational analyses that controlled for the shared variance between the mediators and CP at T2. Overall, the findings support an intervention model of coaching parents of high-CP children to promote moral self-regulatory competencies while concurrently applying behavioral methods that directly target CP.

  8. Interventional effects for mediation analysis with multiple mediators

    OpenAIRE

    Vansteelandt, Stijn; Daniel, Rhian M.

    2017-01-01

    The mediation formula for the identification of natural (in)direct effects has facilitated mediation analyses that better respect the nature of the data, with greater consideration of the need for confounding control. The default assumptions on which it relies are strong, however. In particular, they are known to be violated when confounders of the mediator–outcome association are affected by the exposure. This complicates extensions of counterfactual-based mediation analysis to settings that...

  9. Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

    Science.gov (United States)

    Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

    2015-11-01

    The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  10. General gauge mediation

    International Nuclear Information System (INIS)

    Meade, Patrick; Seiberg, Nathan; Shih, David

    2009-01-01

    We give a general definition of gauge mediated supersymmetry breaking which encompasses all the known gauge mediation models. In particular, it includes both models with messengers as well as direct mediation models. A formalism for computing the soft terms in the generic model is presented. Such a formalism is necessary in strongly-coupled direct mediation models where perturbation theory cannot be used. It allows us to identify features of the entire class of gauge mediation models and to distinguish them from specific signatures of various subclasses. (author)

  11. Peroxidase-mediated binding of aromatic amine carcinogens to tissue DNA

    International Nuclear Information System (INIS)

    Wise, R.W.; Lakshmi, V.M.; Zenser, T.V.; Davis, B.B.

    1986-01-01

    Benzidine is a aromatic amine bladder carcinogen in man and dog which requires endogenous metabolic activation. Dog bladder microsomes activate benzidine to bind glutathione and DNA by a peroxidatic but not a mixed-function oxidase mediated pathway. Prostaglandin H synthase was responsible for peroxidatic metabolism. This study was designed to assess benzidine metabolism in a whole cell system. Rabbit renal medullary slices (100 mg/ml) were incubated for 60 min. in Krebs-Ringer bicarbonate buffer containing 100 μM 3 H-benzidine and 250 μM arachidonic acid. Arachidonic acid increased 3-(glutathione-S-yL)-benzidine, a product of peroxidatically activated benzidine, (6-fold) and 3 H-benzidine binding to endogenous DNA (4-fold). Indomethacin (100 μM) completely inhibited arachidonic acid-mediated increases in conjugate formation and DNA binding. HPLC analysis of the media demonstrated benzidine (95% of total 3 H), 3-(glutathion-S-yL)-benzidine (1%) and two unidentified peaks (4%). These results are consistent with the hydroperoxidase activity of prostaglandin H synthase mediating metabolic activation of benzidine to bind tissue nucleophiles in a whole cell system. Inhibition of peroxidatic activation of aromatic amines to bind DNA may prevent initiation of bladder cancer

  12. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Science.gov (United States)

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  13. Regioselectivity of tributyltin ether mediated alkylations. A 119Sn and 13C NMR study

    International Nuclear Information System (INIS)

    Cruzado, C.; Bernabe, M.; Martin-Lomas, M.

    1989-01-01

    The 119 Sn and 13 C NMR spectra of the stannylated species resulting from the treatment of conformationally rigid polyhydroxylated compounds with bis(tributyltin) oxide have been determined and the effect of N-methylimidazole, added as catalyst in tributyltin ether mediated regioselective alkylations, has been investigated. The observed signal intensity changes, upfield shifts, signal broadenings, and the results of variable temperature experiments have been interpreted as indicative of the selective formation of pentacoordinated tin species, involving conveniently disposed adjacent hydroxyl groups, on addition of the catalyst. On these bases, a mechanistic hypothesis for the observed regioselectivity of N-methylimidazole-catalyzed tributyltin ether mediated benzylations is proposed. 13 references, 5 tables

  14. Plant Hormone Homeostasis, Signaling, and Function during Adventitious Root Formation in Cuttings.

    Science.gov (United States)

    Druege, Uwe; Franken, Philipp; Hajirezaei, Mohammad R

    2016-01-01

    Adventitious root (AR) formation in cuttings is a multiphase developmental process, resulting from wounding at the cutting site and isolation from the resource and signal network of the whole plant. Though, promotive effects of auxins are widely used for clonal plant propagation, the regulation and function of plant hormones and their intricate signaling networks during AR formation in cuttings are poorly understood. In this focused review, we discuss our recent publications on the involvement of polar auxin transport (PAT) and transcriptional regulation of auxin and ethylene action during AR formation in petunia cuttings in a broad context. Integrating new findings on cuttings of other plant species and general models on plant hormone networks, a model on the regulation and function of auxin, ethylene, and jasmonate in AR formation of cuttings is presented. PAT and cutting off from the basipetal auxin drain are considered as initial principles generating early accumulation of IAA in the rooting zone. This is expected to trigger a self-regulatory process of auxin canalization and maximization to responding target cells, there inducing the program of AR formation. Regulation of auxin homeostasis via auxin influx and efflux carriers, GH3 proteins and peroxidases, of flavonoid metabolism, and of auxin signaling via AUX/IAA proteins, TOPLESS, ARFs, and SAUR-like proteins are postulated as key processes determining the different phases of AR formation. NO and H2O2 mediate auxin signaling via the cGMP and MAPK cascades. Transcription factors of the GRAS-, AP2/ERF-, and WOX-families link auxin signaling to cell fate specification. Cyclin-mediated governing of the cell cycle, modifications of sugar metabolism and microtubule and cell wall remodeling are considered as important implementation processes of auxin function. Induced by the initial wounding and other abiotic stress factors, up-regulation of ethylene biosynthesis, and signaling via ERFs and early accumulation of

  15. Plant hormone homeostasis, signaling and function during adventitious root formation in cuttings

    Directory of Open Access Journals (Sweden)

    Uwe eDruege

    2016-03-01

    Full Text Available Adventitious root (AR formation in cuttings is a multiphase developmental process, resulting from wounding at the cutting site and isolation from the resource and signal network of the whole plant. Though promotive effects of auxins are widely used for clonal plant propagation, the regulation and function of plant hormones and their intricate signaling networks during AR formation in cuttings are poorly understood. In this focused review, we discuss our recent publications on the involvement of polar auxin transport (PAT and transcriptional regulation of auxin and ethylene action during AR formation in petunia cuttings in a broad context. Integrating new findings on cuttings of other plant species and general models on plant hormone networks, a model on the regulation and function of auxin, ethylene and jasmonate in AR formation of cuttings is presented. PAT and cutting off from the basipetal auxin drain are considered as initial principles generating early accumulation of IAA in the rooting zone. This is expected to trigger a self-regulatory process of auxin canalization and maximization to responding target cells, there inducing the program of AR formation. Regulation of auxin homeostasis via auxin influx and efflux carriers, GH3 proteins and peroxidases, of flavonoid metabolism and of auxin signaling via AUX/IAA proteins, TOPLESS, ARFs and SAUR-like proteins are postulated as key processes determining the different phases of AR formation. NO and H2O2 mediate auxin signaling via the cGMP and MAPK cascades. Transcription factors of the GRAS-, AP2/ERF- and WOX-families link auxin signaling to cell fate specification. Cyclin-mediated governing of the cell cycle, modifications of sugar metabolism and microtubule and cell wall remodeling are considered as important implementation processes of auxin function. Induced by the initial wounding and other abiotic stress factors, up-regulation of ethylene biosynthesis and signaling via ERFs and early

  16. Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps.

    Science.gov (United States)

    Juneau, Richard A; Pang, Bing; Weimer, Kristin E D; Armbruster, Chelsie E; Swords, W Edward

    2011-01-01

    Nontypeable Haemophilus influenzae (NTHI) is a leading cause of otitis media infections, which are often chronic and/or recurrent in nature. NTHI and other bacterial species persist in vivo within biofilms during otitis media and other persistent infections. These biofilms have a significant host component that includes neutrophil extracellular traps (NETs). These NETs do not mediate clearance of NTHI, which survives within NET structures by means of specific subpopulations of lipooligosaccharides on the bacterial surface that are determinants of biofilm formation in vitro. In this study, the ability of NTHI and NTHI components to initiate NET formation was examined using an in vitro model system. Both viable and nonviable NTHI strains were shown to promote NET formation, as did preparations of bacterial DNA, outer membrane proteins, and lipooligosaccharide (endotoxin). However, only endotoxin from a parental strain of NTHI exhibited equivalent potency in NET formation to that of NTHI. Additional studies showed that NTHI entrapped within NET structures is resistant to both extracellular killing within NETs and phagocytic killing by incoming neutrophils, due to oligosaccharide moieties within the lipooligosaccharides. Thus, we concluded that NTHI elicits NET formation by means of multiple pathogen-associated molecular patterns (most notably endotoxin) and is highly resistant to killing within NET structures. These data support the conclusion that, for NTHI, formation of NET structures may be a persistence determinant by providing a niche within the middle-ear chamber.

  17. Better theory-of-mind skills in children hearing voices mitigate the risk of secondary delusion formation

    NARCIS (Netherlands)

    Bartels-Velthuis, A. A.; Blijd-Hoogewys, E. M. A.; van Os, J.

    Objective: To examine the social cognitive vulnerabilities mediating delusion formation in children presenting with hallucinatory experiences. Method: A sample of 259 12- and 13-year-old children, from a baseline case-control sample of children with and without auditory hallucinations (AH), were

  18. MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms.

    Science.gov (United States)

    Joosten, Sander P J; Zeilstra, Jurrit; van Andel, Harmen; Mijnals, R Clinton; Zaunbrecher, Joost; Duivenvoorden, Annet A M; van de Wetering, Marc; Clevers, Hans; Spaargaren, Marcel; Pals, Steven T

    2017-10-01

    /Met fl/fl /LacZ mice. Lgr5 Creert2 /Met fl/fl /LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5 Creert2 /Met fl/fl /Apc fl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (Ah Cre /Met fl/fl /Apc fl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44 -/- mice did not expand to the same extent as crypts from Cd44 +/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  19. Carbon–carbon bond cleavage for Cu-mediated aromatic trifluoromethylations and pentafluoroethylations

    Directory of Open Access Journals (Sweden)

    Tsuyuka Sugiishi

    2015-12-01

    Full Text Available This short review highlights the copper-mediated fluoroalkylation using perfluoroalkylated carboxylic acid derivatives. Carbon–carbon bond cleavage of perfluoroalkylated carboxylic acid derivatives takes place in fluoroalkylation reactions at high temperature (150–200 °C or under basic conditions to generate fluoroalkyl anion sources for the formation of fluoroalkylcopper species. The fluoroalkylation reactions, which proceed through decarboxylation or tetrahedral intermediates, are useful protocols for the synthesis of fluoroalkylated aromatics.

  20. Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds.

    Science.gov (United States)

    Johannes, C; Majcherczyk, A; Hüttermann, A

    1996-10-01

    Laccase of Trametes versicolor was generally able to oxidize anthracene in vitro. After 72 h incubation about 35% of the anthracene was transformed stoichiometrically to 9,10-anthraquinone. Transformation of anthracene increased rapidly in the presence of different mediators that readily generate stable radicals: 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1-hydroxybenzotriazole. For the reaction, the presence of both the laccase and the mediator was necessary. In the presence of 0.005 mM 1-hydroxybenzotriazole this conversion had removed 47% of the anthracene after 72 h; 75% of the substrate was oxidized during this period when ABTS (1 mM) was used as mediator. In contrast to reactions without or with only low concentrations of a mediator, there was a discrepancy between the disappearance of anthracene and the formation of 9,10-anthraquinone in mediator-forced reactions. Coupling-products of mediators with anthracene degradation products were found. Anthracene disappeared nearly completely after incubation for 72 h with laccase in a 0.1 mM solution of 1-hydroxybenzotriazole and was transformed to 9,10-anthraquinone in about 80% yield; 90% of the substrate was transformed in the presence of ABTS (2.0 mM) resulting again in 80% quinone. Phenothiazine was not effective in this system.

  1. Tetraspanin 7 regulates sealing zone formation and the bone-resorbing activity of osteoclasts

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Jun-Oh; Lee, Yong Deok; Kim, Haemin; Kim, Min Kyung; Song, Min-Kyoung; Lee, Zang Hee; Kim, Hong-Hee, E-mail: hhbkim@snu.ac.kr

    2016-09-02

    Tetraspanin family proteins regulate morphology, motility, fusion, and signaling in various cell types. We investigated the role of the tetraspanin 7 (Tspan7) isoform in the differentiation and function of osteoclasts. Tspan7 was up-regulated during osteoclastogenesis. When Tspan7 expression was reduced in primary precursor cells by siRNA-mediated gene knock-down, the generation of multinuclear osteoclasts was not affected. However, a striking cytoskeletal abnormality was observed: the formation of the podosome belt structure was inhibited and the microtubular network were disrupted by Tspan7 knock-down. Decreases in acetylated microtubules and levels of phosphorylated Src and Pyk2 in Tspan7 knock-down cells supported the involvement of Tspan7 in cytoskeletal rearrangement signaling in osteoclasts. This cytoskeletal defect interfered with sealing zone formation and subsequently the bone-resorbing activity of mature osteoclasts on dentin surfaces. Our results suggest that Tspan7 plays an important role in cytoskeletal organization required for the bone-resorbing function of osteoclasts by regulating signaling to Src, Pyk2, and microtubules. - Highlights: • Tspan7 expression is up-regulated during osteoclastogenesis. • Tspan7 regulates podosome belt organization in osteoclasts. • Tspan7 is crucial for sealing zone formation and bone-resorption by osteoclasts. • Src and Pyk2 phosphorylation and microtubule acetylation mediate Tspan7 function.

  2. O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation

    International Nuclear Information System (INIS)

    Ascenzi, Paolo; Gullotta, Francesca; Gioia, Magda; Coletta, Massimo; Fasano, Mauro

    2011-01-01

    Research highlights: → Human serum heme-albumin displays globin-like properties. → O 2 -mediated oxidation of ferrous nitrosylated human serum heme-albumin. → Allosteric modulation of human serum heme-albumin reactivity. → Rifampicin is an allosteric effector of human serum heme-albumin. → Human serum heme-albumin is a ROS and NOS scavenger. -- Abstract: Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O 2 -mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O 2 -mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k = 9.8 x 10 -5 and 8.3 x 10 -4 s -1 , and h = 1.3 x 10 -4 and 8.5 x 10 -4 s -1 , in the absence and presence of rifampicin, respectively, at pH = 7.0 and T = 20.0 o C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O 2 -mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O 2 does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O 2 -mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

  3. Highly Stable Double-Stranded DNA Containing Sequential Silver(I)-Mediated 7-Deazaadenine/Thymine Watson-Crick Base Pairs.

    Science.gov (United States)

    Santamaría-Díaz, Noelia; Méndez-Arriaga, José M; Salas, Juan M; Galindo, Miguel A

    2016-05-17

    The oligonucleotide d(TX)9 , which consists of an octadecamer sequence with alternating non-canonical 7-deazaadenine (X) and canonical thymine (T) as the nucleobases, was synthesized and shown to hybridize into double-stranded DNA through the formation of hydrogen-bonded Watson-Crick base pairs. dsDNA with metal-mediated base pairs was then obtained by selectively replacing W-C hydrogen bonds by coordination bonds to central silver(I) ions. The oligonucleotide I adopts a duplex structure in the absence of Ag(+) ions, and its stability is significantly enhanced in the presence of Ag(+) ions while its double-helix structure is retained. Temperature-dependent UV spectroscopy, circular dichroism spectroscopy, and ESI mass spectrometry were used to confirm the selective formation of the silver(I)-mediated base pairs. This strategy could become useful for preparing stable metallo-DNA-based nanostructures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Visual attention and emotional memory: recall of aversive pictures is partially mediated by concurrent task performance.

    Science.gov (United States)

    Pottage, Claire L; Schaefer, Alexandre

    2012-02-01

    The emotional enhancement of memory is often thought to be determined by attention. However, recent evidence using divided attention paradigms suggests that attention does not play a significant role in the formation of memories for aversive pictures. We report a study that investigated this question using a paradigm in which participants had to encode lists of randomly intermixed negative and neutral pictures under conditions of full attention and divided attention followed by a free recall test. Attention was divided by a highly demanding concurrent task tapping visual processing resources. Results showed that the advantage in recall for aversive pictures was still present in the DA condition. However, mediation analyses also revealed that concurrent task performance significantly mediated the emotional enhancement of memory under divided attention. This finding suggests that visual attentional processes play a significant role in the formation of emotional memories. PsycINFO Database Record (c) 2012 APA, all rights reserved

  5. Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Telma Blanca Lombardo Bedran

    2013-01-01

    Full Text Available Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.

  6. Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots.

    Science.gov (United States)

    Zubairova, Laily D; Nabiullina, Roza M; Nagaswami, Chandrasekaran; Zuev, Yuriy F; Mustafin, Ilshat G; Litvinov, Rustem I; Weisel, John W

    2015-12-04

    Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure, and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure, and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1-0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure, and resistance to fibrinolysis.

  7. Negative regulation of NOD1 mediated angiogenesis by PPARγ-regulated miR-125a

    International Nuclear Information System (INIS)

    Kang, Hyesoo; Park, Youngsook; Lee, Aram; Seo, Hyemin; Kim, Min Jung; Choi, Jihea; Jo, Ha-neul; Jeong, Ha-neul; Cho, Jin Gu; Chang, Woochul; Lee, Myeong-Sok; Jeon, Raok; Kim, Jongmin

    2017-01-01

    Infection with pathogens activates the endothelial cell and its sustained activation may result in impaired endothelial function. Endothelial dysfunction contributes to the pathologic angiogenesis that is characteristic of infection-induced inflammatory pathway activation. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a protein receptor which recognizes bacterial molecules and stimulates an immune reaction in various cells; however, the underlying molecular mechanisms in the regulation of inflammation-triggered angiogenesis are not fully understood. Here we report that peroxisome proliferator-activated receptor gamma (PPARγ)-mediated miR-125a serves as an important regulator of NOD1 agonist-mediated angiogenesis in endothelial cells by directly targeting NOD1. Treatment of human umbilical vein endothelial cells with natural PPARγ ligand, 15-Deoxy-Delta12,14-prostaglandin J2, led to inhibition of NOD1 expression; contrarily, protein levels of NOD1 were significantly increased by PPARγ knockdown. We report that PPARγ regulation of NOD1 expression is a novel microRNA-mediated regulation in endothelial cells. MiR-125a expression was markedly decreased in human umbilical vein endothelial cells subjected to PPARγ knockdown while 15-Deoxy-Delta12,14-prostaglandin J2 treatment increased the level of miR-125a. In addition, NOD1 is closely regulated by miR-125a, which directly targets the 3′ untranslated region of NOD1. Moreover, both overexpression of miR-125a and PPARγ activation led to inhibition of NOD1 agonist-induced tube formation in endothelial cells. Finally, NOD1 agonist increased the formation of cranial and subintestinal vessel plexus in zebrafish, and this effect was abrogated by concurrent PPARγ activation. Overall, these findings identify a PPARγ-miR-125a-NOD1 signaling axis in endothelial cells that is critical in the regulation of inflammation-mediated angiogenesis. - Highlights: • Expression of NOD1 is regulated by

  8. Systemic immunological tolerance to ocular antigens is mediated by TNF-related apoptosis-inducing ligand (TRAIL)-expressing CD8+ T cells*

    OpenAIRE

    Griffith, Thomas S.; Brincks, Erik L.; Gurung, Prajwal; Kucaba, Tamara A.; Ferguson, Thomas A.

    2010-01-01

    Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required FasL-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by C...

  9. Bayesian Mediation Analysis

    OpenAIRE

    Yuan, Ying; MacKinnon, David P.

    2009-01-01

    This article proposes Bayesian analysis of mediation effects. Compared to conventional frequentist mediation analysis, the Bayesian approach has several advantages. First, it allows researchers to incorporate prior information into the mediation analysis, thus potentially improving the efficiency of estimates. Second, under the Bayesian mediation analysis, inference is straightforward and exact, which makes it appealing for studies with small samples. Third, the Bayesian approach is conceptua...

  10. Ratio-of-Mediator-Probability Weighting for Causal Mediation Analysis in the Presence of Treatment-by-Mediator Interaction

    Science.gov (United States)

    Hong, Guanglei; Deutsch, Jonah; Hill, Heather D.

    2015-01-01

    Conventional methods for mediation analysis generate biased results when the mediator--outcome relationship depends on the treatment condition. This article shows how the ratio-of-mediator-probability weighting (RMPW) method can be used to decompose total effects into natural direct and indirect effects in the presence of treatment-by-mediator…

  11. Technology-Use Mediation

    DEFF Research Database (Denmark)

    Bansler, Jørgen P.; Havn, Erling C.

    2003-01-01

    This study analyzes how a group of ‘mediators’ in a large, multinational company adapted a computer-mediated communication technology (a ‘virtual workspace’) to the organizational context (and vice versa) by modifying features of the technology, providing ongoing support for users, and promoting...... appropriate conventions of use. Our findings corroborate earlier research on technology-use mediation, which suggests that such mediators can exert considerable influence on how a particular technology will be established and used in an organization. However, this study also indicates that the process...... of technology-use mediation is more complex and indeterminate than earlier literature suggests. In particular, we want to draw attention to the fact that advanced computer-mediated communication technologies are equivocal and that technology-use mediation consequently requires ongoing sensemaking (Weick 1995)....

  12. Nitrosoureas Inhibit the Stathmin Mediated Migration and Invasion of Malignant Glioma Cells

    OpenAIRE

    Liang, Xing-Jie; Choi, Yong; Sackett, Dan L.; Park, John K.

    2008-01-01

    Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify...

  13. Micro dynamics in mediation

    OpenAIRE

    Boserup, Hans

    2014-01-01

    The author has identified a number of styles in mediation, which lead to different processes and different outcomes. Through discourse and conversation analysis he examines the micro dynamics in three of these, the postmodern styles: systemic, transformative and narrative mediation. The differences between the three mediation ideologies and practice is illustrated through role play scripts enacted in each style. Mediator and providers of mediation and trainers in mediation are encouraged to a...

  14. Steps in the design, development and formative evaluation of obesity prevention-related behavior change trials

    Directory of Open Access Journals (Sweden)

    Baranowski Janice

    2009-01-01

    Full Text Available Abstract Obesity prevention interventions through dietary and physical activity change have generally not been effective. Limitations on possible program effectiveness are herein identified at every step in the mediating variable model, a generic conceptual framework for understanding how interventions may promote behavior change. To minimize these problems, and thereby enhance likely intervention effectiveness, four sequential types of formative studies are proposed: targeted behavior validation, targeted mediator validation, intervention procedure validation, and pilot feasibility intervention. Implementing these studies would establish the relationships at each step in the mediating variable model, thereby maximizing the likelihood that an intervention would work and its effects would be detected. Building consensus among researchers, funding agencies, and journal editors on distinct intervention development studies should avoid identified limitations and move the field forward.

  15. Bayesian dynamic mediation analysis.

    Science.gov (United States)

    Huang, Jing; Yuan, Ying

    2017-12-01

    Most existing methods for mediation analysis assume that mediation is a stationary, time-invariant process, which overlooks the inherently dynamic nature of many human psychological processes and behavioral activities. In this article, we consider mediation as a dynamic process that continuously changes over time. We propose Bayesian multilevel time-varying coefficient models to describe and estimate such dynamic mediation effects. By taking the nonparametric penalized spline approach, the proposed method is flexible and able to accommodate any shape of the relationship between time and mediation effects. Simulation studies show that the proposed method works well and faithfully reflects the true nature of the mediation process. By modeling mediation effect nonparametrically as a continuous function of time, our method provides a valuable tool to help researchers obtain a more complete understanding of the dynamic nature of the mediation process underlying psychological and behavioral phenomena. We also briefly discuss an alternative approach of using dynamic autoregressive mediation model to estimate the dynamic mediation effect. The computer code is provided to implement the proposed Bayesian dynamic mediation analysis. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  16. Response of humic acid formation to elevated nitrate during chicken manure composting.

    Science.gov (United States)

    Shi, Mingzi; Wei, Zimin; Wang, Liqin; Wu, Junqiu; Zhang, Duoying; Wei, Dan; Tang, Yu; Zhao, Yue

    2018-06-01

    Nitrate can stimulate microbes to degrade aromatic compounds, whereas humic acid (HA) as a high molecular weight aromatic compound, its formation may be affected by elevated nitrate during composting. Therefore, this study is conducted to determine the effect of elevated nitrate on HA formation. Five tests were executed by adding different nitrate concentrations to chicken manure composting. Results demonstrate that the concentration of HA in treatment group is significantly decreased compared with control group (p < 0.05), especially in the highest nitrate concentration group. RDA indicates that the microbes associated with HA and environmental parameters are influenced by elevated nitrate. Furthermore, structural equation model reveals that elevated nitrate reduces HA formation by mediating microbes directly, or by affecting ammonia and pH as the indirect drivers to regulate microbial community structure. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Discovery and characterization of a novel lachrymatory factor synthase in Petiveria alliacea and its influence on alliinase-mediated formation of biologically active organosulfur compounds.

    Science.gov (United States)

    Musah, Rabi A; He, Quan; Kubec, Roman

    2009-11-01

    A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two alpha-subunits (68.8 kD each), one gamma-subunit (22.5 kD), and one delta-subunit (11.9 kD). The two alpha-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

  18. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  19. Causal mediation analysis with multiple mediators in the presence of treatment noncompliance.

    Science.gov (United States)

    Park, Soojin; Kürüm, Esra

    2018-05-20

    Randomized experiments are often complicated because of treatment noncompliance. This challenge prevents researchers from identifying the mediated portion of the intention-to-treated (ITT) effect, which is the effect of the assigned treatment that is attributed to a mediator. One solution suggests identifying the mediated ITT effect on the basis of the average causal mediation effect among compliers when there is a single mediator. However, considering the complex nature of the mediating mechanisms, it is natural to assume that there are multiple variables that mediate through the causal path. Motivated by an empirical analysis of a data set collected in a randomized interventional study, we develop a method to estimate the mediated portion of the ITT effect when both multiple dependent mediators and treatment noncompliance exist. This enables researchers to make an informed decision on how to strengthen the intervention effect by identifying relevant mediators despite treatment noncompliance. We propose a nonparametric estimation procedure and provide a sensitivity analysis for key assumptions. We conduct a Monte Carlo simulation study to assess the finite sample performance of the proposed approach. The proposed method is illustrated by an empirical analysis of JOBS II data, in which a job training intervention was used to prevent mental health deterioration among unemployed individuals. Copyright © 2018 John Wiley & Sons, Ltd.

  20. Mediators and Metaphorical Analysis: A Phenomenological Study of Florida Family Court Mediators

    Science.gov (United States)

    Storrow, Rebecca A.

    2012-01-01

    Florida family court mediation programs have typically been assessed with quantitative analysis. To understand the complexity of the experience of being a family mediator, it was necessary to explore how mediators practiced through qualitative research. Metaphors have been considered to be representations of mediators' mental models regarding…

  1. Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Rafiei, Shahrzad; Komarova, Svetlana V

    2013-01-01

    Advanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood. We studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes. Soluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis. This study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches

  2. Integrating hospital information systems in healthcare institutions: a mediation architecture.

    Science.gov (United States)

    El Azami, Ikram; Cherkaoui Malki, Mohammed Ouçamah; Tahon, Christian

    2012-10-01

    Many studies have examined the integration of information systems into healthcare institutions, leading to several standards in the healthcare domain (CORBAmed: Common Object Request Broker Architecture in Medicine; HL7: Health Level Seven International; DICOM: Digital Imaging and Communications in Medicine; and IHE: Integrating the Healthcare Enterprise). Due to the existence of a wide diversity of heterogeneous systems, three essential factors are necessary to fully integrate a system: data, functions and workflow. However, most of the previous studies have dealt with only one or two of these factors and this makes the system integration unsatisfactory. In this paper, we propose a flexible, scalable architecture for Hospital Information Systems (HIS). Our main purpose is to provide a practical solution to insure HIS interoperability so that healthcare institutions can communicate without being obliged to change their local information systems and without altering the tasks of the healthcare professionals. Our architecture is a mediation architecture with 3 levels: 1) a database level, 2) a middleware level and 3) a user interface level. The mediation is based on two central components: the Mediator and the Adapter. Using the XML format allows us to establish a structured, secured exchange of healthcare data. The notion of medical ontology is introduced to solve semantic conflicts and to unify the language used for the exchange. Our mediation architecture provides an effective, promising model that promotes the integration of hospital information systems that are autonomous, heterogeneous, semantically interoperable and platform-independent.

  3. Direct detection signatures of self-interacting dark matter with a light mediator

    International Nuclear Information System (INIS)

    Nobile, Eugenio Del; Kaplinghat, Manoj; Yu, Hai-Bo

    2015-01-01

    Self-interacting dark matter (SIDM) is a simple and well-motivated scenario that could explain long-standing puzzles in structure formation on small scales. If the required self-interaction arises through a light mediator (with mass ∼ 10 MeV) in the dark sector, this new particle must be unstable to avoid overclosing the universe. The decay of the light mediator could happen due to a weak coupling of the hidden and visible sectors, providing new signatures for direct detection experiments. The SIDM nuclear recoil spectrum is more peaked towards low energies compared to the usual case of contact interactions, because the mediator mass is comparable to the momentum transfer of nuclear recoils. We show that the SIDM signal could be distinguished from that of DM particles with contact interactions by considering the time-average energy spectrum in experiments employing different target materials, or the average and modulated spectra in a single experiment. Using current limits from LUX and SuperCDMS, we also derive strong bounds on the mixing parameter between hidden and visible sector

  4. Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis

    DEFF Research Database (Denmark)

    Ballabeni, Andrea; Melixetian, Marina; Zamponi, Raffaella

    2004-01-01

    -mediated degradation by inhibiting its ubiquitination. In particular, Geminin ensures basal levels of CDT1 during S phase and its accumulation during mitosis. Consistently, inhibition of Geminin synthesis during M phase leads to impairment of pre-RC formation and DNA replication during the following cell cycle....... Moreover, we show that inhibition of CDK1 during mitosis, and not Geminin depletion, is sufficient for premature formation of pre-RCs, indicating that CDK activity is the major mitotic inhibitor of licensing in human cells. Taken together with recent data from our laboratory, our results demonstrate...

  5. Chromosomal instability can be induced by the formation of breakage-prone chromosome rearrangement junctions

    International Nuclear Information System (INIS)

    Allen, R.N.; Ritter, L.; Moore, S.R.; Grosovsky, A.J.

    2003-01-01

    Full text: Studies in our lab have led to the hypothesis that chromosomal rearrangements can generate novel breakage-prone sites, resulting in chromosomal instability acting predominantly in cis. For example, specific breakage of large blocks of centromeric region heterochromatin on chromosome 16q by treatment with 2,6-diaminopurine (DAP) is associated with repeated rearrangement of chromosome 16q during outgrowth of DAP-treated clones, thereby establishing a link between the initial site of damage and the occurrence of persistent chromosomal instability. Similarly, karyotypic analysis of gamma ray induced instability demonstrated that chromosomal rearrangements in sub-clones were significantly clustered near the site of previously identified chromosomal rearrangement junctions in unstable parental clones. This study investigates the hypothesis that integration of transfected sequences into host chromosomes could create breakage-prone junction regions and persistent genomic instability without exposure to DNA-damage agents. These junctions may mimic the unstable chromosomal rearrangements induced by DAP or radiation, and thus provide a test of the broader hypothesis that instability can to some extent be attributed to the formation of novel chromosomal breakage hot spots. These experiments were performed using human-hamster hybrid AL cells containing a single human chromosome 11, which was used to monitor instability in a chromosomal painting assay. AL cells were transfected with a 2.5 Kb fragment containing multiple copies of the 180 bp human alpha heterochromatic repeat, which resulted in chromosomal instability in 41% of the transfected clones. Parallel exposure to gamma-radiation resulted in a similar level of chromosomal instability, although control transfections with plasmid alone did not lead to karyotypic instability. Chromosomal instability induced by integration of alpha heterochromatic repeats was also frequently associated with delayed reproductive

  6. The importance of a hot-sequential mechanism in triplet-state formation by charge recombination in reaction centers of bacterial photosynthesis

    International Nuclear Information System (INIS)

    Saito, K.; Mukai, K.; Sumi, H.

    2006-01-01

    In photosynthesis, pigment-excitation energies in the antenna system produced by light harvesting are transferred among antenna pigments toward the core antenna, where they are captured by the reaction center and initially fixed in the form of a charge separation. Primary charge separation between an oxidized special pair (P + ) and a reduced bacteriopheohytin (H - ) is occasionally intervened by recombination, and a spin-triplet state ( 3 P*) is formed on P in the bacterial reaction center. The 3 P* state is harmful to bio-organisms, inducing the formation of the highly damaging singlet oxygen species. Therefore, understanding the 3 P*-formation mechanism is important. The 3 P* formation is mediated by a state |m> of intermediate charge separation between P and the accessory chlorophyll, which is located between P and H. It will be shown theoretically in the present work that at room temperature, not only the mechanism of superexchange by quantum-mechanical virtual mediation at |m>, but also a hot-sequential mechanism contributes to the mediation. In the latter, although |m> is produced as a real state, the final state 3 P* is quickly formed during thermalization of phonons in the protein matrix in |m>. In the former, the final state is formed more quickly before dephasing-thermalization of phonons in |m>. 3 P* is unistep formed from the charge-separated state in the both mechanisms

  7. Mediation of information and educational mediation: conceptual discussions

    Directory of Open Access Journals (Sweden)

    Helena Célia de Souza Sacerdote

    2016-04-01

    Full Text Available Introduction: This is systematization of theoretical and methodological contributions related to the concepts of mediation information and pedagogical mediation in the literature. Objective: To understand possible intersection of information science and Online Education with regard to these concepts to check that both can be considered as analogous in its essence and practice. Methodology: Literature review based on literature by consulting the scientific productions selected in search of SciELO.ORG databases and EBSCO Host, the portal of CAPES / MEC and Google Scholar. Results: The most cited concepts in information science and education were de Almeida Junior (2009 and Masetto (2013, respectively. Conclusion: It is observed that the concept of mediation can move interchangeably between both areas. This is because the evidence found in the productions of the last five years indicate that the concept of information of mediation seems to have found its bases in education (educational psychology.

  8. Entropy-driven crystal formation on highly strained substrates

    KAUST Repository

    Savage, John R.

    2013-05-20

    In heteroepitaxy, lattice mismatch between the deposited material and the underlying surface strongly affects nucleation and growth processes. The effect of mismatch is well studied in atoms with growth kinetics typically dominated by bond formation with interaction lengths on the order of one lattice spacing. In contrast, less is understood about how mismatch affects crystallization of larger particles, such as globular proteins and nanoparticles, where interparticle interaction energies are often comparable to thermal fluctuations and are short ranged, extending only a fraction of the particle size. Here, using colloidal experiments and simulations, we find particles with short-range attractive interactions form crystals on isotropically strained lattices with spacings significantly larger than the interaction length scale. By measuring the free-energy cost of dimer formation on monolayers of increasing uniaxial strain, we show the underlying mismatched substrate mediates an entropy-driven attractive interaction extending well beyond the interaction length scale. Remarkably, because this interaction arises from thermal fluctuations, lowering temperature causes such substrate-mediated attractive crystals to dissolve. Such counterintuitive results underscore the crucial role of entropy in heteroepitaxy in this technologically important regime. Ultimately, this entropic component of lattice mismatched crystal growth could be used to develop unique methods for heterogeneous nucleation and growth of single crystals for applications ranging from protein crystallization to controlling the assembly of nanoparticles into ordered, functional superstructures. In particular, the construction of substrates with spatially modulated strain profiles would exploit this effect to direct self-assembly, whereby nucleation sites and resulting crystal morphology can be controlled directly through modifications of the substrate.

  9. Olfactory memory formation in Drosophila: from molecular to systems neuroscience.

    Science.gov (United States)

    Davis, Ronald L

    2005-01-01

    The olfactory nervous system of insects and mammals exhibits many similarities, which suggests that the mechanisms for olfactory learning may be shared. Molecular genetic investigations of Drosophila learning have uncovered numerous genes whose gene products are essential for olfactory memory formation. Recent studies of the products of these genes have continued to expand the range of molecular processes known to underlie memory formation. Recent research has also broadened the neuroanatomical areas thought to mediate olfactory learning to include the antennal lobes in addition to a previously accepted and central role for the mushroom bodies. The roles for neurons extrinsic to the mushroom body neurons are becoming better defined. Finally, the genes identified to participate in Drosophila olfactory learning have conserved roles in mammalian organisms, highlighting the value of Drosophila for gene discovery.

  10. Objective and subjective indicators of happiness in Brazil: the mediating role of social class.

    Science.gov (United States)

    Islam, Gazi; Wills-Herrera, Eduardo; Hamilton, Marilyn

    2009-04-01

    The authors tested the proposition that monetary household income affects subjective well-being (E. Deiner, E. M. Suh, R. E. Lucas, & H. L. Smith, 1999) through the mediating mechanisms of objective and subjective social classes. The present authors drew a representative sample in a door-to-door survey format from a Brazilian urban center. Using a back-translated version of E. Diener, R. A. Emmons, R. J. Larson, and S. Griffin's (1985) Satisfaction With Life Scale, the present authors demonstrated a significant relation with income. However, this effect was mediated by objectively and subjectively measured social classes. These effects reinforce, extend, and internationally generalize the Person x Situation perspective elaborated by E. Diener et al. (1999).

  11. CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

    NARCIS (Netherlands)

    Marcos, Veronica; Zhou, Zhe; Yildirim, Ali Onder; Bohla, Alexander; Hector, Andreas; Vitkov, Ljubomir; Wiedenbauer, Eva-Maria; Krautgartner, Wolf Dietrich; Stoiber, Walter; Belohradsky, Bernd H.; Rieber, Nikolaus; Kormann, Michael; Koller, Barbara; Roscher, Adelbert; Roos, Dirk; Griese, Matthias; Eickelberg, Oliver; Döring, Gerd; Mall, Marcus A.; Hartl, Dominik

    2010-01-01

    Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases.

  12. Bayesian Mediation Analysis

    Science.gov (United States)

    Yuan, Ying; MacKinnon, David P.

    2009-01-01

    In this article, we propose Bayesian analysis of mediation effects. Compared with conventional frequentist mediation analysis, the Bayesian approach has several advantages. First, it allows researchers to incorporate prior information into the mediation analysis, thus potentially improving the efficiency of estimates. Second, under the Bayesian…

  13. DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini).

    Science.gov (United States)

    Rocha, Marla Piumbini; Pompolo, Silvia Das Graças; Dergam, Jorge Abdala; Fernandes, Anderson; Campos, Lucio Antonio De Oliveira

    2002-01-01

    We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.

  14. Selectins mediate small cell lung cancer systemic metastasis.

    Directory of Open Access Journals (Sweden)

    Franziska Heidemann

    Full Text Available Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181. However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

  15. Stearoyl-CoA Desaturase-1 Protects Cells against Lipotoxicity-Mediated Apoptosis in Proximal Tubular Cells

    Directory of Open Access Journals (Sweden)

    Tamaki Iwai

    2016-11-01

    Full Text Available Saturated fatty acid (SFA-related lipotoxicity is a pathogenesis of diabetes-related renal proximal tubular epithelial cell (PTEC damage, closely associated with a progressive decline in renal function. This study was designed to identify a free fatty acid (FFA metabolism-related enzyme that can protect PTECs from SFA-related lipotoxicity. Among several enzymes involved in FFA metabolism, we identified stearoyl-CoA desaturase-1 (SCD1, whose expression level significantly decreased in the kidneys of high-fat diet (HFD-induced diabetic mice, compared with non-diabetic mice. SCD1 is an enzyme that desaturates SFAs, converting them to monounsaturated fatty acids (MUFAs, leading to the formation of neutral lipid droplets. In culture, retrovirus-mediated overexpression of SCD1 or MUFA treatment significantly ameliorated SFA-induced apoptosis in PTECs by enhancing intracellular lipid droplet formation. In contrast, siRNA against SCD1 exacerbated the apoptosis. Both overexpression of SCD1 and MUFA treatment reduced SFA-induced apoptosis via reducing endoplasmic reticulum stress in cultured PTECs. Thus, HFD-induced decrease in renal SCD1 expression may play a pathogenic role in lipotoxicity-induced renal injury, and enhancing SCD1-mediated desaturation of SFA and subsequent formation of neutral lipid droplets may become a promising therapeutic target to reduce SFA-induced lipotoxicity. The present study provides a novel insight into lipotoxicity in the pathogenesis of diabetic nephropathy.

  16. Sheared flow layer formation in tokamak plasmas with reversed magnetic shear

    International Nuclear Information System (INIS)

    Dong, J.Q.; Long, Y.X.; Mou, Z.Z.; Zhang, J.H.; Li, J.Q.

    2005-01-01

    Sheared flow layer (SFL) formation due to magnetic energy release through tearing-reconnections in tokamak plasmas is investigated. The characteristics of the SFLs created in the development of double tearing mode, mediated by electron viscosity in configurations with non-monotonic safety factor q profiles and, therefore, two rational flux surfaces of same q value, are analyzed in detail as an example. Quasi-linear simulations demonstrate that the sheared flows induced by the mode have desirable characteristics (lying at the boundaries of the magnetic islands), and sufficient levels required for internal transport barrier (ITB) formation. A possible correlation of the SFLs with experimental observations, that double transport barrier structures are preferentially formed in proximity of the two rational surfaces, is also proffered. (author)

  17. Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

    DEFF Research Database (Denmark)

    Danovi, Davide; Meulmeester, Erik; Pasini, Diego

    2004-01-01

    has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRas(V12...

  18. Variación heterocromática en Proechimys semispinosus (Rodentia: Echimyidae de la región pacífica colombiana

    Directory of Open Access Journals (Sweden)

    Bueno Martha Lucía

    1993-12-01

    Full Text Available The presence oftwo chromosomal races is reported in a comparative study of populatlons of Proechimys semlspinosus from the Pacific lowlands of Colombia. These races are differenciated by the constitutive heterochromatin found using the C-band technlque. The population from Nariño locality lacks centromeric heterochromatin and has large blocks of telomeric heterochromatin in several chromosomes while the population from Chocó loca lity has small blocks of centromeric heterochromatin in the majority of the chromosomes and small and medium size blocks of telomeric heterochromatin only in three pairs of chromosomes. Thesimilarity between the heterochromatin pattem in the Gorgona island (Gómez-Laverde et al" 1990 and Nariño populations suggests a very close relation ship and supports the affinities ofthe island's biota with the southwestem region of Colombia and northem region of Ecuador previously reported in others biological groups.Mediante un estudio comparativo en poblaciones de Proechimys semispinosus del pacífico colombiano se registra la presencia de dos razas cromosómicas; estas se diferencian por el patrón de heterocromatina constitutiva detectable mediante las técnicas de bandas C. La población de la localidad de Nariño presenta ausencia casi total de heterocromatina centromérica y presencia de bloques grandes de heterocromatina telomérica en varios cromosomas, mientras que la población de la localidad del Chocó presenta bloques muy pequeños de heterocromatina centromérica en la mayoría de los cromosomas y bloques pequeños y medianos teloméricos solo en tres pares cromosómlcos. La similitud del patrón de heterocromatina, entre las poblaciones de la isla Gorgona (Gómez-Laverde et al" 1990 y de Nariño, sugiere una estrecha relación entre ellas; este hecho se suma a las evidencias sobre la afinidad de la biota de la isla con la región su roccidental de Colombia y del norte del Ecuador, reportada en otros grupos biológicos.

  19. The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

    Science.gov (United States)

    Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

    2014-01-01

    Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

  20. Mediation Works: An Action Research Study Evaluating the Peer Mediation Program from the Eyes of Mediators and Faculty

    Science.gov (United States)

    Cook, Jacqueline Yvonne; Boes, Susan R.

    2013-01-01

    A literature review was conducted to understand how mediators and faculty view a Peer Mediation Program (PMP). The review identified four subgroups: mediators, teachers, administrators, and school counselors as well as their views on the success or lack of success of PMPs. The research also reflects how to best engage stakeholders in the mediation…

  1. Functional analysis of Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Hein, Estrid; Honoré, Christian Le Fèvre; Skjoedt, Mikkel-Ole

    2010-01-01

    assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition...... was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides...

  2. The virulence regulator PrfA promotes biofilm formation by Listeria monocytogenes.

    Science.gov (United States)

    Lemon, Katherine P; Freitag, Nancy E; Kolter, Roberto

    2010-08-01

    Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.

  3. How the adoption of impression management goals alters impression formation.

    Science.gov (United States)

    Gibson, Bryan; Poposki, Elizabeth M

    2010-11-01

    Five experiments (N = 390) tested the hypothesis that adopting an impression management goal leads the impression manager to view an interaction partner as having less of the trait he or she is attempting to express. This hypothesis was confirmed for the impression management goals of appearing introverted, extraverted, smart, confident, and happy. Experiment 2 shows that adoption of the impression goal could alter judgments even when participants could not act on the goal. Experiment 3 provides evidence that adopting an impression management goal prompted a comparison mind-set and that this comparison mind-set activation mediated target judgments. Experiment 4 rules out a potential alternative explanation and provides more direct evidence that comparison of the impression manager's self-concept mediates the impression of the target. Experiment 5 eliminates a potential confound and extends the effect to another impression goal. These experiments highlight the dynamic interplay between impression management and impression formation.

  4. Osteoclast TGF-β Receptor Signaling Induces Wnt1 Secretion and Couples Bone Resorption to Bone Formation

    Science.gov (United States)

    Weivoda, Megan M; Ruan, Ming; Pederson, Larry; Hachfeld, Christine; Davey, Rachel A; Zajac, Jeffrey D; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Osteoblast-mediated bone formation is coupled to osteoclast-mediated bone resorption. These processes become uncoupled with age, leading to increased risk for debilitating fractures. Therefore, understanding how osteoblasts are recruited to sites of resorption is vital to treating age-related bone loss. Osteoclasts release and activate TGF-β from the bone matrix. Here we show that osteoclastspecific inhibition of TGF-β receptor signaling in mice results in osteopenia due to reduced osteoblast numbers with no significant impact on osteoclast numbers or activity. TGF-β induced osteoclast expression of Wnt1, a protein crucial to normal bone formation, and this response was blocked by impaired TGF-β receptor signaling. Osteoclasts in aged murine bones had lower TGF-β signaling and Wnt1 expression in vivo. Ex vivo stimulation of osteoclasts derived from young or old mouse bone marrow macrophages showed no difference in TGF-β–induced Wnt1 expression. However, young osteoclasts expressed reduced Wnt1 when cultured on aged mouse bone chips compared to young mouse bone chips, consistent with decreased skeletal TGF-β availability with age. Therefore, osteoclast responses to TGF-β are essential for coupling bone resorption to bone formation, and modulating this pathway may provide opportunities to treat age-related bone loss. PMID:26108893

  5. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    Energy Technology Data Exchange (ETDEWEB)

    Jan, Yi-Hua [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Baker, Angela A. [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

  6. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    International Nuclear Information System (INIS)

    Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

  7. Calcium signaling mediates five types of cell morphological changes to form neural rosettes.

    Science.gov (United States)

    Hříbková, Hana; Grabiec, Marta; Klemová, Dobromila; Slaninová, Iva; Sun, Yuh-Man

    2018-02-12

    Neural rosette formation is a critical morphogenetic process during neural development, whereby neural stem cells are enclosed in rosette niches to equipoise proliferation and differentiation. How neural rosettes form and provide a regulatory micro-environment remains to be elucidated. We employed the human embryonic stem cell-based neural rosette system to investigate the structural development and function of neural rosettes. Our study shows that neural rosette formation consists of five types of morphological change: intercalation, constriction, polarization, elongation and lumen formation. Ca 2+ signaling plays a pivotal role in the five steps by regulating the actions of the cytoskeletal complexes, actin, myosin II and tubulin during intercalation, constriction and elongation. These, in turn, control the polarizing elements, ZO-1, PARD3 and β-catenin during polarization and lumen production for neural rosette formation. We further demonstrate that the dismantlement of neural rosettes, mediated by the destruction of cytoskeletal elements, promotes neurogenesis and astrogenesis prematurely, indicating that an intact rosette structure is essential for orderly neural development. © 2018. Published by The Company of Biologists Ltd.

  8. Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2017-07-12

    The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

  9. Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration.

    Science.gov (United States)

    Kempuraj, Duraisamy; Thangavel, Ramasamy; Selvakumar, Govindhasamy P; Zaheer, Smita; Ahmed, Mohammad E; Raikwar, Sudhanshu P; Zahoor, Haris; Saeed, Daniyal; Natteru, Prashant A; Iyer, Shankar; Zaheer, Asgar

    2017-01-01

    Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1-42 (Aβ1-42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can

  10. 45 CFR 16.18 - Mediation.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Mediation. 16.18 Section 16.18 Public Welfare... BOARD § 16.18 Mediation. (a) In cases pending before the Board. If the Board decides that mediation... mediation techniques and will provide or assist in selecting a mediator. The mediator may take any steps...

  11. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    Science.gov (United States)

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  12. Rapid one-pot propargylamine synthesis by plasmon mediated catalysis with gold nanoparticles on ZnO under ambient conditions.

    Science.gov (United States)

    González-Béjar, María; Peters, Kate; Hallett-Tapley, Geniece L; Grenier, Michel; Scaiano, Juan C

    2013-02-28

    Surface plasmon excitation of gold nanoparticles on ZnO in the presence of an aldehyde, an amine and phenylacetylene led to rapid and selective formation of propargylamines with good yields (50-95%) at room temperature. Plasmon mediated catalysis is the best available route for this ternary coupling.

  13. Seed Mediated Growth of Gold Nanoparticles Based on Liquid Arc Discharge

    International Nuclear Information System (INIS)

    Ashkarran, Ali Akbar

    2013-01-01

    We report studies on the growth of gold nanoparticles by a seed-mediated approach in solution. The synthetic method is adapted from one we published earlier (Ashkarran et al. Appl. Phys. A 2009, 96, 423). The synthesized gold nanoparticles were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), UV-Vis spectroscopy, optical imaging and atomic force microscopy (AFM). Optical absorption spectroscopy of the prepared samples at 15 A arc current in HAuCl 4 solution shows a surface plasmon resonance around 520 nm. It is found that sodium citrate acts as a stabilizer and surface capping agent of the colloidal nanoparticles. The intensity of the plasmonic peak of the prepared gold nanoparticles for 1 minute arc duration gradually increases due to seed mediation for up to 6 hours. The formation time of gold nanoparticles at higher seed concentrations is less than that at lower seed concentrations. (plasma technology)

  14. Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment

    DEFF Research Database (Denmark)

    Qu, Xiaoling; Afelik, Solomon; Jensen, Jan Nygaard

    2013-01-01

    of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated...... involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation...

  15. Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation.

    Science.gov (United States)

    Janissen, Richard; Murillo, Duber M; Niza, Barbara; Sahoo, Prasana K; Nobrega, Marcelo M; Cesar, Carlos L; Temperini, Marcia L A; Carvalho, Hernandes F; de Souza, Alessandra A; Cotta, Monica A

    2015-04-20

    Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

  16. Specific role of taurine in the 8-brominated-2'-deoxyguanosine formation.

    Science.gov (United States)

    Asahi, Takashi; Nakamura, Yoshimasa; Kato, Yoji; Osawa, Toshihiko

    2015-11-15

    At the sites of inflammation, hypohalous acids, such as hypochlorous acid and hypobromous acid (HOBr), are produced by myeloperoxidase. These hypohalous acids rapidly react with the primary amino groups to produce haloamines, which are relatively stable and can diffuse long distances and cross the plasma membrane. In this study, we examined the effects of taurine, the most abundant free amino acid in the leukocyte cytosol, on the hypohalous acid-dependent formation of 8-chloro-2'-deoxyguanosine (8-CldG) and 8-bromo-2'-deoxyguanosine (8-BrdG). The reaction of taurine with HOBr yielded taurine bromamine, which is the most stable among other bromamines of amino acids. Taurine also enhanced the bromination of only dG among the four 2'-deoxynucleosides, whereas it inhibited the 8-CldG formation. The specificity of taurine for the enhanced formation of halogenated dG is completely different from that of nicotine, an enhancer of chlorination. The amount of dibrominated taurine (taurine dibromamine) closely correlated with the formation of 8-BrdG, suggesting that taurine dibromamine might be a plausible mediator for the dG bromination in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Cell type-specific modulation of lipid mediator's formation in murine adipose tissue by omega-3 fatty acids

    Czech Academy of Sciences Publication Activity Database

    Kuda, Ondřej; Rombaldová, Martina; Janovská, Petra; Flachs, Pavel; Kopecký, Jan

    2016-01-01

    Roč. 469, č. 3 (2016), s. 731-736 ISSN 0006-291X R&D Projects: GA ČR(CZ) GP13-04449P; GA ČR(CZ) GA13-00871S; GA MŠk(CZ) LH14040 Institutional support: RVO:67985823 Keywords : adipose tissue macrophages * omega-3 PUFA * protectin D1 * lipid mediators * lipidomics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.466, year: 2016

  18. Mediation by Demonstration and Dialogue. An Evaluation of Practices. Deliverable D12

    International Nuclear Information System (INIS)

    Elam, Mark; Sundqvist, Goeran; Soneryd, Linda

    2009-10-01

    Two basic forms of mediation have been identified and analysed: the public mediation of radioactive waste management by demonstration and by dialogue. The former coincides firstly with the showing and visualizing radioactive waste management issues and solutions for public inspection and recognition. The latter, on the other hand, corresponds firstly with the establishment of different styles of public discussion, deliberation and inquiry for elaborating further on policy issues and solutions. Both forms of mediation provide the basis for the collection and collation of significant bodies of public evidence and testimony which can be used to continuously inform and guide decision-making processes. Principles and Guidelines of Mediation 1. Both mediation by demonstration and mediation by dialogue should be understood as indispensable in the formation of arenas of risk governance in radioactive waste management. 2. As the two basic forms of mediation each has its own part to play in advancing radioactive waste management solutions, neither one should be automatically privileged over the other in any policy process 3. Both mediation by demonstration and mediation by dialogue can be expected to generate large bodies of public evidence and testimony which can be used to help inform and guide decision-making processes. Historically, evidence deriving from mediation by demonstration has been accorded greater prominence in the radioactive waste management field than evidence deriving from mediation by dialogue. For this reason, new ways of effectively combining evidence and testimony deriving from both forms of mediation should be explored in policy processes in future. 4. Because mediation by demonstration builds upon a clear division between those who demonstrate and those who are being asked to see and evaluate what is being shown, mediation by dialogue should be conceived and constructed as an opportunity to unsettle and destabilize these established roles. 5. Because

  19. Formation of Gd coordination polymer with 1D chains mediated by Bronsted acidic ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Qianqian; Han, Ying [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai (China); Lin, Hechun, E-mail: hclin@ee.ecnu.edu.cn [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai (China); Zhang, Yuanyuan; Duan, Chungang [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai (China); Peng, Hui, E-mail: hpeng@ee.ecnu.edu.cn [Key Laboratory of Polar Materials and Devices, Ministry of Education, East China Normal University, Shanghai (China); Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan, Shanxi 030006 (China)

    2017-03-15

    One dimensional coordination polymer Gd[(SO{sub 4})(NO{sub 3})(C{sub 2}H{sub 6}SO){sub 2}] (1) is prepared through the mediation of Bronsted acid ionic liquid, which crystallized in the monoclinic space of C2/c. In this polymer, adjacent Gd atoms are linked by two SO{sub 4}{sup 2-} ions to generate a 1-D chain, and all oxygen atoms in SO{sub 4}{sup 2-} groups are connected to three nearest Gd atoms in µ{sup 3}:η{sup 1}:η{sup 1}:η{sup 2} fashion. Gd, S and N from SO{sub 4}{sup 2-} and NO{sub 3}{sup -} are precisely coplanar. The planar is coordinated by a pair of DMSO molecules, which is parallel and linked by hydrogen bonding to form a three-dimensional supramolecular network. Magnetic susceptibility measurement of 1 reveals weak antiferromagnetic interactions between the Gd (III) ions. It exhibits relatively large magneto-caloric effect with –ΔS{sub m}=28.8 J Kg{sup −1} K{sup −1} for ΔH=7 T. - Graphical abstract: Coordination polymer Gd[(SO{sub 4})(NO{sub 3})(C{sub 2}H{sub 6}SO){sub 2}] was obtained mediated by Bronsted acid Ionic Liquid, which presents a 1-D chains collected by SO{sub 4}{sup 2-} groups. Magnetic susceptibility of the polymer reveals weak antiferromagnetic interactions between the Gd(III) ions with the relatively large magneto-caloric effect of –ΔS{sub m}=28.8 J Kg{sup −1} K{sup −1} for ΔH= 7T.

  20. Bioactive Phytochemicals: Bioactivity, Sources, Preparations, and/or Modifications via Silver Tetrafluoroborate Mediation

    Directory of Open Access Journals (Sweden)

    Matthew C. Achilonu

    2015-01-01

    Full Text Available This review provides an overview of the biological activities, natural occurrences, and the silver tetrafluoroborate- (AgBF4- mediated synthesis of proanthocyanidins, glycosides, N-heterocyclic alkaloid analogues (of pyrrole, morphine, quinoline, isoquinoline, and indole, furan analogues, and halocompounds. AgBF4 has been reviewed as an effective reaction promoter, used extensively in the synthesis of relevant biologically active compounds via carbon-carbon and carbon-heteroatom bonds formation. The literatures from 1979 to April 2014 were reviewed.