Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

KAUST Repository

Jiang, Hanlun

2016-12-06

MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches.

Science.gov (United States)

Jiang, Hanlun; Zhu, Lizhe; Héliou, Amélie; Gao, Xin; Bernauer, Julie; Huang, Xuhui

2017-01-01

MicroRNA (miRNA) and Argonaute (AGO) protein together form the RNA-induced silencing complex (RISC) that plays an essential role in the regulation of gene expression. Elucidating the underlying mechanism of AGO-miRNA recognition is thus of great importance not only for the in-depth understanding of miRNA function but also for inspiring new drugs targeting miRNAs. In this chapter we introduce a combined computational approach of molecular dynamics (MD) simulations, Markov state models (MSMs), and protein-RNA docking to investigate AGO-miRNA recognition. Constructed from MD simulations, MSMs can elucidate the conformational dynamics of AGO at biologically relevant timescales. Protein-RNA docking can then efficiently identify the AGO conformations that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms of molecular recognition between large, flexible, and complex biomolecules.

Molecular composition of IMP1 ribonucleoprotein granules

DEFF Research Database (Denmark)

Jønson, Lars; Vikesaa, Jonas; Krogh, Anders

2007-01-01

Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in mo......Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization...

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus

International Nuclear Information System (INIS)

Reimer, G.; Raska, I.; Scheer, U.; Tan, E.M.

1988-01-01

Certain autoimmune sera contain antibodies against a nucleolar ribonucleotprotein particle associated with 7-2-RNA. In this study, the authors showed by immunofluorescence microscopy that antibodies reactive with 7-2-ribonucleoprotein immunolocalized in the granular regions of actinomycin D and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)--segregated nucleoli from Vero cells. By electron microscopic immunocytochemistry, antigen-antibody complexes were located in the granular component of transcriptionally active nucleoli from rat liver hepatocytes and HeLa cells. Anti-7-2-RNP antibodies from two autoimmune sera immunoprecipitated a major protein of M r 40,000 from [ 35 S] methionine-labeled HeLa cell extract. The immunolocalization data suggest that 7-2-ribonucleoprotein may be involved in stages of ribosome biogenesis which take place in the granular component of the nucleolus, i.e., assembly, maturation, and/or transport of preribosomes

Dicer and Argonaute Genes Involved in RNA Interference in the Entomopathogenic Fungus Metarhizium robertsii.

Science.gov (United States)

Meng, Huimin; Wang, Zhangxun; Wang, Yulong; Zhu, Hong; Huang, Bo

2017-04-01

RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer ( M. robertsii dcl1 [ Mrdcl1 ] and Mrdcl2 ) and Argonaute ( Mrago1 and Mrago2 ) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δ dcl2 and Δ ago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δ dcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δ dcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δ dcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsii IMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role

Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins

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Katherine S. Bridge

2017-07-01

Full Text Available As core components of the microRNA-induced silencing complex (miRISC, Argonaute (AGO proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390 on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1.

Science.gov (United States)

Csorba, Tibor; Lózsa, Rita; Hutvágner, György; Burgyán, József

2010-05-01

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double-stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein-protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA-induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre-programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high-molecular-weight complex, suggesting the existence of a multi-protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA-RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co-expressed AGO1 in both the presence and absence of P0.

Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

Science.gov (United States)

Joseph, Thomas T; Osman, Roman

2012-01-01

In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA) is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC) that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the cumulative effects of

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates.

Science.gov (United States)

Flores-Jasso, C Fabián; Salomon, William E; Zamore, Phillip D

2013-02-01

Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies

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Aniello Russo

2010-02-01

Full Text Available RNA interference (RNAi is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2 protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA. The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and/or B metal ion binding, doesn’t affect the enzymatic activity, suggesting that this residue doesn’t belong to the active site.

Convergent transmission of RNAi guide-target mismatch information across Argonaute internal allosteric network.

Directory of Open Access Journals (Sweden)

Thomas T Joseph

Full Text Available In RNA interference, a guide strand derived from a short dsRNA such as a microRNA (miRNA is loaded into Argonaute, the central protein in the RNA Induced Silencing Complex (RISC that silences messenger RNAs on a sequence-specific basis. The positions of any mismatched base pairs in an miRNA determine which Argonaute subtype is used. Subsequently, the Argonaute-guide complex binds and silences complementary target mRNAs; certain Argonautes cleave the target. Mismatches between guide strand and the target mRNA decrease cleavage efficiency. Thus, loading and silencing both require that signals about the presence of a mismatched base pair are communicated from the mismatch site to effector sites. These effector sites include the active site, to prevent target cleavage; the binding groove, to modify nucleic acid binding affinity; and surface allosteric sites, to control recruitment of additional proteins to form the RISC. To examine how such signals may be propagated, we analyzed the network of internal allosteric pathways in Argonaute exhibited through correlations of residue-residue interactions. The emerging network can be described as a set of pathways emanating from the core of the protein near the active site, distributed into the bulk of the protein, and converging upon a distributed cluster of surface residues. Nucleotides in the guide strand "seed region" have a stronger relationship with the protein than other nucleotides, concordant with their importance in sequence selectivity. Finally, any of several seed region guide-target mismatches cause certain Argonaute residues to have modified correlations with the rest of the protein. This arises from the aggregation of relatively small interaction correlation changes distributed across a large subset of residues. These residues are in effector sites: the active site, binding groove, and surface, implying that direct functional consequences of guide-target mismatches are mediated through the

Radioimmunoassay for antibodies to rubella virus and its ribonucleoprotein component

International Nuclear Information System (INIS)

Ho-Terry, L.; Cohen, A.

1979-01-01

Using a radioimmune precipitation technique, the antibody response to intact rubella virus and its ribonucleoprotein component was measured. The method was very sensitive and reproducible, and did not require preliminary serum fractionation for the identification of antibodies of different immunoglobulin classes. The results showed that the IgA and IgG antibodies against the intact virus persisted in the sera of patients long after the initial infection. In contrast, IgA and IgG antibodies against the ribonucleoprotein component of rubella virus were detected only in sera of patients after recent rubella infection. This observation suggested that a test for antibodies to the ribonucleoprotein component may provide additional evidence in the diagnosis of recent rubella infection. This could be potentially a useful test particularly in the management of pregnant patients. (U.K.)

Argonaute: The executor of small RNA function.

Science.gov (United States)

Azlan, Azali; Dzaki, Najat; Azzam, Ghows

2016-08-20

The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Torsin Mediates Primary Envelopment of Large Ribonucleoprotein Granules at the Nuclear Envelope

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Vahbiz Jokhi

2013-04-01

Full Text Available A previously unrecognized mechanism through which large ribonucleoprotein (megaRNP granules exit the nucleus is by budding through the nuclear envelope (NE. This mechanism is akin to the nuclear egress of herpes-type viruses and is essential for proper synapse development. However, the molecular machinery required to remodel the NE during this process is unknown. Here, we identify Torsin, an AAA-ATPase that in humans is linked to dystonia, as a major mediator of primary megaRNP envelopment during NE budding. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the messenger RNAs contained within fail to reach synaptic sites, preventing normal synaptic protein synthesis and thus proper synaptic bouton development. These studies begin to establish the cellular machinery underlying the exit of megaRNPs via budding, offer an explanation for the “nuclear blebbing” phenotype found in dystonia models, and provide an important link between Torsin and the synaptic phenotypes observed in dystonia.

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

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Wibke J Meyer

2006-08-01

Full Text Available Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P

Science.gov (United States)

Jarrous, Nayef; Wolenski, Joseph S.; Wesolowski, Donna; Lee, Christopher; Altman, Sidney

1999-01-01

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. PMID:10444065

The Argonauts in Slovenian Literature for Children and Youth

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Milena Mileva Blažić

2015-12-01

Full Text Available Ljubljana has been associated with literature since its beginning. The founda- tion of the city Emona, for example, which – according to myth – dates back to the 13th  century BC, is associated with the myth of the Argonauts, Jason and the Golden Fleece. In the process of literary reception, the ancient myth evolved into the fairy tale type ATU 513A, according to H.-J. Uther’s motif index of fairy tales. The motif of the Golden Fleece became a symbol of mythic search beyond time and space in Slovenian literature, including children’s literature: in the picture books by Dane Zajc and Miroslav Šuput: The Argonauts (Argonavti, Anton Komat: The Lake Fairy (Vila Jezerka, Desa Muck: The Giant Hen (Kokoš velikanka, Andreja Peklar: The Boy with the Red Cap (Fant z rdečo kapico. It is used with different meanings and in combination with various mythic, fabulous and realistic animal symbols.

Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress

International Nuclear Information System (INIS)

Hayakawa, Hiroshi; Fujikane, Aya; Ito, Riyoko; Matsumoto, Masaki; Nakayama, Keiichi I.; Sekiguchi, Mutsuo

2010-01-01

Research highlights: → We performed comprehensive survey for proteins that bind to oxidized RNA. → HNRNPD and HNRNPC proteins were identified as oxidized RNA binding proteins. → Knockdown of HNRNPD/C expression caused increased sensitivity to H 2 O 2 . → Amounts of HNRNPD protein rapidly decreased when cells were exposed to H 2 O 2 . -- Abstract: Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression.

Argonaute-2-null embryonic stem cells are retarded in self-renewal ...

Indian Academy of Sciences (India)

Present address: Institute of Stem Cells and Regenerative Medicine, Bangalore, India ... [Chandra Shekar P, Naim A, Partha Sarathi D and Kumar S 2011 Argonaute-2-null embryonic stem cells are retarded in self-renewal ..... Research, India.

Effects of Argonaute on Gene Expression in Thermus thermophilus.

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Daan C Swarts

Full Text Available Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo.To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago, and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels.In the absence of exogenous DNA (plasmid, TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.

The ribonucleoprotein Csr network.

Science.gov (United States)

Seyll, Ethel; Van Melderen, Laurence

2013-11-08

Ribonucleoprotein complexes are essential regulatory components in bacteria. In this review, we focus on the carbon storage regulator (Csr) network, which is well conserved in the bacterial world. This regulatory network is composed of the CsrA master regulator, its targets and regulators. CsrA binds to mRNA targets and regulates translation either negatively or positively. Binding to small non-coding RNAs controls activity of this protein. Expression of these regulators is tightly regulated at the level of transcription and stability by various global regulators (RNAses, two-component systems, alarmone). We discuss the implications of these complex regulations in bacterial adaptation.

The Ribonucleoprotein Csr Network

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Ethel Seyll

2013-11-01

Full Text Available Ribonucleoprotein complexes are essential regulatory components in bacteria. In this review, we focus on the carbon storage regulator (Csr network, which is well conserved in the bacterial world. This regulatory network is composed of the CsrA master regulator, its targets and regulators. CsrA binds to mRNA targets and regulates translation either negatively or positively. Binding to small non-coding RNAs controls activity of this protein. Expression of these regulators is tightly regulated at the level of transcription and stability by various global regulators (RNAses, two-component systems, alarmone. We discuss the implications of these complex regulations in bacterial adaptation.

The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment

Science.gov (United States)

2017-10-01

for prostate cancer , the work may impact development of diagnostic /prognostic products based on pThoc1. The presence of the THO ribonucleoprotin...AWARD NUMBER: W81XWH-14-1-0475 TITLE: The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate Cancer Treatment Assignment PRINCIPAL...15Sept 2016 - 14Sep2017 4. TITLE AND SUBTITLE The Thoc1 Ribonucleoprotein as a Novel Biomarker for Prostate 5a. CONTRACT NUMBER Cancer Treatment

Analysis of credible accidents for Argonaut reactors. Report for October 1980-April 1981

International Nuclear Information System (INIS)

Hawley, S.C.; Kathren, R.L.; Robkin, M.A.

1981-04-01

Five areas of potential accidents have been evaluated for the Argonaut-UTR reactors. They are: insertion of excess reactivity, catastrophic rearrangement of the core, explosive chemical reaction, graphite fire, and a fuel-handling accident

Time and myth: the Argonauts in Ljubljana

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Ana María Sánchez Tarrío

2012-12-01

Full Text Available This papers gives a breif overview of research in its initial phases, which examines, from the perspective of the construction of national identity, the traditional theme of the enduring and timeless nature of Classical myths and in particular the ancient myth, which is the subject of Borges’s sharp irony in his short-story “The immortal”. The reception of the Argonauts myth in Slovenian culture, initiated by the work of Janez Vajkard or Johann Weichard Freiherr von Valvasor (1641–1693, offers a significant case-study, which also highlights the relevance of Humanist and Baroque culture in the critical history of European nationalism. Writing before the emergence in Europe of the Hegelian conception of “Volkgeist”, the polymath writer applied a humanistic approach to patriotic themes, revealing Slovene culture, hybrid from its origins, to the wider world. A characteristic feature of his approach was the fusion of earlier textual tradition with contemporary oral material. Both in Slovenia and in the rest of Europe the comparison of the nineteenth century treatment of material dealing with national identity with its earlier treatment and transmission by humanist writers highlights the importance of the 16th and 17th centuries in the configuration of the different national faces of Europe as well as the significant role of common Greek and Latin roots. As a result, the myth of the Argonauts in Ljubljana, against the backdrop of idealistic or essentialist nationalist faiths, has the not inconsiderable virtue of underscoring the contaminatio that is characteristic of the construction of national identity.

A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii.

Directory of Open Access Journals (Sweden)

Laurence Braun

2010-05-01

Full Text Available In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA, which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and

Protection of germline gene expression by the C. elegans Argonaute CSR-1.

Science.gov (United States)

Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

2013-12-23

In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog

Czech Academy of Sciences Publication Activity Database

Ma, W.; Gabriel, T.S.; Martis, M.M.; Gursinsky, T.; Schubert, V.; Vrána, Jan; Doležel, Jaroslav; Grundlach, H.; Altschmied, L.; Scholz, U.; Himmelbach, A.; Behrens, S.E.; Banaei-Moghaddam, A.M.; Houben, A.

2017-01-01

Roč. 213, č. 2 (2017), s. 916-928 ISSN 0028-646X R&D Projects: GA MŠk(CZ) LO1204; GA ČR GBP501/12/G090 Institutional support: RVO:61389030 Keywords : rna-polymerase-ii * ribosomal-rna * accessory chromosomes * plant argonautes * gene-expression * evolution * transcription * pseudogenes * sequences * identification * Argonaute * B chromosomes * B-located genes * gene erosion. gene expression * pseudogenization * Secale cereale Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 7.330, year: 2016

A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

KAUST Repository

Jin, Hailing; Zhu, Jian-Kang

2010-01-01

RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host's essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

Making RISC.

Science.gov (United States)

Kawamata, Tomoko; Tomari, Yukihide

2010-07-01

It is well established that 20- to 30-nt small RNAs, including small interfering RNAs, microRNAs and Piwi-interacting RNAs, play crucial roles in regulating gene expression and control a surprisingly diverse array of biological processes. These small RNAs cannot work alone: they must form effector ribonucleoprotein complexes - RNA-induced silencing complexes (RISCs) - to exert their function. Thus, RISC assembly is a key process in small RNA-mediated silencing. Recent biochemical analyses of RISC assembly, together with new structural studies of Argonaute, the core protein component of RISC, suggest a revised view of how mature RISC, which contains single-stranded guide RNA, is built from small RNAs that are born double-stranded. Copyright 2010 Elsevier Ltd. All rights reserved.

The Siemens-Argonaut reactor as a driver zone for a high-temperature reactor cell. Der Siemens-Argonaut-Reaktor als Treiberzone fuer eine Hochtemperaturreaktorzelle

Energy Technology Data Exchange (ETDEWEB)

Mueller, H; Schuerrer, F; Ninaus, W; Oswald, K; Rabitsch, H; Kreiner, H [Technische Univ., Graz (Austria). Inst. fuer Theoretische Physik und Reaktorphysik; Neef, R D [Kernforschungsanlage Juelich G.m.b.H. (Germany, F.R.). Inst. fuer Reaktorentwicklung

1984-12-15

To enable a validation of neutron physics calculation methods for pebble bed reactors the inner reflector of an Argonaut research reactor was substituted by a full of about 1200 fuel elements of the AVR-Juelich type. The report describes the measuring instruments and the reactor physical layout of the arrangement by the code packages GAMTEREX, ZUT-D.G.L. and MUPO. Comparison of calculated reaction rates with measurements show good agreement. Application of the codes to high-temperature reactors in abnormal states is envisaged. (Author, translated by G.Q.)

Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements

Directory of Open Access Journals (Sweden)

van der Oost John

2009-08-01

Full Text Available Abstract Background In eukaryotes, RNA interference (RNAi is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding and PIWI (active or inactivated nuclease domains, the prokaryotic Argonaute homologs (pAgos fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative nucleases. Given these observations, the apparent extensive

A viral suppressor protein inhibits host RNA silencing by hooking up with Argonautes

KAUST Repository

Jin, Hailing

2010-05-01

RNA viruses are particularly vulnerable to RNAi-based defenses in the host, and thus have evolved specific proteins, known as viral suppressors of RNA silencing (VSRs), as a counterdefense. In this issue of Genes & Development, Azevedo and colleagues (pp. 904-915) discovered that P38, the VSR of Turnip crinkle virus, uses its glycine/tryptophane (GW) motifs as an ARGONAUTE (AGO) hook to attract and disarm the host\\'s essential effector of RNA silencing. Several GW motif-containing cellular proteins are known to be important partners of AGOs in RNA silencing effector complexes in yeast, plants, and animals. The GW motif appears to be a versatile and effective tool for regulating the activities of RNA silencing pathways, and the use of GW mimicry to compete for and inhibit host AGOs may be a strategy used by many pathogens to counteract host RNAi-based defenses. © 2010 by Cold Spring Harbor Laboratory Press.

Evaluation of performance of Son Tek Argonaut acoustic doppler velocity log in tow tank and sea

Digital Repository Service at National Institute of Oceanography (India)

Joseph, A; Madhan, R.; Mascarenhas, A.A; Desai, R.G.P.; VijayKumar, K.; Dias, M.; Tengali, S.; Methar, A

Performance of a 500-kHz, 3-beam downward-looking Sontex Argonaut acoustic Doppler velocity log (DVL) based on measurements at tow-tank and sea is addressed. Its accuracy and linearity under tow-tank measurements were largely scattered...

Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins?

Science.gov (United States)

Nakanishi, Kotaro

2016-09-01

RNA silencing is a eukaryote-specific phenomenon in which microRNAs and small interfering RNAs degrade messenger RNAs containing a complementary sequence. To this end, these small RNAs need to be loaded onto an Argonaute protein (AGO protein) to form the effector complex referred to as RNA-induced silencing complex (RISC). RISC assembly undergoes multiple and sequential steps with the aid of Hsc70/Hsp90 chaperone machinery. The molecular mechanisms for this assembly process remain unclear, despite their significance for the development of gene silencing techniques and RNA interference-based therapeutics. This review dissects the currently available structures of AGO proteins and proposes models and hypotheses for RISC assembly, covering the conformation of unloaded AGO proteins, the chaperone-assisted duplex loading, and the slicer-dependent and slicer-independent duplex separation. The differences in the properties of RISC between prokaryotes and eukaryotes will also be clarified. WIREs RNA 2016, 7:637-660. doi: 10.1002/wrna.1356 For further resources related to this article, please visit the WIREs website. © 2016 The Authors. WIREs RNA published by Wiley Periodicals, Inc.

microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

OpenAIRE

Pinder, Benjamin D; Smibert, Craig A

2012-01-01

Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices.

Science.gov (United States)

Jannot, Guillaume; Vasquez-Rifo, Alejandro; Simard, Martin J

2011-01-01

During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.

Natural variation of the amino-terminal glutamine-rich domain in Drosophila argonaute2 is not associated with developmental defects.

Directory of Open Access Journals (Sweden)

Daniel Hain

2010-12-01

Full Text Available The Drosophila argonaute2 (ago2 gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs. We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex, is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution.

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown

International Nuclear Information System (INIS)

Asai, Tomohiro; Suzuki, Yuko; Matsushita, Saori; Yonezawa, Sei; Yokota, Junichi; Katanasaka, Yasufumi; Ishida, Tatsuhiro; Dewa, Takehisa; Kiwada, Hiroshi; Nango, Mamoru; Oku, Naoto

2008-01-01

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis

Substrate recognition by ribonucleoprotein ribonuclease MRP.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

ATP-dependent human RISC assembly pathways.

Science.gov (United States)

Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

2010-01-01

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

Science.gov (United States)

Zheng, Chun-Lei; Sumizawa, Tomoyuki; Che, Xiao-Fang; Tsuyama, Shinichiro; Furukawa, Tatsuhiko; Haraguchi, Misako; Gao, Hui; Gotanda, Takenari; Jueng, Hei-Cheul; Murata, Fusayoshi; Akiyama, Shin-Ichi

2005-01-07

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

Structural insights into RNA processing by the human RISC-loading complex.

Science.gov (United States)

Wang, Hong-Wei; Noland, Cameron; Siridechadilok, Bunpote; Taylor, David W; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A; Nogales, Eva

2009-11-01

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

Directory of Open Access Journals (Sweden)

Mark A Bernard

Full Text Available Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS and hypoxia-inducible factor 1-α subunit (HIF1A. Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM with substrate inhibition kinetics (Ki=105 nM, demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER bound in the active site of DICER may undergo direct transfer (as AGO2 substrate to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29, suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a

Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

KAUST Repository

Jiang, Hanlun

2015-07-16

Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.

Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

KAUST Repository

Jiang, Hanlun; Sheong, Fu Kit; Zhu, Lizhe; Gao, Xin; Bernauer, Julie; Huang, Xuhui

2015-01-01

Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.

Understanding the core of RNA interference: The dynamic aspects of Argonaute-mediated processes

KAUST Repository

Zhu, Lizhe

2016-10-05

At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms.

miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity

DEFF Research Database (Denmark)

Moore, Michael J; Scheel, Troels K H; Luna, Joseph M

2015-01-01

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modifie...

Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

Directory of Open Access Journals (Sweden)

Cédric Schelcher

2016-06-01

Full Text Available RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

Human gliomas contain morphine

DEFF Research Database (Denmark)

Olsen, Peter; Rasmussen, Mads; Zhu, Wei

2005-01-01

BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogeno...... of the solutions used in the study nor was it present as a residual material in blank HPLC runs. CONCLUSIONS: Morphine is present in human gliomas, suggesting that it may exert an action that effects tumour physiology/pathology.......BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...

Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods

DEFF Research Database (Denmark)

Heegaard, N H; Larsen, Martin Røssel; Muncrief, T

2000-01-01

ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein...

Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins

Directory of Open Access Journals (Sweden)

Ki-Eun Park

2016-05-01

Full Text Available The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT. By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

Functions of Heterogeneous Nuclear Ribonucleoproteins in Stem Cell Potency and Differentiation

Directory of Open Access Journals (Sweden)

Qishan Chen

2013-01-01

Full Text Available Stem cells possess huge importance in developmental biology, disease modelling, cell replacement therapy, and tissue engineering in regenerative medicine because they have the remarkable potential for self-renewal and to differentiate into almost all the cell types in the human body. Elucidation of molecular mechanisms regulating stem cell potency and differentiation is essential and critical for extensive application. Heterogeneous nuclear ribonucleoproteins (hnRNPs are modular proteins consisting of RNA-binding motifs and auxiliary domains characterized by extensive and divergent functions in nucleic acid metabolism. Multiple roles of hnRNPs in transcriptional and posttranscriptional regulation enable them to be effective gene expression regulators. More recent findings show that hnRNP proteins are crucial factors implicated in maintenance of stem cell self-renewal and pluripotency and cell differentiation. The hnRNPs interact with certain sequences in target gene promoter regions to initiate transcription. In addition, they recognize 3′UTR or 5′UTR of specific gene mRNA forming mRNP complex to regulate mRNA stability and translation. Both of these regulatory pathways lead to modulation of gene expression that is associated with stem cell proliferation, cell cycle control, pluripotency, and committed differentiation.

Structural basis for substrate placement by an archaeal box C/D ribonucleoprotein particle.

Science.gov (United States)

Xue, Song; Wang, Ruiying; Yang, Fangping; Terns, Rebecca M; Terns, Michael P; Zhang, Xinxin; Maxwell, E Stuart; Li, Hong

2010-09-24

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs. Copyright © 2010 Elsevier Inc. All rights reserved.

AGO2: a new Argonaute compromising plant virus accumulation

Directory of Open Access Journals (Sweden)

Veria Y Alvarado

2012-01-01

Full Text Available Plant viruses use several strategies to transport their nucleic acid genomes throughout the plants. Regardless of the movement mechanism, a universal major block to uninterrupted viral trafficking is the induction of antiviral silencing that degrades viral RNA. To counteract this defense, viruses encode suppressors that block certain steps in the RNA silencing pathway, and consequently these proteins allow viral spread to proceed. There is a constant battle between plants and viruses and sometimes viruses will succeed and invade the plants and in other cases the RNA silencing mechanism will override the virus. A key role in the silencing versus suppression conflict between plants and viruses is played by one or more members of the ARGONAUTE protein (AGO family encoded by plants. Here we review the mechanisms and effects of antiviral silencing with an emphasis on the contribution of AGOs, especially the recently discovered role of AGO2.

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

Science.gov (United States)

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins

International Nuclear Information System (INIS)

Hoffman, D.W.; Query, C.C.; Golden, B.L.; White, S.W.; Keene, J.D.

1991-01-01

An RNA recognition motif (RRM) of ∼80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel β-strands and two α-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent β-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface to the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins

Human gliomas contain morphine

DEFF Research Database (Denmark)

Olsen, Peter; Rasmussen, Mads; Zhu, Wei

2005-01-01

BACKGROUND: Morphine has been found in cancer cell lines originating from human and animal cells. Thus, it became important to demonstrate whether or not actual tumours contain this opiate alkaloid. MATERIAL/METHODS: Human glioma tissues were biochemically treated to isolate and separate endogenous...

Neutronic calculation of the next fuel elements for the Argonaut reactor

International Nuclear Information System (INIS)

Oliveira, C.R.E.; Brito Aghina, L.O. de

1981-01-01

The best parameters of the next fuel elements of the Argonaut reactor, at IEN (Instituto de Engenharia Nuclear - Brazil), were determined. The next fuel elements will be rods of an uranium mixture (19.98% enriched), graphite and bakelite. The parameters to be determined are: mixture density, percentage of uranium in the mixture, pellet radius, rod material and elements arrangement (step). The calculations routines consisted in the analysis of several steps, using the LEOPARD computer code for cell calculations and RMAT1D for one dimensional spatial calculations (criticality) with four energy groups. Finally a neutronic study of the Argounat reactors present configuration was done, using the HAMMER computer code (cell), the EXTERMINATOR computer code (two-dimensional calculations) and RAMAT1D. (Author) [pt

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

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C. Soldani

2010-05-01

Full Text Available Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP-containing components (PANA, hnRNP-core proteins, fibrillarin or RNP-associated nuclear proteins (SC-35 splicing factor. Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures.

Identification and characterization of argonaute protein, Ago2 and its associated small RNAs in Schistosoma japonicum.

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Pengfei Cai

Full Text Available BACKGROUND: The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC, have not yet been fully explored. METHODOLOGY/PRINCIPAL FINDINGS: Two monoclonal antibodies (mAbs specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2, but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs generated from transposable elements (TEs, especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons. CONCLUSIONS/SIGNIFICANCE: In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived

Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

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Fumitaka Momose

Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Protein composition of catalytically active human telomerase from immortal cells

DEFF Research Database (Denmark)

Cohen, Scott B; Graham, Mark E; Lovrecz, George O

2007-01-01

Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on th...

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro.

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Somarelli, Jason A; Mesa, Annia; Rodriguez, Carol E; Sharma, Shalini; Herrera, Rene J

2014-04-25

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5' splice sites at exon/intron boundaries. U1 snRNAs associate with 5' splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the 'Smith antigen', or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs. Copyright © 2014 Elsevier B.V. All rights reserved.

Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer.

Science.gov (United States)

Sand, Michael; Skrygan, Marina; Georgas, Dimitrios; Arenz, Christoph; Gambichler, Thilo; Sand, Daniel; Altmeyer, Peter; Bechara, Falk G

2012-11-01

The microprocessor complex mediates intranuclear biogenesis of precursor microRNAs from the primary microRNA transcript. Extranuclear, mature microRNAs are incorporated into the RNA-induced silencing complex (RISC) before interaction with complementary target mRNA leads to transcriptional repression or cleavage. In this study, we investigated the expression profiles of the microprocessor complex subunit DiGeorge syndrome critical region gene 8 (DGCR8) and the RISC components argonaute-1 (AGO1), argonaute-2 (AGO2), as well as double-stranded RNA-binding proteins PACT, TARBP1, and TARBP2 in epithelial skin cancer and its premalignant stage. Patients with premalignant actinic keratoses (AK, n = 6), basal cell carcinomas (BCC, n = 15), and squamous cell carcinomas (SCC, n = 7) were included in the study. Punch biopsies were harvested from the center of the tumors (lesional), from healthy skin sites (intraindividual controls), and from healthy skin sites in a healthy control group (n = 16; interindividual control). The DGCR8, AGO1, AGO2, PACT, TARBP1, and TARBP2 mRNA expression levels were detected by quantitative real-time reverse transcriptase polymerase chain reaction. The DGCR8, AGO1, AGO2, PACT, and TARBP1 expression levels were significantly higher in the AK, BCC, and SCC groups than the healthy controls (P  0.05). This study indicates that major components of the miRNA pathway, such as the microprocessor complex and RISC, are dysregulated in epithelial skin cancer. Copyright © 2011 Wiley Periodicals, Inc.

Comparison of the ribonucleoproteins of different rabies virus serotypes by radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Bruns, M; Dietzschold, B; Schneider, L G; Cox, J H [Federal Research Inst. for Animal Virus Diseases, Tuebingen (Germany, F.R.)

1977-12-01

Radioimmunoassay (RIA) provides a sensitive serological procedure for detecting rabies virus ribonucleoprotein (RNP) as well as its specific antibodies. RIA was carried out using highly purified RNPs labelled by the chloramine-T method. This paper describes optimal conditions for iodination of RNP with high specific activity. The optimal concentrations of /sup 125/I, RNP, chloramine-T, and reducing agent as well as the effect of pH on the reaction were investigated. RIA proved to be extremely sensitive for detection of homologous antibodies. In competition experiments the part-relationship of the group-specific RNPs of the three rabies virus serotypes (HEP, MOK, and LBV) was confirmed.

Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

Science.gov (United States)

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

2006-12-20

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

Science.gov (United States)

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Host factors that interact with the pestivirus N-terminal protease, Npro, are components of the ribonucleoprotein complex.

Science.gov (United States)

Jefferson, Matthew; Donaszi-Ivanov, Andras; Pollen, Sean; Dalmay, Tamas; Saalbach, Gerhard; Powell, Penny P

2014-09-01

The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to

The slicer activity of ARGONAUTE1 is required specifically for the phasing, not production, of trans-acting short interfering RNAs in Arabidopsis

DEFF Research Database (Denmark)

Arribas Hernandez, Laura; Marchais, Antonin; Poulsen, Christian

2016-01-01

ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. miRNA-guided s......ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. mi...... is required for assembly of active AGO1-siRNA complexes in vivo, and many AGO1-bound siRNAs are trimmed in the absence of slicer activity. Remarkably, seedlings defective in AGO1 slicer activity produce abundant siRNAs from tasiRNA loci in vivo. These siRNAs depend on RDR6 and SUPPRESSOR OF GENE SILENCING3...

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition.

Science.gov (United States)

Lostalé-Seijo, Irene; Louzao, Iria; Juanes, Marisa; Montenegro, Javier

2017-12-01

The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.

Functional diversification of Argonautes in nematodes: an expanding universe.

Science.gov (United States)

Buck, Amy H; Blaxter, Mark

2013-08-01

In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism.

Decommissioning the Jason Argonaut research reactor at a world heritage site

Energy Technology Data Exchange (ETDEWEB)

Lockwood, R.J.S.; Beeley, P.A. [HMS Sultan, Gosport, Hampshire (United Kingdom)

2001-07-01

The Jason low power Argonaut type, water and graphite moderated reactor was located in King William Building, which is a Grade 1 listed building within the Royal Naval College, Greenwich, London. The College itself is a Scheduled Ancient Monument with World Heritage Site status and is situated about a mile from the Greenwich Dome. The decision to decommission Jason to International Atomic Energy Agency Stage 3 status (unrestricted site use) was taken in 1996. All physical decommissioning work was completed by October 1999, site radiological clearance was obtained in November 1999, the site license was withdrawn and the site was handed over for future unrestricted use on 9 December 1999. The Jason decommissioning project was safely completed to time, cost and quality by the Millennium [2] without any adverse effects on World Heritage aspects of the site. In this paper details are provided about the Jason fuel removal phase and an outline of the other phases of the project.

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

Science.gov (United States)

Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

1992-01-01

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly.

Science.gov (United States)

Endo, Yayoi; Iwakawa, Hiro-oki; Tomari, Yukihide

2013-07-01

Plant ARGONAUTE7 (AGO7) assembles RNA-induced silencing complex (RISC) specifically with miR390 and regulates the auxin-signalling pathway via production of TAS3 trans-acting siRNAs (tasiRNAs). However, how AGO7 discerns miR390 among other miRNAs remains unclear. Here, we show that the 5' adenosine of miR390 and the central region of miR390/miR390* duplex are critical for the specific interaction with AGO7. Furthermore, despite the existence of mismatches in the seed and central regions of the duplex, cleavage of the miR390* strand is required for maturation of AGO7-RISC. These findings suggest that AGO7 uses multiple checkpoints to select miR390, thereby circumventing promiscuous tasiRNA production.

N-methylation of the heterogeneous nuclear ribonucleoproteins in HeLa cells

International Nuclear Information System (INIS)

Rieker, J.P.

1984-01-01

Several of the core proteins on the 40S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from HeLa cells contain N/sup G/,N/sup G/-dimethyl-L-arginine (uDMA). 3-deazaadenosine (c 3 Ado), an inhibitor of and substrate for s-adenosyl-L-homocysteine hydrolase, has been used to study the methylation patterns of the individual polypeptides. Trimethyllysine and uDMA formation in total cellular protein were inhibited in the presence of the drug while other methylated basic amino acids were unaffected. This inhibition was reversed within 60 min after removal of the drug from the medium. Monolayer HeLa cultures were incubated with [methyl- 3 H]-L-methoinine for 12 hours in the presence of 50 uM c 3 Ado. Purified particles were obtained by centrifugation of nuclear extracts on sucrose density gradients. The core proteins were isolated by two-dimensional gel electrophoresis, acid hydrolyzed and analyzed for radioactivity incorporated into methionine and methylated basic amino acids. The ratio of radioactivity incorporated into uDMA relative to that into methionine for the two major particle proteins with molecular weights of 31,000 (A 1 ) and 43,000 (A 2 ) was about 2.0 and 0.2 in control cultures. In the presence of c 3 Ado, these ratios were depressed 60 to 80%. Results of pulse-chase experiments suggested that A 1 and A 2 are metabolically stable proteins (t/sub 0.5/ > 75 hr), whether or not the proteins were undermethylated. Monomethyl-L-arginine may be a precursor in the formation of u-DMA

Synthesis and Gene Silencing Properties of siRNAs Containing Terminal Amide Linkages

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Maria Gaglione

2014-01-01

Full Text Available The active components of the RNAi are 21 nucleotides long dsRNAs containing a 2 nucleotide overhang at the 3′ end, carrying 5′-phosphate and 3′-hydroxyl groups (siRNAs. Structural analysis revealed that the siRNA is functionally bound at both ends to RISC. Terminal modifications are considered with interest as the introduction of chemical moieties interferes with the 3′ overhang recognition by the PAZ domain and the 5′-phosphate recognition by the MID and PIWI domains of RISC. Herein, we report the synthesis of modified siRNAs containing terminal amide linkages by introducing hydroxyethylglycine PNA (hegPNA moieties at 5′, and at 3′ positions and on both terminals. Results of gene silencing studies highlight that some of these modifications are compatible with the RNAi machinery and markedly increase the resistance to serum-derived nucleases even after 24 h of incubation. Molecular docking simulations were attained to give at atomistic level a clearer picture of the effect of the most performing modifications on the interactions with the human Argonaute 2 PAZ, MID, and PIWI domains. This study adds another piece to the puzzle of the heterogeneous chemical modifications that can be attained to enhance the silencing efficiency of siRNAs.

Argonaute 2 and nasopharyngeal carcinoma: a genetic association study and functional analysis

International Nuclear Information System (INIS)

Li, Peiyao; Meng, Jinfeng; Zhai, Yun; Zhang, Hongxing; Yu, Lixia; Wang, Zhifu; Zhang, Xiaoai; Cao, Pengbo; Chen, Xi; Han, Yuqing; Zhang, Yang; Chen, Huipeng; Ling, Yan; Li, Yuxia; Cui, Ying; Bei, Jin-Xin; Zeng, Yi-Xin; He, Fuchu; Zhou, Gangqiao

2015-01-01

Argonaute 2 (AGO2), a central component of RNA-induced silencing complex, plays critical roles in cancer. We examined whether the single nucleotide polymorphisms (SNPs) of AGO2 were related to the risk of nasopharyngeal carcinoma (NPC). Twenty-five tag SNPs within AGO2 were genotyped in Guangxi population consisting of 855 NPC patients and 1036 controls. The SNPs significantly associated with NPC were further replicated in Guangdong population consisting of 996 NPC patients and 972 controls. Functional experiments were conducted to examine the biologic roles of AGO2 in NPC. A significantly increased risk of advanced lymph node metastasis of NPC was identified for the AGO2 rs3928672 GA + AA genotype compared with GG genotype in both the Guangxi and Guangdong populations (combined odd ratio = 2.08, 95 % confidence interval = 1.44-3.01, P = 8.60 × 10 −5 ). Moreover, the AGO2 protein expression levels of rs3928672 GA + AA genotype carriers were higher than the GG genotype carriers in the NPC tissues (P = 0.041), and AGO2 was significantly over-expressed in NPC tissues compared with non-cancerous nasopharyngeal tissues (P = 0.011). In addition, AGO2 knockdown reduced cell proliferation, induced apoptosis, and inhibited migration of NPC cells. Furthermore, gene expression microarray showed that genes altered following AGO2 knockdown were clustered in tumorigenesis and metastasis relevant pathways. Our findings suggest that the genetic polymorphism in AGO2 may be a risk factor for the advanced lymph node metastasis of NPC in Chinese populations, and AGO2 acts as an oncogene in the development of NPC. The online version of this article (doi:10.1186/s12885-015-1895-4) contains supplementary material, which is available to authorized users

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

DEFF Research Database (Denmark)

Honoré, B; Rasmussen, H H; Vorum, H

1995-01-01

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share...... epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75......%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used...

Recovery of enriched Uranium (20% U-235) from wastes obtained in the preparation of fuel elements for argonaut type reactors

International Nuclear Information System (INIS)

Uriarte, A.; Ramos, L.; Estrada, J.; del Val, J. L.

1962-01-01

Results obtained with the two following installations for recovering enriched uranium (20% U-235) from wastes obtained in the preparation of fuel elements for Argonaut type reactors are presented. Ion exchange unit to recover uranium form mother liquors resulting from the precipitation ammonium diuranate (ADU) from UO 2 F 2 solutions. Uranium recovery unit from solid wastes from the process of manufacture of fuel elements, consisting of a) waste dissolution, and b) extraction with 10% (v/v) TBP. (Author) 9 refs

Recovery of enriched Uranium (20% U-235) from wastes obtained in the preparation of fuel elements for argonaut type reactors

Energy Technology Data Exchange (ETDEWEB)

Uriarte, A; Ramos, L; Estrada, J; Val, J L. del

1962-07-01

Results obtained with the two following installations for recovering enriched uranium (20% U-235) from wastes obtained in the preparation of fuel elements for Argonaut type reactors are presented. Ion exchange unit to recover uranium form mother liquors resulting from the precipitation ammonium diuranate (ADU) from UO{sub 2}F{sub 2} solutions. Uranium recovery unit from solid wastes from the process of manufacture of fuel elements, consisting of a) waste dissolution, and b) extraction with 10% (v/v) TBP. (Author) 9 refs.

The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.

Science.gov (United States)

Beckmann, Benedikt M; Castello, Alfredo; Medenbach, Jan

2016-06-01

Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

Report on the interpretation of critical experiments in the Siemens-Argonaut-Reactor Graz to study water ingress into spherical elements. Ergebnisbericht zur Auslegung kritischer Experimente am Siemens-Argonaut-Reaktor Graz zum Studium des Wassereinbruches im Kugelhaufen

Energy Technology Data Exchange (ETDEWEB)

Schuerrer, F [Technische Univ., Graz (Austria). Inst. fuer Theoretische Physik und Reaktorphysik; Neef, R D [Kernforschungsanlage Juelich G.m.b.H. (Germany, F.R.). Inst. fuer Reaktorentwicklung

1979-04-15

The experiments described are of interest in the study of water contamination in HTR fuel elements. The Siemens Argonaut Reactor (SAR) has been considered as a research tool for a simulation experiment. Following a brief description of the SAR, planned programs are discussed in 'dry' and 'wet' cores. Detector foil types and locations are noted. A theoretical model is developed and nuclide concentrations estimated in the various spectral zones. Reactivity calculations have been made and are summarised for various H{sub 2}O percentage concentrations. The discussion is supported by simplified core layout diagrams and graphs of core flux distributions. Neutron diffusion and spectra calculations are referenced to computer programs used by KFA-Juelich, published elsewhere, and include GAM, THERMOS, MUPO and EXTERMINATOR-2. (G.C.)

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

Science.gov (United States)

Kenesi, Erzsébet; Carbonell, Alberto; Lózsa, Rita; Vértessy, Beáta; Lakatos, Lóránt

2017-07-27

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

Science.gov (United States)

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein.

Science.gov (United States)

Pinder, Benjamin D; Smibert, Craig A

2013-01-01

Argonaute (Ago) proteins are typically recruited to target messenger RNAs via an associated small RNA such as a microRNA (miRNA). Here, we describe a new mechanism of Ago recruitment through the Drosophila Smaug RNA-binding protein. We show that Smaug interacts with the Ago1 protein, and that Ago1 interacts with and is required for the translational repression of the Smaug target, nanos mRNA. The Ago1/nanos mRNA interaction does not require a miRNA, but it does require Smaug. Taken together, our data suggest a model whereby Smaug directly recruits Ago1 to nanos mRNA in a miRNA-independent manner, thereby repressing translation.

CmRBP50 protein phosphorylation is essential for assembly of a stable phloem-mobile high-affinity ribonucleoprotein complex.

Science.gov (United States)

Li, Pingfang; Ham, Byung-Kook; Lucas, William J

2011-07-01

RNA-binding proteins (RBPs) form ribonucleoprotein (RNP) complexes that play crucial roles in RNA processing for gene regulation. The angiosperm sieve tube system contains a unique population of transcripts, some of which function as long-distance signaling agents involved in regulating organ development. These phloem-mobile mRNAs are translocated as RNP complexes. One such complex is based on a phloem RBP named Cucurbita maxima RNA-binding protein 50 (CmRBP50), a member of the polypyrimidine track binding protein family. The core of this RNP complex contains six additional phloem proteins. Here, requirements for assembly of this CmRBP50 RNP complex are reported. Phosphorylation sites on CmRBP50 were mapped, and then coimmunoprecipitation and protein overlay studies established that the phosphoserine residues, located at the C terminus of CmRBP50, are critical for RNP complex assembly. In vitro pull-down experiments revealed that three phloem proteins, C. maxima phloem protein 16, C. maxima GTP-binding protein, and C. maxima phosphoinositide-specific phospholipase-like protein, bind directly with CmRBP50. This interaction required CmRBP50 phosphorylation. Gel mobility-shift assays demonstrated that assembly of the CmRBP50-based protein complex results in a system having enhanced binding affinity for phloem-mobile mRNAs carrying polypyrimidine track binding motifs. This property would be essential for effective long-distance translocation of bound mRNA to the target tissues.

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

International Nuclear Information System (INIS)

Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

2005-01-01

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

Staufen- and FMRP-Containing Neuronal RNPs Are Structurally and Functionally Related to Somatic P Bodies

OpenAIRE

Barbee, Scott A.; Estes, Patricia S.; Cziko, Anne-Marie; Hillebrand, Jens; Luedeman, Rene A.; Coller, Jeff M.; Johnson, Nick; Howlett, Iris C.; Geng, Cuiyun; Ueda, Ryu; Brand, Andrea H.; Newbury, Sarah F.; Wilhelm, James E.; Levine, Richard B.; Nakamura, Akira

2006-01-01

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRPcontaining RNPs in Drosophila neurons contain proteins also present in somatic “P bodies,” including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (ar...

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

Directory of Open Access Journals (Sweden)

Lia Carolina Soares Medeiros

2017-11-01

Full Text Available Trypanosomatids (order Kinetoplastida, including the human pathogens Trypanosoma cruzi (agent of Chagas disease, Trypanosoma brucei, (African sleeping sickness, and Leishmania (leishmaniasis, affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9 technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9, but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9, and in vitro-transcribed single guide RNAs (sgRNAs results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major. RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.

Human placenta secretes apolipoprotein B-100-containing lipoproteins

DEFF Research Database (Denmark)

Munk-Madsen, Eva; Lindegaard, Marie Louise Skakkebæk; Andersen, Claus B

2004-01-01

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very...... lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway...

Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos

International Nuclear Information System (INIS)

Zhang, Ping; Wang, Ningling; Lin, Xianhua; Jin, Li; Xu, Hong; Li, Rong; Huang, Hefeng

2016-01-01

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ping [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Wang, Ningling [Department of Assisted Reproduction, Shanghai Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Lin, Xianhua; Jin, Li [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Xu, Hong, E-mail: xuhong1168@126.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Li, Rong [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China); Huang, Hefeng, E-mail: huanghefg@hotmail.com [The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai (China)

2016-02-26

Heterogeneous nuclear ribonucleoprotein K (hnRNP K), an evolutionarily conserved protein, is involved in several important cellular processes that are relevant to cell proliferation, differentiation, apoptosis, and cancer development. However, details of hnRNP K expression during mammalian oogenesis and preimplantation embryo development are lacking. The present study investigates the expression and cellular localization of K protein in the mouse ovaries and preimplantation embryos using immunostaining. We demonstrate, for the first time, that hnRNP K is abundantly expressed in the nuclei of mouse oocytes in primordial, primary and secondary follicles. In germ vesicle (GV)-stage oocytes, hnRNP K accumulates in the germinal vesicle in a spot distribution manner. After germinal vesicle breakdown, speckled hnRNP K is diffusely distributed in the cytoplasm. However, after fertilization, the K protein relocates into the female and male pronucleus and persists in the blastomere nuclei. Localization of K protein in the human ovary and ovarian granulosa cell tumor (GCT) was also investigated. Overall, this study provides important morphological evidence to better understand the possible roles of hnRNP K in mammalian oogenesis and early embryo development. - Highlights: • HnRNP K localizes in the nucleus of GV-stage oocyte in a punctate distribution. • HnRNP K strongly accumulates in zygotic pronuclei as condensed spots. • The localization of hnRNP K during oogenesis and embryogenesis is characteristic. • HnRNP K might have an important role in oogenesis and embryonic development.

The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation.

Science.gov (United States)

Baumberger, Nicolas; Tsai, Ching-Hsui; Lie, Miranda; Havecker, Ericka; Baulcombe, David C

2007-09-18

Plant and animal viruses encode suppressor proteins of an adaptive immunity mechanism in which viral double-stranded RNA is processed into 21-25 nt short interfering (si)RNAs. The siRNAs guide ARGONAUTE (AGO) proteins so that they target viral RNA. Most viral suppressors bind long dsRNA or siRNAs and thereby prevent production of siRNA or binding of siRNA to AGO. The one exception is the 2b suppressor of Cucumoviruses that binds to and inhibits AGO1. Here we describe a novel suppressor mechanism in which a Polerovirus-encoded F box protein (P0) targets the PAZ motif and its adjacent upstream sequence in AGO1 and mediates its degradation. F box proteins are components of E3 ubiquitin ligase complexes that add polyubiquitin tracts on selected lysine residues and thereby mark a protein for proteasome-mediated degradation. With P0, however, the targeted degradation of AGO is insensitive to inhibition of the proteasome, indicating that the proteasome is not involved. We also show that P0 does not block a mobile signal of silencing, indicating that the signal molecule does not have AGO protein components. The ability of P0 to block silencing without affecting signal movement may contribute to the phloem restriction of viruses in the Polerovirus group.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

Science.gov (United States)

Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

2016-06-01

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

Complete cDNA sequence coding for human docking protein

Energy Technology Data Exchange (ETDEWEB)

Hortsch, M; Labeit, S; Meyer, D I

1988-01-11

Docking protein (DP, or SRP receptor) is a rough endoplasmic reticulum (ER)-associated protein essential for the targeting and translocation of nascent polypeptides across this membrane. It specifically interacts with a cytoplasmic ribonucleoprotein complex, the signal recognition particle (SRP). The nucleotide sequence of cDNA encoding the entire human DP and its deduced amino acid sequence are given.

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology.

Directory of Open Access Journals (Sweden)

Ezequiel Nazer

2018-03-01

Full Text Available Drosophila Argonaute2 (AGO2 has been shown to regulate expression of certain loci in an RNA interference (RNAi-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs. Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht, which is immersed in a LAD located within a repressive topologically-associated domain (TAD. Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Science.gov (United States)

Saliou, Jean-Michel; Manival, Xavier; Tillault, Anne-Sophie; Atmanene, Cédric; Bobo, Claude; Branlant, Christiane; Van Dorsselaer, Alain; Charpentier, Bruno; Cianférani, Sarah

2015-08-01

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Construction of retroviral recombinant containing human tissue ...

African Journals Online (AJOL)

USER

2010-03-29

Mar 29, 2010 ... Recombinant retroviral vector containing human tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) gene was ..... heavy metal ions, the protein could be express in an .... involves adhesion, degradation and movement. To.

Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus

Science.gov (United States)

Zhou, Zhuo; Cao, Mengmeng; Guo, Yang; Zhao, Lili; Wang, Jingfeng; Jia, Xue; Li, Jianguo; Wang, Conghui; Gabriel, Gülsah; Xue, Qinghua; Yi, Yonghong; Cui, Sheng; Jin, Qi; Wang, Jianwei; Deng, Tao

2014-02-01

The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.

Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs.

Directory of Open Access Journals (Sweden)

Federica Collino

Full Text Available BACKGROUND: Cell-derived microvesicles (MVs have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs and liver resident stem cells (HLSCs were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation. CONCLUSIONS: This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem

Isolation and characterization of human apolipoprotein M-containing lipoproteins

DEFF Research Database (Denmark)

Christoffersen, Christina; Nielsen, Lars Bo; Axler, Olof

2006-01-01

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma...... with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apo...

A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

Science.gov (United States)

Nicolás, Francisco E.; Moxon, Simon; de Haro, Juan P.; Dalmay, Tamas; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

2013-01-01

The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1 − M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi. PMID:23935973

76 FR 65210 - Certain Products and Pharmaceutical Compositions Containing Recombinant Human Erythropoetin...

Science.gov (United States)

2011-10-20

... INTERNATIONAL TRADE COMMISSION [Investigation No. 337-TA-568] Certain Products and Pharmaceutical Compositions Containing Recombinant Human Erythropoetin; Termination of Investigation on the Basis of... after importation of certain products and pharmaceutical compositions containing recombinant human...

Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles

International Nuclear Information System (INIS)

Kroemer, A.

1987-01-01

Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles U1 to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights ≅ 150,000 and 115,000 respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or U10 RNA

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

Science.gov (United States)

Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2013-08-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

International Nuclear Information System (INIS)

Loucaides, Eva M.; Kirchbach, Johann C. von; Foeglein, Agnes; Sharps, Jane; Fodor, Ervin; Digard, Paul

2009-01-01

The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

Science.gov (United States)

Lemieux, Bruno; Laterreur, Nancy; Perederina, Anna; Noël, Jean-François; Dubois, Marie-Line; Krasilnikov, Andrey S; Wellinger, Raymund J

2016-05-19

Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

The Argonaute CSR-1 and its 22G-RNA cofactors are required for holocentric chromosome segregation.

Science.gov (United States)

Claycomb, Julie M; Batista, Pedro J; Pang, Ka Ming; Gu, Weifeng; Vasale, Jessica J; van Wolfswinkel, Josien C; Chaves, Daniel A; Shirayama, Masaki; Mitani, Shohei; Ketting, René F; Conte, Darryl; Mello, Craig C

2009-10-02

RNAi-related pathways regulate diverse processes, from developmental timing to transposon silencing. Here, we show that in C. elegans the Argonaute CSR-1, the RNA-dependent RNA polymerase EGO-1, the Dicer-related helicase DRH-3, and the Tudor-domain protein EKL-1 localize to chromosomes and are required for proper chromosome segregation. In the absence of these factors chromosomes fail to align at the metaphase plate and kinetochores do not orient to opposing spindle poles. Surprisingly, the CSR-1-interacting small RNAs (22G-RNAs) are antisense to thousands of germline-expressed protein-coding genes. Nematodes assemble holocentric chromosomes in which continuous kinetochores must span the expressed domains of the genome. We show that CSR-1 interacts with chromatin at target loci but does not downregulate target mRNA or protein levels. Instead, our findings support a model in which CSR-1 complexes target protein-coding domains to promote their proper organization within the holocentric chromosomes of C. elegans.

Efficient Recreation of t(11;22 EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9

Directory of Open Access Journals (Sweden)

Raul Torres-Ruiz

2017-05-01

Full Text Available Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22 translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1 using end-joining DNA processing factors involved in repair mechanisms, or (2 ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3 all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22 is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22 in human stem cells opens up new avenues in modeling Ewing sarcoma.

The C. elegans CSR-1 argonaute pathway counteracts epigenetic silencing to promote germline gene expression.

Science.gov (United States)

Seth, Meetu; Shirayama, Masaki; Gu, Weifeng; Ishidate, Takao; Conte, Darryl; Mello, Craig C

2013-12-23

Organisms can develop adaptive sequence-specific immunity by reexpressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piwi-interacting RNA (piRNA) pathway recruits RNA-dependent RNA polymerase (RdRP) to foreign sequences to amplify a transgenerational small-RNA-induced epigenetic silencing signal (termed RNAe). Here, we provide evidence that, in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self-mRNAs. We refer to this mechanism, which can prevent or reverse RNAe, as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a transgenerational CSR-1 memory that recognizes and protects self-mRNAs, allowing piRNAs to recognize foreign sequences innately, without the need for prior exposure

A Flexible Domain-Domain Hinge Promotes an Induced-fit Dominant Mechanism for the Loading of Guide-DNA into Argonaute Protein in Thermus thermophilus

KAUST Repository

Zhu, Lizhe

2016-02-24

Argonaute proteins (Ago) are core components of the RNA Induced Silencing Complex (RISC) that load and utilize small guide nucleic acids to silence mRNAs or cleave foreign DNAs. Despite the essential role of Ago in gene regulation and defense against virus, the molecular mechanism of guide-strand loading into Ago remains unclear. We explore such a mechanism in the bacterium Thermus thermophilus Ago (TtAgo), via a computational approach combining molecular dynamics, bias-exchange metadynamics, and protein-DNA docking. We show that apo TtAgo adopts multiple closed states that are unable to accommodate guide-DNA. Conformations able to accommodate the guide are beyond the reach of thermal fluctuations from the closed states. These results suggest an induced-fit dominant mechanism for guide-strand loading in TtAgo, drastically different from the two-step mechanism for human Ago 2 (hAgo2) identified in our previous study. Such a difference between TtAgo and hAgo2 is found to mainly originate from the distinct rigidity of their L1-PAZ hinge. Further comparison among known Ago structures from various species indicates that the L1-PAZ hinge may be flexible in general for prokaryotic Agos but rigid for eukaryotic Agos. © 2016 American Chemical Society.

A Flexible Domain-Domain Hinge Promotes an Induced-fit Dominant Mechanism for the Loading of Guide-DNA into Argonaute Protein in Thermus thermophilus.

Science.gov (United States)

Zhu, Lizhe; Jiang, Hanlun; Sheong, Fu Kit; Cui, Xuefeng; Gao, Xin; Wang, Yanli; Huang, Xuhui

2016-03-17

Argonaute proteins (Ago) are core components of the RNA Induced Silencing Complex (RISC) that load and utilize small guide nucleic acids to silence mRNAs or cleave foreign DNAs. Despite the essential role of Ago in gene regulation and defense against virus, the molecular mechanism of guide-strand loading into Ago remains unclear. We explore such a mechanism in the bacterium Thermus thermophilus Ago (TtAgo), via a computational approach combining molecular dynamics, bias-exchange metadynamics, and protein-DNA docking. We show that apo TtAgo adopts multiple closed states that are unable to accommodate guide-DNA. Conformations able to accommodate the guide are beyond the reach of thermal fluctuations from the closed states. These results suggest an induced-fit dominant mechanism for guide-strand loading in TtAgo, drastically different from the two-step mechanism for human Ago 2 (hAgo2) identified in our previous study. Such a difference between TtAgo and hAgo2 is found to mainly originate from the distinct rigidity of their L1-PAZ hinge. Further comparison among known Ago structures from various species indicates that the L1-PAZ hinge may be flexible in general for prokaryotic Ago's but rigid for eukaryotic Ago's.

The macronutrients in human milk change after storage in various containers.

Science.gov (United States)

Chang, Yu-Chuan; Chen, Chao-Huei; Lin, Ming-Chih

2012-06-01

The concentrations of macronutrients in human milk can be influenced by various processes, such as storage, freezing, and thawing, that are performed by lactating working mothers and breast milk banks. We evaluated the impact of various containers on the nutrient concentrations in human milk. A total of 42 breast milk samples from 18 healthy lactating mothers were collected. A baseline macronutrient concentration was determined for each sample. Then, the breast milk samples were divided and stored in nine different commercial milk containers. After freezing at -20°C for 2 days, the milk samples were thawed and analyzed again. A midinfrared human milk analyzer (HMA) was used to measure the protein, fat, and carbohydrate contents. There was a significant decrease in the fat content following the storage, freezing, and thawing processes, ranging from 0.27-0.30 g/dL (p=0.02), but no significant decrease in energy content (p=0.069) was noted in the nine different containers. There were statistically significant increases in protein and carbohydrate concentrations in all containers (p=0.021 and 0.001, respectively), however there were no significant differences between the containers in terms of fat, protein, carbohydrate, or energy contents. Human milk, when subjected to storage, freezing, and thawing processes, demonstrated a significant decrease in fat content (up to 9% reduction) in various containers. It is better for infants to receive milk directly from the mother via breastfeeding. More studies are warranted to evaluate the effects of milk storage on infant growth and development. Copyright © 2012. Published by Elsevier B.V.

Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

Science.gov (United States)

Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

2018-01-01

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

Does human pancreas contain salivary-type isoamylase?

Science.gov (United States)

Shimamura, J; Fridhandler, L; Berk, J E

1975-01-01

Amylase isoenzyme analysis was made of extracts of normal human pancreatic tissue by first conducting ion exchange chromatography of the purified material. This gave evidence of only pancreatic type (P-type) isoamylase for all purposes. However, when effluent fractions in which salivary type isoamylase would ordinarily be expected to be present were harvested, pooled, concentrated, and rechromatographed, the pancreatic extracts were found to contain some salivary type (S-type) isoamylase. The latter accounted for approximately 0-8 to 1-7% of the total recovered amylase activity. This finding of S-type isoamylase in normal human pancreas potentially has important bearing on the interpretation of isamylase analysis. PMID:1218813

HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages.

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Valentina Vongrad

Full Text Available MiRNAs and other small noncoding RNAs (sncRNAs are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM.The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP, which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs.PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.

A Polypyrimidine Tract Binding Protein, Pumpkin RBP50, Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex[W

Science.gov (United States)

Ham, Byung-Kook; Brandom, Jeri L.; Xoconostle-Cázares, Beatriz; Ringgold, Vanessa; Lough, Tony J.; Lucas, William J.

2009-01-01

RNA binding proteins (RBPs) are integral components of ribonucleoprotein (RNP) complexes and play a central role in RNA processing. In plants, some RBPs function in a non-cell-autonomous manner. The angiosperm phloem translocation stream contains a unique population of RBPs, but little is known regarding the nature of the proteins and mRNA species that constitute phloem-mobile RNP complexes. Here, we identified and characterized a 50-kD pumpkin (Cucurbita maxima cv Big Max) phloem RNA binding protein (RBP50) that is evolutionarily related to animal polypyrimidine tract binding proteins. In situ hybridization studies indicated a high level of RBP50 transcripts in companion cells, while immunolocalization experiments detected RBP50 in both companion cells and sieve elements. A comparison of the levels of RBP50 present in vascular bundles and phloem sap indicated that this protein is highly enriched in the phloem sap. Heterografting experiments confirmed that RBP50 is translocated from source to sink tissues. Collectively, these findings established that RBP50 functions as a non-cell-autonomous RBP. Protein overlay, coimmunoprecipitation, and cross-linking experiments identified the phloem proteins and mRNA species that constitute RBP50-based RNP complexes. Gel mobility-shift assays demonstrated that specificity, with respect to the bound mRNA, is established by the polypyrimidine tract binding motifs within such transcripts. We present a model for RBP50-based RNP complexes within the pumpkin phloem translocation stream. PMID:19122103

Climate: a planet and men: what human influence on climate warming?

International Nuclear Information System (INIS)

Orsenna, E.; Petit, M.; Chabreuil, A. and others

2011-01-01

The authors, all members of the 'Argonauts club', have decided with other scientists, to write this book in order to allow the reader to make his own opinion about the influence of human activities on global warming. The book presents and supplies precise answers to the questions about the climate machine and its complex mechanisms according to the present day state of scientific knowledge. It takes stock of the actual climate situation and of the overall possible means at our disposal to limit the extent of the harmful anthropic impact. (J.S.)

Circular chromatin complexes in human lymphocytes high-resolution autoradiography

International Nuclear Information System (INIS)

Becak, M.L.; Fukuda-Pizzocaro, K.; Santos, R. de C.S. dos; Brunner, O.

1985-01-01

Transcriptionally active chromatin fibers were observed in chromosomes presenting the loops/scaffold configuration. The active fibers showed altered nucleosomes and presented multiforked aspects which led to the formation of ring complexes. The ribonucleoprotein transcripts (RNP) appeared as networks of 0.1 μm or multiples tandemly disposed along the fiber. It is suggested that the ring complexes belong to the human genome. The possibility that these circular structures come from a prokaryote is also considered. (author) [pt

Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9.

Science.gov (United States)

Torres-Ruiz, Raul; Martinez-Lage, Marta; Martin, Maria C; Garcia, Aida; Bueno, Clara; Castaño, Julio; Ramirez, Juan C; Menendez, Pablo; Cigudosa, Juan C; Rodriguez-Perales, Sandra

2017-05-09

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Human Splicing Finder: an online bioinformatics tool to predict splicing signals.

Science.gov (United States)

Desmet, François-Olivier; Hamroun, Dalil; Lalande, Marine; Collod-Béroud, Gwenaëlle; Claustres, Mireille; Béroud, Christophe

2009-05-01

Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-beta Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5' and 3' splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.

Human placenta secretes apolipoprotein B-100-containing lipoproteins

DEFF Research Database (Denmark)

Munk-Madsen, Eva; Lindegaard, Marie Louise Skakkebæk; Andersen, Claus B

2004-01-01

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very...... of lipid transfer from the mother to the developing fetus....

Binding of the heterogeneous ribonucleoprotein K (hnRNP K to the Epstein-Barr virus nuclear antigen 2 (EBNA2 enhances viral LMP2A expression.

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Henrik Gross

Full Text Available The Epstein-Barr Virus (EBV -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively. EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2 Type 1. The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3 which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K. Specific binding of the ADMA- antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.

Spinal muscular atrophy: Selective motor neuron loss and global defect in the assembly of ribonucleoproteins.

Science.gov (United States)

Beattie, Christine E; Kolb, Stephen J

2018-08-15

Spinal muscular atrophy is caused by deletions or mutations in the SMN1 gene that result in reduced expression of the SMN protein. The SMN protein is an essential molecular chaperone that is required for the biogenesis of multiple ribonucleoprotein (RNP) complexes including spliceosomal small nuclear RNPs (snRNPs). Reductions in SMN expression result in a reduced abundance of snRNPs and to downstream RNA splicing alterations. SMN is also present in axons and dendrites and appears to have important roles in the formation of neuronal mRNA-protein complexes during development or neuronal repair. Thus, SMA is an exemplar, selective motor neuron disorder that is caused by defects in fundamental RNA processing events. A detailed molecular understanding of how motor neurons fail, and why other neurons do not, in SMA will yield important principals about motor neuron maintenance and neuronal specificity in neurodegenerative diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests.

Science.gov (United States)

Gillet, François-Xavier; Garcia, Rayssa A; Macedo, Leonardo L P; Albuquerque, Erika V S; Silva, Maria C M; Grossi-de-Sa, Maria F

2017-01-01

Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil ( Anthonomus grandis ), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests

Directory of Open Access Journals (Sweden)

François-Xavier Gillet

2017-04-01

Full Text Available Genetically modified (GM crops producing double-stranded RNAs (dsRNAs are being investigated largely as an RNA interference (RNAi-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis, we showed that the chimeric protein PTD-DRBD (peptide transduction domain—dsRNA binding domain combined with dsRNA forms a ribonucleoprotein particle (RNP that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

Regulatory RNPs: a novel class of ribonucleoproteins that potentially contribute to ribosome heterogeneity

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Aaron R. Poole

2017-09-01

Full Text Available Many ribonucleoproteins (RNPs, which are comprised of noncoding RNA and associated proteins, are involved in essential cellular processes such as translation and pre-mRNA splicing. One class of RNP is the small Cajal body-specific RNP (scaRNP, which contributes to the biogenesis of small nuclear RNPs (snRNPs that are central components of the spliceosome. Three scaRNAs are internally processed, generating stable nucleolus-enriched RNAs of unknown function. Here, we provide data that show that these RNAs become part of RNPs we term regulatory RNPs (regRNPs. Most modifications within rRNA (predominantly pseudouridylation and ribose 2′-O-methylation are conducted by small nucleolar RNPs (snoRNPs, and we provide evidence that the activity of at least some of these snoRNPs is under the control of regRNPs. Because modifications within rRNA can vary in different physiological or pathological situations, rRNA modifications are thought to be the major source of ribosome heterogeneity. Our identification of regRNPs thus provides a potential mechanism for how ribosome heterogeneity may be accomplished. This work also provides additional functional connections between the Cajal body and the nucleolus.

Poly(A-Specific Ribonuclease Mediates 3′-End Trimming of Argonaute2-Cleaved Precursor MicroRNAs

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Mayuko Yoda

2013-11-01

Full Text Available MicroRNAs (miRNAs are typically generated as ∼22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago proteins to form an RNA-induced silencing complex (RISC. However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3′ arm of the pre-miR-451 precursor hairpin by Ago2. The 3′ end of the Ago2-cleaved pre-miR-451 intermediate is then trimmed to the mature length by an unknown nuclease. Here, using a classical chromatographic approach, we identified poly(A-specific ribonuclease (PARN as the enzyme responsible for the 3′–5′ exonucleolytic trimming of Ago2-cleaved pre-miR-451. Surprisingly, our data show that trimming of Ago2-cleaved precursor miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than the mature length. Our findings define the maturation step in the miRNA biogenesis pathway that depends on Ago2-mediated cleavage.

A Nuclear Role for miR-9 and Argonaute Proteins in Balancing Quiescent and Activated Neural Stem Cell States

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Shauna Katz

2016-10-01

Full Text Available Throughout life, adult neural stem cells (NSCs produce new neurons and glia that contribute to crucial brain functions. Quiescence is an essential protective feature of adult NSCs; however, the establishment and maintenance of this state remain poorly understood. We demonstrate that in the adult zebrafish pallium, the brain-enriched miR-9 is expressed exclusively in a subset of quiescent NSCs, highlighting a heterogeneity within these cells, and is necessary to maintain NSC quiescence. Strikingly, miR-9, along with Argonaute proteins (Agos, is localized to the nucleus of quiescent NSCs, and manipulating their nuclear/cytoplasmic ratio impacts quiescence. Mechanistically, miR-9 permits efficient Notch signaling to promote quiescence, and we identify the RISC protein TNRC6 as a mediator of miR-9/Agos nuclear localization in vivo. We propose a conserved non-canonical role for nuclear miR-9/Agos in controlling the balance between NSC quiescence and activation, a key step in maintaining adult germinal pools.

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

Science.gov (United States)

Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

2007-09-18

Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.

The co-chaperones Fkbp4/5 control Argonaute2 expression and facilitate RISC assembly.

Science.gov (United States)

Martinez, Natalia J; Chang, Hao-Ming; Borrajo, Jacob de Riba; Gregory, Richard I

2013-11-01

Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.

The developmental outcomes of P0-mediated ARGONAUTE destabilization in tomato.

Science.gov (United States)

Hendelman, Anat; Kravchik, Michael; Stav, Ran; Zik, Moriyah; Lugassi, Nitsan; Arazi, Tzahi

2013-01-01

The plant protein ARGONAUTE1 (AGO1) functions in multiple RNA-silencing pathways, including those of microRNAs, key regulators of growth and development. Genetic analysis of ago1 mutants with informative defects has provided valuable insights into AGO1's biological functions. Tomato encodes two AGO1 homologs (SlAGO1s), but mutants have not been described to date. To analyze SlAGO1s' involvement in development, we confirmed that both undergo decay in the presence of the Polerovirus silencing suppressor P0 and produce a transgenic responder line (OP:P0HA) that, upon transactivation, expresses P0 C-terminally fused to a hemagglutinin (HA) tag (P0HA) and destabilizes SlAGO1s at the site of expression. By crossing OP:P0HA with a battery of driver lines, constitutive as well as organ- and stage-specific SlAGO1 downregulation was induced in the F1 progeny. Activated plants exhibited various developmental phenotypes that partially overlapped with those of Arabidopsis ago1 mutants. Plants that constitutively expressed P0HA had reduced SlAGO1 levels and increased accumulation of miRNA targets, indicating compromised SlAGO1-mediated silencing. Consistent with this, they exhibited pleiotropic morphological defects and their growth was arrested post-germination. Transactivation of P0HA in young leaf and floral organ primordia dramatically modified corresponding organ morphology, including the radialization of leaflets, petals and anthers, suggesting that SlAGO1s' activities are required for normal lateral organ development and polarity. Overall, our results suggest that the OP:P0HA responder line can serve as a valuable tool to suppress SlAGO1 silencing pathways in tomato. The suppression of additional SlAGOs by P0HA and its contribution to the observed phenotypes awaits investigation.

Functional organization of the Sm core in the crystal structure of human U1 snRNP.

OpenAIRE

Weber, G.; Trowitzsch, S.; Kastner, B.; Lührmann, R.; Wahl, M.

2010-01-01

The U1 small nuclear ribonucleoprotein initiates the assembly of the spliceosome. Here, the structure of the natively purified U1 small nuclear ribonucleoprotein particle reveals the core Sm protein ring and its interactions with the Sm site in the small nuclear RNA.

Purification of ribonucleoproteins by a novel approach: isolation of the SSB1 ribonucleoprotein from yeast and demonstration that it has no role in mRNA splicing.

Science.gov (United States)

Cusick, M E

1992-12-29

A novel approach is described to purify potential ribonucleoproteins (RNP) of yeast. The method assays a yeast RNP complex, assembled in vitro on actin pre-mRNA, by low-ionic strength acrylamide gel electrophoresis. The minimal protein components of this RNP complex were three proteins, one of 30 kDa and two at 42-44 kDa, defined by formation of the complex on biotinylated-RNA, binding of this complex to avidin-agarose, and salt elution of the protein in the biotinylated-RNP complex. Using the assay for RNP complex formation, an RNP protein was purified to homogeneity on the basis of its affinity towards single-stranded DNA and RNA. This RNP protein turned out to be identical to a known RNP protein, the single-stranded binding protein 1 (ssb1) of yeast, on the basis of identical gel electrophoretic migration, antibody cross-reactivity, and identical properties on the gel complex formation assay. In vitro mRNA splicing was normal in extracts made from a yeast strain missing ssb1 (ssb1- strain). Addition of anti-ssb1 antibody to splicing extracts made from a wild type strain did not inhibit or diminish splicing. Instead, mRNA splicing was reproducibly stimulated several fold, indicating competition between ssb1 and splicing factors for binding to single-stranded RNA in the extracts. RNP complexes still formed in the ssb1- strain, demonstrating that it would be possible to purify other RNP proteins from this strain using the gel complex formation assay.

Evaluation of Triple Containment Method for Air Transport of Contaminated Human

National Research Council Canada - National Science Library

Neville, J

2003-01-01

A triple containment system intended for transport of biologically contaminated human remains was tested for its ability to maintain integrity during exposure to altitude changes representative of air transport...

Human luteinized granulosa cells secrete apoB100-containing lipoproteins

NARCIS (Netherlands)

Gautier, Thomas; Becker, Steffi; Drouineaud, Veronique; Menetrier, Franck; Sagot, Paul; Nofer, Jerzy-Roch; von Otte, Soeren; Lagrost, Laurent; Masson, David; Tietge, Uwe J. F.

Thus far, liver, intestine, heart, and placenta have been shown to secrete apolipoprotein (apo) B-containing lipoproteins. In the present study, we first investigated lipoproteins in human follicular fluid (FF), surrounding developing oocytes within the ovary, as well as in corresponding plasma

DNA double-strand braks serve as a major factor for the expression of Arabidopsis Argonaute 2

Energy Technology Data Exchange (ETDEWEB)

Lee, Sung Beom; Chung, Moon Soo; Lee, Gun Woong; Chung, Byung Yeoup [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2017-02-15

Argonaute 2 (AtAGO2) is a well characterized effector protein in Arabidopsis for its functionalities associated with DNA double-strand break (DSB)-induced small RNAs (diRNAs) and for its inducible expression upon γ-irradiation. However, its transcriptional regulation depending on the recovery time after the irradiation and on the specific response to DSBs has been poorly understood. We analyzed the 1,313 bp promoter sequence of the AtAGO2 gene (1.3kb{sub pro}) to characterize the transcriptional regulation of AtAGO2 at various recovery times after γ-irradiation. A stable transformant harboring 1.3kbpro fused with GUS gene showed that the AtAGO2 is highly expressed in response to γ-irradiation, after which the expression of the gene is gradually decreased until 5 days of DNA damage recovery. We also confrm that the AtAGO2 expression patterns are similar to that of γ-irradiation after the treatments of radiomimetic genotoxins (bleomycin and zeocin). However, methyl methanesulfonate and mitomycin C, which are associated with the inhibition of DNA replication, do not induce the expression of the AtAGO2, suggesting that the expression of the AtAGO2 is closely related with DNA DSBs rather than DNA replication.

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts.

Science.gov (United States)

Lisse, Thomas S; Vadivel, Kanagasabai; Bajaj, S Paul; Chun, Rene F; Hewison, Martin; Adams, John S

2014-01-01

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing. More recently hnRNP C has also been shown to function as a DNA binding protein exerting a dominant-negative effect on transcriptional responses to the vitamin D hormone,1,25-dihydroxyvitamin D (1,25(OH) 2 D), via interaction in cis with vitamin D response elements (VDREs). The physiologically active form of human hnRNPC is a tetramer of hnRNPC1 (huC1) and C2 (huC2) subunits known to be critical for specific RNA binding activity in vivo , yet the requirement for heterodimerization of huC1 and C2 in DNA binding and downstream action is not well understood. While over-expression of either huC1 or huC2 alone in mouse osteoblastic cells did not suppress 1,25(OH) 2 D-induced transcription, over-expression of huC1 and huC2 in combination using a bone-specific polycistronic vector successfully suppressed 1,25(OH) 2 D-mediated induction of osteoblast target gene expression. Over-expression of either huC1 or huC2 in human osteoblasts was sufficient to confer suppression of 1,25(OH) 2 D-mediated transcription, indicating the ability of transfected huC1 and huC2 to successfully engage as heterodimerization partners with endogenously expressed huC1 and huC2. The failure of the chimeric combination of mouse and human hnRNPCs to impair 1,25(OH) 2 D-driven gene expression in mouse cells was structurally predicted, owing to the absence of the last helix in the leucine zipper (LZ) heterodimerization domain of hnRNPC gene product in lower species, including the mouse. These results confirm that species-specific heterodimerization of hnRNPC1 and hnRNPC2 is a necessary prerequisite for DNA binding and down-regulation of 1,25(OH) 2 D-VDR-VDRE-directed gene transactivation in osteoblasts.

Myosin Va associates with mRNA in ribonucleoprotein particles present in myelinated peripheral axons and in the central nervous system.

Science.gov (United States)

Calliari, Aldo; Farías, Joaquina; Puppo, Agostina; Canclini, Lucía; Mercer, John A; Munroe, David; Sotelo, José R; Sotelo-Silveira, José R

2014-03-01

Sorting of specific mRNAs to particular cellular locations and regulation of their translation is an essential mechanism underlying cell polarization. The transport of RNAs by kinesins and dyneins has been clearly established in several cell models, including neurons in culture. A similar role appears to exist in higher eukaryotes for the myosins. Myosin Va (Myo5a) has been described as a component of ribonucleoprotein particles (RNPs) in the adult rat nervous system and associated to ZBP1 and ribosomes in ribosomal periaxoplasmic plaques (PARPs), making it a likely candidate for mediating some aspects of RNA transport in neurons. To test this hypothesis, we have characterized RNPs containing Myo5a in adult brains of rats and mice. Microarray analysis of RNAs co-immunoprecipitated with Myo5a indicates that this motor may associate with a specific subpopulation of neuronal mRNAs. We found mRNAs encoding α-synuclein and several proteins with functions in translation in these RNPs. Immunofluorescence analyses of RNPs showed apparent co-localization of Myo5a with ribosomes, mRNA and RNA-binding proteins in discrete structures present both in axons of neurons in culture and in myelinated fibers of medullary roots. Our data suggest that PARPs include RNPs bearing the mRNA coding for Myo5a and are equipped with kinesin and Myo5a molecular motors. In conclusion, we suggest that Myo5a is involved in mRNA trafficking both in the central and peripheral nervous systems. Copyright © 2013 Wiley Periodicals, Inc.

Biological role of copper and copper-containing proteins in human and animal organism

OpenAIRE

ANTONYAK H.L.; VAZHNENKO A.V.; PANAS N.E.

2011-01-01

Current scientific data related to copper metabolism and functional activity of Cu-containing proteins in human and animal cells are reviewed in the article. Important functional role of this essential element in human and animal organism is analyzed.

An association between RBMX, a heterogeneous nuclear ribonucleoprotein, and ARTS-1 regulates extracellular TNFR1 release

International Nuclear Information System (INIS)

Adamik, Barbara; Islam, Aminul; Rouhani, Farshid N.; Hawari, Feras I.; Zhang Jing; Levine, Stewart J.

2008-01-01

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1β-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1β-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

Science.gov (United States)

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

Science.gov (United States)

Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

2018-01-01

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Structural analysis of the complex between influenza B nucleoprotein and human importin-α.

Science.gov (United States)

Labaronne, Alice; Milles, Sigrid; Donchet, Amélie; Jensen, Malene Ringkjøbing; Blackledge, Martin; Bourhis, Jean-Marie; Ruigrok, Rob W H; Crépin, Thibaut

2017-12-07

Influenza viruses are negative strand RNA viruses that replicate in the nucleus of the cell. The viral nucleoprotein (NP) is the major component of the viral ribonucleoprotein. In this paper we show that the NP of influenza B has a long N-terminal tail of 70 residues with intrinsic flexibility. This tail contains the Nuclear Location Signal (NLS). The nuclear trafficking of the viral components mobilizes cellular import factors at different stages, making these host-pathogen interactions promising targets for new therapeutics. NP is imported into the nucleus by the importin-α/β pathway, through a direct interaction with importin-α isoforms. Here we provide a combined nuclear magnetic resonance and small-angle X-ray scattering (NMR/SAXS) analysis to describe the dynamics of the interaction between influenza B NP and the human importin-α. The NP of influenza B does not have a single NLS nor a bipartite NLS but our results suggest that the tail harbors several adjacent NLS sequences, located between residues 30 and 71.

Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents

International Nuclear Information System (INIS)

Albertsen, H.M.; Abderrahim, H.; Cann, H.M.; Dausset, J.; Le Paslier, D.; Cohen, D.

1990-01-01

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, the authors performed a series of preliminary experiments aimed at developing a suitable protocol. They found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes

Sensitivity evaluation of human factors for reliability of the containment spray system

International Nuclear Information System (INIS)

Tsujimura, Yasuhiro; Suzuki, Eiji

1988-01-01

Evaluation of the human reliability is one of the most difficult problems that deal with the safety and reliability of large systems, especially of the Engineered Safety Features (ESF) of the nuclear power plant. Influences of human factors on the reliability of the Containment Spray System in the ESF were estimated by using the FTA method in this paper. As a result, the adequacy of the system structure and the effects of human factors on variations of the design of the system structure were explained. (author)

A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein

Energy Technology Data Exchange (ETDEWEB)

Chang, So Young; Park, Ji Hoon [Respiratory Viruses Research Laboratory, Discovery Biology Department, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 Beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400 (Korea, Republic of); Kim, Young Ho; Kang, Jong Seong [College of Pharmacy, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Min, Ji-Young, E-mail: jiyoung.min@ip-korea.org [Respiratory Viruses Research Laboratory, Discovery Biology Department, Institut Pasteur Korea, 16, Daewangpangyo-ro 712 Beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, 463-400 (Korea, Republic of)

2016-03-04

The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action. - Highlights: • The plant-derived natural product TGBG has broad-spectrum antiviral activity against seasonal influenza A and B viruses. • TGBG has a novel anti-viral mechanism of action that from differs from the currently available anti-influenza drugs. • TGBG hinders nuclear export of the influenza virus ribonucleoprotein (v

A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein

International Nuclear Information System (INIS)

Chang, So Young; Park, Ji Hoon; Kim, Young Ho; Kang, Jong Seong; Min, Ji-Young

2016-01-01

The need to develop anti-influenza drugs with novel antiviral mechanisms is urgent because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. We identified a novel anti-influenza molecule by screening 861 plant-derived natural components using a high-throughput image-based assay that measures inhibition of the influenza virus infection. 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (TGBG) from Euphorbia humifusa Willd showed broad-spectrum anti-influenza activity against two seasonal influenza A strains, A/California/07/2009 (H1N1) and A/Perth/16/2009 (H3N2), and seasonal influenza B strain B/Florida/04/2006. We investigated the mode of action of TGBG using neuraminidase activity inhibition and time-of-addition assays, which evaluate the viral release and entry steps, respectively. We found that TGBG exhibits a novel antiviral mechanism that differs from the FDA-approved anti-influenza drugs oseltamivir which inhibits viral release, and amantadine which inhibits viral entry. Immunofluorescence assay demonstrated that TGBG significantly inhibits nuclear export of influenza nucleoproteins (NP) during the early stages of infection causing NP to accumulate in the nucleus. In addition, influenza-induced activation of the Akt signaling pathway was suppressed by TGBG in a dose-dependent manner. These data suggest that a putative mode of action of TGBG involves inhibition of viral ribonucleoprotein (vRNP) export from the nucleus to the cytoplasm consequently disrupting the assembly of progeny virions. In summary, TGBG has potential as novel anti-influenza therapeutic with a novel mechanism of action. - Highlights: • The plant-derived natural product TGBG has broad-spectrum antiviral activity against seasonal influenza A and B viruses. • TGBG has a novel anti-viral mechanism of action that from differs from the currently available anti-influenza drugs. • TGBG hinders nuclear export of the influenza virus ribonucleoprotein (v

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

OpenAIRE

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics ...

The Vasa Homolog RDE-12 engages target mRNA and multiple argonaute proteins to promote RNAi in C. elegans.

Science.gov (United States)

Shirayama, Masaki; Stanney, William; Gu, Weifeng; Seth, Meetu; Mello, Craig C

2014-04-14

Argonaute (AGO) proteins are key nuclease effectors of RNAi. Although purified AGOs can mediate a single round of target RNA cleavage in vitro, accessory factors are required for small interfering RNA (siRNA) loading and to achieve multiple-target turnover. To identify AGO cofactors, we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi, including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 colocalizes with WAGO-1 in germline P granules and in cytoplasmic and perinuclear foci in somatic cells. These findings and our genetic studies suggest that RDE-12 is first recruited to target mRNA by upstream AGOs (RDE-1 and ERGO-1), where it promotes small RNA amplification and/or WAGO-1 loading. Downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 and may thus have additional functions in target mRNA surveillance and silencing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Elucidating Mechanisms of Molecular Recognition Between Human Argonaute and miRNA Using Computational Approaches

KAUST Repository

Jiang, Hanlun; Zhu, Lizhe; Hé liou, Amé lie; Gao, Xin; Bernauer, Julie; Huang, Xuhui

2016-01-01

that are geometrically accessible to miRNA. Using our recent work on human AGO2 as an example, we explain the rationale and the workflow of our method in details. This combined approach holds great promise to complement experiments in unraveling the mechanisms

An Estimation of Human Error Probability of Filtered Containment Venting System Using Dynamic HRA Method

Energy Technology Data Exchange (ETDEWEB)

Jang, Seunghyun; Jae, Moosung [Hanyang University, Seoul (Korea, Republic of)

2016-10-15

The human failure events (HFEs) are considered in the development of system fault trees as well as accident sequence event trees in part of Probabilistic Safety Assessment (PSA). As a method for analyzing the human error, several methods, such as Technique for Human Error Rate Prediction (THERP), Human Cognitive Reliability (HCR), and Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) are used and new methods for human reliability analysis (HRA) are under developing at this time. This paper presents a dynamic HRA method for assessing the human failure events and estimation of human error probability for filtered containment venting system (FCVS) is performed. The action associated with implementation of the containment venting during a station blackout sequence is used as an example. In this report, dynamic HRA method was used to analyze FCVS-related operator action. The distributions of the required time and the available time were developed by MAAP code and LHS sampling. Though the numerical calculations given here are only for illustrative purpose, the dynamic HRA method can be useful tools to estimate the human error estimation and it can be applied to any kind of the operator actions, including the severe accident management strategy.

Mg2+ Effect on Argonaute and RNA Duplex by Molecular Dynamics and Bioinformatics Implications

Science.gov (United States)

Nam, Seungyoon; Ryu, Hyojung; Son, Won-joon; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Nephew, Kenneth P.; Lee, Jinhyuk

2014-01-01

RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg2+ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg2+] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg2+ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg2+ influx channel, TRPM7, and one miRNA’s (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg2+ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs. PMID:25330448

Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems.

Science.gov (United States)

Tsuboyama, Kotaro; Tadakuma, Hisashi; Tomari, Yukihide

2018-05-17

Loading of small RNAs into Argonaute, the core protein in RNA silencing, requires the Hsp70/Hsp90 chaperone machinery. This machinery also activates many other clients, including steroid hormone receptors and kinases, but how their structures change during chaperone-dependent activation remains unclear. Here, we utilized single-molecule Förster resonance energy transfer (smFRET) to probe the conformational changes of Drosophila Ago2 mediated by the chaperone machinery. We found that empty Ago2 exists in various closed conformations. The Hsp70 system (Hsp40 and Hsp70) and the Hsp90 system (Hop, Hsp90, and p23) together render Ago2 into an open, active form. The Hsp70 system, but not the Hsp90 system alone, is sufficient for Ago2 to partially populate the open form. Instead, the Hsp90 system is required to extend the dwell time of Ago2 in the open state, which must be transiently primed by the Hsp70 system. Our data uncover distinct and coordinated actions of the chaperone machinery, where the Hsp70 system expands the structural ensembles of Ago2 and the Hsp90 system captures and stabilizes the active form. Copyright © 2018 Elsevier Inc. All rights reserved.

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

International Nuclear Information System (INIS)

Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong; Webster, Keith A.

2010-01-01

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

Energy Technology Data Exchange (ETDEWEB)

Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong [Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, University of Miami Miller School of Medicine, Miami, FL 33136 (United States); Webster, Keith A., E-mail: kwebster@med.miami.edu [Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, University of Miami Miller School of Medicine, Miami, FL 33136 (United States)

2010-06-11

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

Study of miRNA Based Gene Regulation, Involved in Solid Cancer, by the Assistance of Argonaute Protein

Directory of Open Access Journals (Sweden)

Surya Narayan Rath

2016-09-01

Full Text Available Solid tumor is generally observed in tissues of epithelial or endothelial cells of lung, breast, prostate, pancreases, colorectal, stomach, and bladder, where several genes transcription is regulated by the microRNAs (miRNAs. Argonaute (AGO protein is a family of protein which assists in miRNAs to bind with mRNAs of the target genes. Hence, study of the binding mechanism between AGO protein and miRNAs, and also with miRNAs-mRNAs duplex is crucial for understanding the RNA silencing mechanism. In the current work, 64 genes and 23 miRNAs have been selected from literatures, whose deregulation is well established in seven types of solid cancer like lung, breast, prostate, pancreases, colorectal, stomach, and bladder cancer. In silico study reveals, miRNAs namely, miR-106a, miR-21, and miR-29b-2 have a strong binding affinity towards PTEN, TGFBR2, and VEGFA genes, respectively, suggested as important factors in RNA silencing mechanism. Furthermore, interaction between AGO protein (PDB ID-3F73, chain A with selected miRNAs and with miRNAs-mRNAs duplex were studied computationally to understand their binding at molecular level. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding miRNAs based gene silencing mechanism in solid cancer.

Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells.

Science.gov (United States)

Zhou, Rui; Park, Juw Won; Chun, Rene F; Lisse, Thomas S; Garcia, Alejandro J; Zavala, Kathryn; Sea, Jessica L; Lu, Zhi-Xiang; Xu, Jianzhong; Adams, John S; Xing, Yi; Hewison, Martin

2017-01-25

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH) 2 D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH) 2 D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH) 2 D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH) 2 D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH) 2 D-mediated induction of transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Systemic delivery of siRNA in pumpkin by a plant PHLOEM SMALL RNA-BINDING PROTEIN 1-ribonucleoprotein complex.

Science.gov (United States)

Ham, Byung-Kook; Li, Gang; Jia, Weitao; Leary, Julie A; Lucas, William J

2014-11-01

In plants, the vascular system, specifically the phloem, functions in delivery of small RNA (sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex (sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1-sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1-sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Laboratory containment practices for arthropod vectors of human and animal pathogens.

Science.gov (United States)

Tabachnick, Walter J

2006-03-01

Arthropod-borne pathogens have an impact on the health and well-being of humans and animals throughout the world. Research involving arthropod vectors of disease is often dependent on the ability to maintain the specific arthropod species in laboratory colonies. The author reviews current arthropod containment practices and discusses their importance from public health and ecological perspectives.

Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype

International Nuclear Information System (INIS)

Mendlovic, S.; Brocke, S.; Meshorer, A.; Mozes, E.; Shoenfeld, Y.; Bakimer, R.; Ben-Bassat, M.

1988-01-01

Systemic lupus erythematosus (SLE) is considered to be the quintessential autoimmune disease. It has not been possible to induce SLE in animal models by DNA immunization or by challenge with anti-DNA antibodies. The authors report a murine model of SLE-like disease induced by immunization of C3H.SW female mice with a common human monoclonal anti-DNA idiotype (16/6 idiotype). Following a booster injection with the 16/6 idiotype, high levels of murine anti-16/6 and anti-anti-16/6 antibodies (associated with anti-DNA activity) were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with DNA, poly(I), poly(dT), ribonucleoprotein, autoantigens [Sm, SS-A (Ro), and SS-B (La)], and cardiolipin were noted. The serological findings were associated with increased erythrocyte sedimentation rate, leukopenia, proteinuria, immune complex deposition in the glomerular mesangium, and sclerosis of the glomeruli. The immune complexes in the kidneys were shown to contain the 16/6 idiotype. This experimental SLE-like model may be used to elucidate the mechanisms underlying SLE

Normal RNAi response in human fragile x fibroblasts

DEFF Research Database (Denmark)

Madsen, Charlotte; Grønskov, Karen; Brøndum-Nielsen, Karen

2009-01-01

BACKGROUND: Fragile x syndrome is caused by loss of expression of the FMRP protein involved in the control of a large number of mRNA targets. The Drosophila ortholog dFXR interacts with a protein complex that includes Argonaute2, an essential component of the RNA-induced silencing complex (RISC...

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

Science.gov (United States)

Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

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Laura Pitzonka

Full Text Available Co-transcriptionally assembled ribonucleoprotein (RNP complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes.

Science.gov (United States)

Taoka, Masato; Yamauchi, Yoshio; Nobe, Yuko; Masaki, Shunpei; Nakayama, Hiroshi; Ishikawa, Hideaki; Takahashi, Nobuhiro; Isobe, Toshiaki

2009-11-01

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a approximately 21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes.

The human cap-binding complex is functionally connected to the nuclear RNA exosome

DEFF Research Database (Denmark)

Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken Søndergaard

2013-01-01

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis...

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

Science.gov (United States)

Kamina, Anyango D; Williams, Noreen

2017-01-01

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

Impact of consumption of oligosaccharide-containing biscuits on the fecal microbiota of humans

NARCIS (Netherlands)

Tannock, G.W.; Munro, K.; Bibiloni, R.; Simon, M.A.; Hargreaves, P.; Gopal, P.; Harmsen, H.J.M.; Welling, Gjalt

Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups,

Human-like PB2 627K influenza virus polymerase activity is regulated by importin-α1 and -α7.

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Ben Hudjetz

2012-01-01

Full Text Available Influenza A viruses may cross species barriers and transmit to humans with the potential to cause pandemics. Interplay of human- (PB2 627K and avian-like (PB2 627E influenza polymerase complexes with unknown host factors have been postulated to play a key role in interspecies transmission. Here, we have identified human importin-α isoforms (α1 and α7 as positive regulators of human- but not avian-like polymerase activity. Human-like polymerase activity correlated with efficient recruitment of α1 and α7 to viral ribonucleoprotein complexes (vRNPs without affecting subcellular localization. We also observed that human-like influenza virus growth was impaired in α1 and α7 downregulated human lung cells. Mice lacking α7 were less susceptible to human- but not avian-like influenza virus infection. Thus, α1 and α7 are positive regulators of human-like polymerase activity and pathogenicity beyond their role in nuclear transport.

Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins.

Science.gov (United States)

Liang, Zhen; Chen, Kunling; Zhang, Yi; Liu, Jinxing; Yin, Kangquan; Qiu, Jin-Long; Gao, Caixia

2018-03-01

This protocol is an extension to: Nat. Protoc. 9, 2395-2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014In recent years, CRISPR/Cas9 has emerged as a powerful tool for improving crop traits. Conventional plant genome editing mainly relies on plasmid-carrying cassettes delivered by Agrobacterium or particle bombardment. Here, we describe DNA-free editing of bread wheat by delivering in vitro transcripts (IVTs) or ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment. This protocol serves as an extension of our previously published protocol on genome editing in bread wheat using CRISPR/Cas9 plasmids delivered by particle bombardment. The methods we describe not only eliminate random integration of CRISPR/Cas9 into genomic DNA, but also reduce off-target effects. In this protocol extension article, we present detailed protocols for preparation of IVTs and RNPs; validation by PCR/restriction enzyme (RE) and next-generation sequencing; delivery by biolistics; and recovery of mutants and identification of mutants by pooling methods and Sanger sequencing. To use these protocols, researchers should have basic skills and experience in molecular biology and biolistic transformation. By using these protocols, plants edited without the use of any foreign DNA can be generated and identified within 9-11 weeks.

Functional organization of the Sm core in the crystal structure of human U1 snRNP.

Science.gov (United States)

Weber, Gert; Trowitzsch, Simon; Kastner, Berthold; Lührmann, Reinhard; Wahl, Markus C

2010-12-15

U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5'-splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B', and extended internal loops in D2 and B/B' support a four-way RNA junction and a 3'-terminal stem-loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1-specific 70K protein. The intricate, multi-layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo.

Membranous glomerulonephritis in a patient with anti-u1 ribonucleoprotein (RNP antibody-positive mixed connective tissue disease: A case report

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Naoya Toriu

2018-03-01

Full Text Available We report a 33-year-old Japanese man diagnosed with mixed connective tissue disease (MCTD who developed nephrotic proteinuria. Both speckled antinuclear antibody (ANA and anti-U1 ribonucleoprotein (RNP antibody were positive, but anti-double-stranded DNA (dsDNA antibody and anti-Smith (Sm antibody were negative, while complement levels were normal. Renal biopsy revealed membranous glomerulonephritis (MGN with diffuse thickening of the glomerular basement membrane (GBM plus spike and bubble formation. Immunofluorescence demonstrated granular deposits of IgG and C3 along the GBM. Analysis of IgG subclasses showed predominant deposition of IgG1 and IgG4, unlike typical lupus nephritis in which there is predominant deposition of IgG1, IgG2, IgG3, and C1q. Electron microscopy identified numerous large electron-dense deposits (EDD of various types in the subepithelial region of the GBM, but there were no EDD localized in the mesangium or subendothelium. Based on these findings, MGN was considered to be closely related to MCTD in this patient.

A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

Science.gov (United States)

Wang, Dai; Parrish, Colin R.

1999-01-01

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5′ end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5′ end viral genome did not affect replication of the virus. PMID:10438866

In vitro reconstitution of chaperone-mediated human RISC assembly.

Science.gov (United States)

Naruse, Ken; Matsuura-Suzuki, Eriko; Watanabe, Mariko; Iwasaki, Shintaro; Tomari, Yukihide

2018-01-01

To silence target mRNAs, small RNAs and Argonaute (Ago) proteins need to be assembled into RNA-induced silencing complexes (RISCs). Although the assembly of Drosophila melanogaster RISC was recently reconstituted by Ago2, the Dicer-2/R2D2 heterodimer, and five chaperone proteins, the absence of a reconstitution system for mammalian RISC assembly has posed analytical challenges. Here we describe reconstitution of human RISC assembly using Ago2 and five recombinant chaperone proteins: Hsp90β, Hsc70, Hop, Dnaja2, and p23. Our data show that ATP hydrolysis by both Hsp90β and Hsc70 is required for RISC assembly of small RNA duplexes but not for that of single-stranded RNAs. The reconstitution system lays the groundwork for further studies of small RNA-mediated gene silencing in mammals. © 2018 Naruse et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Selection of LNA-containing DNA aptamers against recombinant human CD73

DEFF Research Database (Denmark)

Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G

2015-01-01

tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences......LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX...... with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several...

Centromere Protein (CENP)-W Interacts with Heterogeneous Nuclear Ribonucleoprotein (hnRNP) U and May Contribute to Kinetochore-Microtubule Attachment in Mitotic Cells

Science.gov (United States)

Chun, Younghwa; Kim, Raehyung; Lee, Soojin

2016-01-01

Background Recent studies have shown that heterogeneous nuclear ribonucleoprotein U (hnRNP U), a component of the hnRNP complex, contributes to stabilize the kinetochore-microtubule interaction during mitosis. CENP-W was identified as an inner centromere component that plays crucial roles in the formation of a functional kinetochore complex. Results We report that hnRNP U interacts with CENP-W, and the interaction between hnRNP U and CENP-W mutually increased each other’s protein stability by inhibiting the proteasome-mediated degradation. Further, their co-localization was observed chiefly in the nuclear matrix region and at the microtubule-kinetochore interface during interphase and mitosis, respectively. Both microtubule-stabilizing and microtubule-destabilizing agents significantly decreased the protein stability of CENP-W. Furthermore, loss of microtubules and defects in microtubule organization were observed in CENP-W-depleted cells. Conclusion Our data imply that CENP-W plays an important role in the attachment and interaction between microtubules and kinetochore during mitosis. PMID:26881882

Effects of national culture on human failures in container shipping: the moderating role of Confucian dynamism.

Science.gov (United States)

Lu, Chin-Shan; Lai, Kee-hung; Lun, Y H Venus; Cheng, T C E

2012-11-01

Recent reports on work safety in container shipping operations highlight high frequencies of human failures. In this study, we empirically examine the effects of seafarers' perceptions of national culture on the occurrence of human failures affecting work safety in shipping operations. We develop a model adopting Hofstede's national culture construct, which comprises five dimensions, namely power distance, collectivism/individualism, uncertainty avoidance, masculinity/femininity, and Confucian dynamism. We then formulate research hypotheses from theory and test the hypotheses using survey data collected from 608 seafarers who work on global container carriers. Using a point scale for evaluating seafarers' perception of the five national culture dimensions, we find that Filipino seafarers score highest on collectivism, whereas Chinese and Taiwanese seafarers score highest on Confucian dynamism, followed by collectivism, masculinity, power distance, and uncertainty avoidance. The results also indicate that Taiwanese seafarers have a propensity for uncertainty avoidance and masculinity, whereas Filipino seafarers lean more towards power distance, masculinity, and collectivism, which are consistent with the findings of Hofstede and Bond (1988). The results suggest that there will be fewer human failures in container shipping operations when power distance is low, and collectivism and uncertainty avoidance are high. Specifically, this study finds that Confucian dynamism plays an important moderating role as it affects the strength of associations between some national culture dimensions and human failures. Finally, we discuss our findings' contribution to the development of national culture theory and their managerial implications for reducing the occurrence of human failures in shipping operations. Copyright © 2012 Elsevier Ltd. All rights reserved.

Overexpression of heterogeneous nuclear ribonucleoprotein F stimulates renal Ace-2 gene expression and prevents TGF-β1-induced kidney injury in a mouse model of diabetes.

Science.gov (United States)

Lo, Chao-Sheng; Shi, Yixuan; Chang, Shiao-Ying; Abdo, Shaaban; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao-Ling; Chan, John S D

2015-10-01

We investigated whether heterogeneous nuclear ribonucleoprotein F (hnRNP F) stimulates renal ACE-2 expression and prevents TGF-β1 signalling, TGF-β1 inhibition of Ace-2 gene expression and induction of tubulo-fibrosis in an Akita mouse model of type 1 diabetes. Adult male Akita transgenic (Tg) mice overexpressing specifically hnRNP F in their renal proximal tubular cells (RPTCs) were studied. Non-Akita littermates and Akita mice served as controls. Immortalised rat RPTCs stably transfected with plasmid containing either rat Hnrnpf cDNA or rat Ace-2 gene promoter were also studied. Overexpression of hnRNP F attenuated systemic hypertension, glomerular filtration rate, albumin/creatinine ratio, urinary angiotensinogen (AGT) and angiotensin (Ang) II levels, renal fibrosis and profibrotic gene (Agt, Tgf-β1, TGF-β receptor II [Tgf-βrII]) expression, stimulated anti-profibrotic gene (Ace-2 and Ang 1-7 receptor [MasR]) expression, and normalised urinary Ang 1-7 level in Akita Hnrnpf-Tg mice as compared with Akita mice. In vitro, hnRNP F overexpression stimulated Ace-2 gene promoter activity, mRNA and protein expression, and attenuated Agt, Tgf-β1 and Tgf-βrII gene expression. Furthermore, hnRNP F overexpression prevented TGF-β1 signalling and TGF-β1 inhibition of Ace-2 gene expression. These data demonstrate that hnRNP F stimulates Ace-2 gene transcription, prevents TGF-β1 inhibition of Ace-2 gene transcription and induction of kidney injury in diabetes. HnRNP F may be a potential target for treating hypertension and renal fibrosis in diabetes.

Organelle-specific injury to melanin-containing cells in human skin by pulsed laser irradiation

Energy Technology Data Exchange (ETDEWEB)

Murphy, G.F.; Shepard, R.S.; Paul, B.S.; Menkes, A.; Anderson, R.R.; Parrish, J.A.

1983-12-01

Physical models predict that ultraviolet laser radiation of appropriately brief pulses can selectively alter melanin-containing cellular targets in human skin. Skin of normal human volunteers was exposed to brief (20 nanosecond) 351-nm wave length pulses from a XeF excimer laser, predicting that those cells containing the greatest quantities of melanized melanosomes (lower half of the epidermis) would be selectively damaged. Transmission electron microscopy revealed the earliest cellular alteration to be immediate disruption of melanosomes, both within melanocytes and basal keratinocytes. This disruption was dose dependent and culminated in striking degenerative changes in these cells. Superficial keratinocytes and Langerhans cells were not affected. It was concluded that the XeF excimer laser is capable of organelle-specific injury to melanosomes. These findings may have important clinical implications in the treatment of both benign and malignant pigmented lesions by laser radiations of defined wave lengths and pulse durations.

No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

DEFF Research Database (Denmark)

Kaspersen, Maja Døvling; Bungum, Mona; Fedder, Jens

2013-01-01

It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR......-based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses....

Serum amyloid A is found on ApoB-containing lipoproteins in obese humans with diabetes.

Science.gov (United States)

Jahangiri, Anisa; Wilson, Patricia G; Hou, Tianfei; Brown, Aparna; King, Victoria L; Tannock, Lisa R

2013-05-01

In murine models of obesity/diabetes, there is an increase in plasma serum amyloid A (SAA) levels along with redistribution of SAA from high-density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoprotein particles, namely, low-density lipoprotein and very low-density lipoprotein. The goal of this study was to determine if obesity is associated with similar SAA lipoprotein redistribution in humans. Three groups of obese individuals were recruited from a weight loss clinic: healthy obese (n = 14), metabolic syndrome (MetS) obese (n = 8), and obese with type 2 diabetes (n = 6). Plasma was separated into lipoprotein fractions by fast protein liquid chromatography, and SAA was measured in lipid fractions using enzyme-linked immunosorbent assay and Western blotting. Only the obese diabetic group had SAA detectable in apoB-containing lipoproteins, and SAA reverted back to HDL with active weight loss. In human subjects, SAA is found in apoB-containing lipoprotein particles only in obese subjects with type 2 diabetes, but not in healthy obese or obese subjects with MetS. Copyright © 2012 The Obesity Society.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

Science.gov (United States)

Meccariello, Angela; Monti, Simona Maria; Romanelli, Alessandra; Colonna, Rita; Primo, Pasquale; Inghilterra, Maria Grazia; Del Corsano, Giuseppe; Ramaglia, Antonio; Iazzetti, Giovanni; Chiarore, Antonia; Patti, Francesco; Heinze, Svenia D; Salvemini, Marco; Lindsay, Helen; Chiavacci, Elena; Burger, Alexa; Robinson, Mark D; Mosimann, Christian; Bopp, Daniel; Saccone, Giuseppe

2017-08-30

The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

The putative Leishmania telomerase RNA (LeishTER undergoes trans-splicing and contains a conserved template sequence.

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Elton J R Vasconcelos

Full Text Available Telomerase RNAs (TERs are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER that contains a 5' spliced leader (SL cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs and its role in parasite telomere biology.

Acute effects of interleukin-6 infusion on apo-B-containing lipoprotein subclasses in humans

DEFF Research Database (Denmark)

Bagdade, John; Pedersen, Bente K; Schwenke, Dawn

2011-01-01

B:E) apoB-containing subclasses present in VLDL. Therefore, we have directly measured these subclasses following their isolation by sequential immunoprecipitation in seven healthy male subjects during a 3-h infusion with recombinant human (rh) IL-6. Though plasma TG and apoB-containing particle number were...... unchanged by IL-6, the distribution of TG-rich subclasses was significantly altered. Compared to baseline values, LpB:E + LpB:C:E increased significantly at 0.5 h (p infused controls at 0.5 and 1 h (p

Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

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Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

2015-12-10

D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. Copyright © 2015 Elsevier B.V. All rights reserved.

Hepatitis C virus RNA functionally sequesters miR-122

DEFF Research Database (Denmark)

Luna, Joseph M; Scheel, Troels K H; Danino, Tal

2015-01-01

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during...

Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling.

Science.gov (United States)

Iwaki, Aya; Ohnuki, Shinsuke; Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

2013-01-01

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

Science.gov (United States)

Pakdeepak, Kanet; Pata, Supansa; Chiampanichayakul, Sawitree; Kasinrerk, Watchara; Tatu, Thanusak

2016-01-01

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Science.gov (United States)

Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

2016-06-10

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses*

Science.gov (United States)

Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Mickum, Megan L.; Ashline, David J.; Prasad, B. V. Venkataram; Estes, Mary K.; Reinhold, Vernon N.; Cummings, Richard D.; Smith, David F.

2014-01-01

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures. PMID:25048705

Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs.

Directory of Open Access Journals (Sweden)

Hsiao-Ling Lu

Full Text Available Piwi-interacting RNAs (piRNAs are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.

9 CFR 381.15 - Exemption from definition of “poultry product” of certain human food products containing poultry.

Science.gov (United States)

2010-01-01

... productâ of certain human food products containing poultry. 381.15 Section 381.15 Animals and Animal...; MANDATORY MEAT AND POULTRY PRODUCTS INSPECTION AND VOLUNTARY INSPECTION AND CERTIFICATION POULTRY PRODUCTS INSPECTION REGULATIONS Exemptions § 381.15 Exemption from definition of “poultry product” of certain human...

Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

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Begoña Barriuso

2016-06-01

Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

Photocytotoxicity in human dermal fibroblasts elicited by permanent makeup inks containing titanium dioxide.

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Wamer, Wayne G; Yin, Jun-Jie

2011-01-01

Titanium dioxide (TiO2) is a pigment widely used in decorative tattoo and permanent makeup inks. However, little is known about the risks associated with its presence in these products. We have developed an in vitro assay to identify inks containing TiO2 that are cytotoxic and/or photocytotoxic. The presence of TiO2 in ten permanent makeup inks was established by X-ray fluorescence. Using X-ray diffraction, we found that seven inks contained predominately TiO2 (anatase), the more photocatalytically active crystalline form of TiO2. The remaining inks contained predominately TiO2 (rutile). To identify cytotoxic and/or photocytotoxic inks, human dermal fibroblasts were incubated for 18 h in media containing inks or pigments isolated from inks. Fibroblasts were then irradiated with 10 J/cm2 UVA radiation combined with 45 J/cm2 visible light for determining photocytotoxicity, or kept in the dark for determining cytotoxicity. Toxicity was assessed as inhibition of colony formation. No inks were cytotoxic. However eight inks, and the pigments isolated from these inks, were photocytotoxic. Using ESR, we found that most pigments from photocytotoxic inks generated hydroxyl radicals when photoexcited with UV radiation. Therefore, the possibility of photocytotoxicity should be considered when evaluating the safety of permanent makeup inks containing TiO2.

Transcriptional regulation of Arabidopsis MIR168a and argonaute1 homeostasis in abscisic acid and abiotic stress responses.

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Li, Wei; Cui, Xiao; Meng, Zhaolu; Huang, Xiahe; Xie, Qi; Wu, Heng; Jin, Hailing; Zhang, Dabing; Liang, Wanqi

2012-03-01

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants.

Human tears contain a chemosignal.

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Gelstein, Shani; Yeshurun, Yaara; Rozenkrantz, Liron; Shushan, Sagit; Frumin, Idan; Roth, Yehudah; Sobel, Noam

2011-01-14

Emotional tearing is a poorly understood behavior that is considered uniquely human. In mice, tears serve as a chemosignal. We therefore hypothesized that human tears may similarly serve a chemosignaling function. We found that merely sniffing negative-emotion-related odorless tears obtained from women donors induced reductions in sexual appeal attributed by men to pictures of women's faces. Moreover, after sniffing such tears, men experienced reduced self-rated sexual arousal, reduced physiological measures of arousal, and reduced levels of testosterone. Finally, functional magnetic resonance imaging revealed that sniffing women's tears selectively reduced activity in brain substrates of sexual arousal in men.

Science, practice, and human errors in controlling Clostridium botulinum in heat-preserved food in hermetic containers.

Science.gov (United States)

Pflug, Irving J

2010-05-01

The incidence of botulism in canned food in the last century is reviewed along with the background science; a few conclusions are reached based on analysis of published data. There are two primary aspects to botulism control: the design of an adequate process and the delivery of the adequate process to containers of food. The probability that the designed process will not be adequate to control Clostridium botulinum is very small, probably less than 1.0 x 10(-6), based on containers of food, whereas the failure of the operator of the processing equipment to deliver the specified process to containers of food may be of the order of 1 in 40, to 1 in 100, based on processing units (retort loads). In the commercial food canning industry, failure to deliver the process will probably be of the order of 1.0 x 10(-4) to 1.0 x 10(-6) when U.S. Food and Drug Administration (FDA) regulations are followed. Botulism incidents have occurred in food canning plants that have not followed the FDA regulations. It is possible but very rare to have botulism result from postprocessing contamination. It may thus be concluded that botulism incidents in canned food are primarily the result of human failure in the delivery of the designed or specified process to containers of food that, in turn, result in the survival, outgrowth, and toxin production of C. botulinum spores. Therefore, efforts in C. botulinum control should be concentrated on reducing human errors in the delivery of the specified process to containers of food.

Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers.

Science.gov (United States)

Shimizu, Makiko; Shiraishi, Arisa; Sato, Ayumi; Nagashima, Satomi; Yamazaki, Hiroshi

2015-02-01

Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 μM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 μM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0-50 μM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3. Copyright © 2014 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

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Liu, Hua; Nonomura, Ken-Ichi

2016-10-01

The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis. © 2016. Published by The Company of Biologists Ltd.

Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

International Nuclear Information System (INIS)

Kapp, L.N.; Painter, R.B.

1989-01-01

SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

Permeation studies of novel terbinafine formulations containing hydrophobins through human nails in vitro.

Science.gov (United States)

Vejnovic, Ivana; Huonder, Cornelia; Betz, Gabriele

2010-09-15

Existing treatments of onychomycosis are not satisfactory. Oral therapies have many side effects and topical formulations are not able to penetrate into the human nail plate and deliver therapeutical concentrations of active agent in situ. The purpose of the present study was to determine the amount of terbinafine, which permeates through the human nail plate, from liquid formulations containing enhancers, namely hydrophobins A-C in the concentration of 0.1% (w/v). The used reference solution contained 10% (w/v) of terbinafine in 60% (v/v) ethanol/water without enhancer. Permeability studies have been performed on cadaver nails using Franz diffusion cells modified to mount nail plates and filled with 60% (v/v) ethanol/water in the acceptor chamber. Terbinafine was quantitatively determined by HPLC. The amount of terbinafine remaining in the nail was extracted by 96% ethanol from pulverized nail material after permeation experiment and presented as percentage of the dry nail weight before the milling test. Permeability coefficient (PC) of terbinafine from reference solution was determined to be 1.52E-10 cm/s. Addition of hydrophobins improved PC in the range of 3E-10 to 2E-9 cm/s. Remaining terbinafine reservoir in the nail from reference solution was 0.83% (n=2). An increase of remaining terbinafine reservoir in the nail was observed in two out of three tested formulations containing hydrophobins compared to the reference. In all cases, known minimum inhibitory concentration of terbinafine for dermatophytes (0.003 microg/ml) has been exceeded in the acceptor chamber of the diffusion cells. All tested proteins (hydrophobins) facilitated terbinafine permeation after 10 days of permeation experiment, however one of them achieved an outstanding enhancement factor of 13.05 compared to the reference. Therefore, hydrophobins can be included in the list of potential enhancers for treatment of onychomycosis. Copyright 2010 Elsevier B.V. All rights reserved.

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

Science.gov (United States)

Rust, K; Grosso, L; Zhang, V; Chang, D; Persson, A; Longmore, W; Cai, G Z; Crouch, E

1991-10-01

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.

Science.gov (United States)

Park, June Hyun; Shin, Chanseok

2015-10-30

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1-4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that 'slicer-dependent unwinding' via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that 'slicer-independent unwinding' plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that 'slicer-dependency' for the unwinding was affected primarily by certain parameters such as temperature and Mg(2+). We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

International Nuclear Information System (INIS)

Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

2008-01-01

The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus

Directory of Open Access Journals (Sweden)

Zongyang Mou

2015-11-01

Full Text Available Brain-derived neurotrophic factor (BDNF plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001 and greater adiposity in both adult and pediatric cohorts (p values < 0.05. We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

Science.gov (United States)

Flores, Omar; Kennedy, Edward M; Skalsky, Rebecca L; Cullen, Bryan R

2014-04-01

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

Characterization of human papillomavirus type 16 pseudovirus containing histones.

Science.gov (United States)

Kim, Hyoung Jin; Kwag, Hye-Lim; Kim, Hong-Jin

2016-08-27

Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities. The amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity. Our findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

Science.gov (United States)

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

Science.gov (United States)

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

B-CELL EPITOPE ON THE U1 SNRNP-C AUTOANTIGEN CONTAINS A SEQUENCE SIMILAR TO THAT OF THE HERPES-SIMPLEX VIRUS PROTEIN

NARCIS (Netherlands)

MISAKI, Y; YAMAMOTO, K; YANAGI, K; MIURA, H; ICHIJO, H; KATO, T; MATO, T; WELLINGWESTER, S; NISHIOKA, K; ITO, K

The mechanism of autoantibody production in autoimmune diseases is not well understood. In the present study we performed the B cell epitope mapping of the U1 small nuclear ribonucleoprotein (snRNP)-C, one of the target molecules of anti-nRNP autoantibody to investigate how B cells respond to the

quatre-quart1 is an indispensable U12 intron-containing gene that plays a crucial role in Arabidopsis development.

Science.gov (United States)

Kwak, Kyung Jin; Kim, Bo Mi; Lee, Kwanuk; Kang, Hunseung

2017-05-17

Despite increasing understanding of the importance of the splicing of U12-type introns in plant development, the key question of which U12 intron-containing genes are essential for plant development has not yet been explored. Here, we assessed the functional role of the quatre-quart1 (QQT1) gene, one of the ~230 U12 intron-containing genes in Arabidopsis thaliana. Expression of QQT1 in the U11/U12-31K small nuclear ribonucleoprotein mutant (31k) rescued the developmental-defect phenotypes of the 31k mutant, whereas the miRNA-mediated qqt1 knockdown mutants displayed severe defects in growth and development, including severely arrested stem growth, small size, and the formation of serrated leaves. The structures of the shoot apical meristems in the qqt1 mutants were abnormal and disordered. Identification of QQT1-interacting proteins via a yeast two-hybrid screening and a firefly luciferase complementation-imaging assay revealed that a variety of proteins, including many chloroplast-targeted proteins, interacted with QQT1. Importantly, the levels of chloroplast-targeted proteins in the chloroplast were reduced, and the chloroplast structure was abnormal in the qqt1 mutant. Collectively, these results provide clear evidence that QQT1 is an indispensable U12 intron-containing gene whose correct splicing is crucial for the normal development of Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Alterations in polyribosome and messenger ribonucleic acid metabolism and messenger ribonucleoprotein utilization in osmotically stressed plant seedlings

International Nuclear Information System (INIS)

Mason, H.S.

1986-01-01

Polyribosome aggregation state in growing tissues of barley and wheat leaf of stems of pea and squash was studied in relation to seedling growth and water status of the growing tissue in plants at various levels of osmotic stress. It was found to be highly correlated with water potential and osmotic potential of the growing tissue and with leaf of stem elongation rate. Stress rapidly reduced polyribosome content and water status in growing tissues of barley leaves; changes were slow and slight in the non-growing leaf blade. Membrane-bound and free polyribosomes were equally sensitive to stress-induced disaggregation. Incorporation of 32 PO 4 3- into ribosomal RNA was rapidly inhibited by stress, but stability of poly(A) + RNA relative to ribosomal RNA was similar in stressed and unstressed tissues, with a half-life of about 12 hours. Stress also caused progressive loss of poly(A) + RNA from these tissues. Quantitation of poly(A) and in vitro messenger template activity in polysome gradient fractions showed a shift of activity from the polysomal region to the region of 20-60 S in stressed plants. Messenger RNA in the 20-60 S region coded for the same peptides as mRNA found in the polysomal fraction. Nonpolysomal and polysome-derived messenger ribonucleoprotein complexes (mRNP) were isolated, and characteristic proteins were found associated with either fraction. Polysomal mRNP from stressed or unstressed plants were translated with similar efficiency in a wheat germ cell-free system. It was concluded that no translational inhibitory activity was associated with nonpolysomal mRNP from barley prepared as described

Protein Kinase C-{delta} mediates down-regulation of heterogeneous nuclear ribonucleoprotein K protein: involvement in apoptosis induction

Energy Technology Data Exchange (ETDEWEB)

Gao, Feng-Hou [NO.3 People' s Hospital affiliated to Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 201900 (China); The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Wu, Ying-Li [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Zhao, Meng [Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China); Liu, Chuan-Xu; Wang, Li-Shun [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Chen, Guo-Qiang, E-mail: chengq@shsmu.edu.cn [The Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025 (China); Institute of Health Science, SJTU-SM/Shanghai Institutes for Biological Science, Chinese Academy of Sciences, Shanghai (China)

2009-11-15

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta ({Delta}PKC-{delta}). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the {Delta}PKC-{delta}, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that {Delta}PKC-{delta} mediated the down-regulation of hnRNP K protein during apoptosis: PKC-{delta} inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-{delta}-deficient apoptotic KG1a cells; conditional induction of {Delta}PKC-{delta} in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of {Delta}PKC-{delta}. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-{delta} down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

Revisiting spatial distribution and biochemical composition of calcium-containing crystals in human osteoarthritic articular cartilage.

OpenAIRE

Nguyen, C.; Bazin, D.; Daudon, M.; Chatron-Colliet, A.; Hannouche, D.; Bianchi, A.; Côme, D.; So, A.; Busso, N.; Lioté, F.; Ea, H.K.

2013-01-01

International audience; INTRODUCTION: Calcium-containing (CaC) crystals, including basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate (CPP), are associated with destructive forms of osteoarthritis (OA). We assessed their distribution and biochemical and morphologic features in human knee OA cartilage. METHODS: We prospectively included 20 patients who underwent total knee replacement (TKR) for primary OA. CaC crystal characterization and identification involved Fourier-transfor...

Transcriptional Regulation of Arabidopsis MIR168a and ARGONAUTE1 Homeostasis in Abscisic Acid and Abiotic Stress Responses1[W

Science.gov (United States)

Li, Wei; Cui, Xiao; Meng, Zhaolu; Huang, Xiahe; Xie, Qi; Wu, Heng; Jin, Hailing; Zhang, Dabing; Liang, Wanqi

2012-01-01

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants. PMID:22247272

PRMT1 methylates the single Argonaute of Toxoplasma gondii and is important for the recruitment of Tudor nuclease for target RNA cleavage by antisense guide RNA

Science.gov (United States)

Musiyenko, Alla; Majumdar, Tanmay; Andrews, Joel; Adams, Brian; Barik, Sailen

2013-01-01

Summary Argonaute (Ago) plays a central role in RNA interference in metazoans, but its status in lower organisms remains ill-defined. We report on the Ago complex of the unicellular protozoan, Toxoplasma gondii (Tg), an obligatory pathogen of mammalian hosts. The PIWI-like domain of TgAgo lacked the canonical DDE/H catalytic triad, explaining its weak target RNA cleavage activity. However, TgAgo associated with a stronger RNA slicer, a Tudor staphylococcal nuclease (TSN), and with a protein Arg methyl transferase, PRMT1. Mutational analysis suggested that the N-terminal RGG-repeat domain of TgAgo was methylated by PRMT1, correlating with the recruitment of TSN. The slicer activity of TgAgo was Mg2+-dependent and required perfect complementarity between the guide RNA and the target. In contrast, the TSN activity was Ca2+-dependent and required an imperfectly paired guide RNA. Ago knockout parasites showed essentially normal growth, but in contrast, the PRMT1 knockouts grew abnormally. Chemical inhibition of Arg-methylation also had an anti-parasitic effect. These results suggest that the parasitic PRMT1 plays multiple roles, and its loss affects the recruitment of a more potent second slicer to the parasitic RNA silencing complex, the exact mechanism of which remains to be determined. PMID:22309152

Telomerase activation by the E6 gene product of human papillomavirus type 16.

Science.gov (United States)

Klingelhutz, A J; Foster, S A; McDougall, J K

1996-03-07

Activation of telomerase, a ribonucleoprotein complex that synthesizes telomere repeat sequences, is linked to cell immortalization and is characteristic of most cell lines and tumours. Here we show that expression of the human papillomavirus type 16 (HPV-16) E6 protein activates telomerase in early-passage human keratinocytes and mammary epithelial cells. This activation was observed in cells pre-crisis, that is, before they became immortal, and occurred within one passage of retroviral infection with vectors expressing HPV-16 E6. Studies using HPV-16 E6 mutants showed that there was no correlation between the ability of the mutants to activate telomerase and their ability to target p53 for degradation, suggesting that telomerase activation by HPV-16 E6 is p53 independent. Keratinocytes expressing wild-type HPV-16 E6 have an extended lifespan, but do not become immortal, indicating that telomerase activation and E6-mediate degradation of p53 are insufficient for their immortalization. These results show that telomerase activation is an intrinsic, but insufficient, component of transformation by HPV.

Telomere elongation in immortal human cells without detectable telomerase activity.

Science.gov (United States)

Bryan, T M; Englezou, A; Gupta, J; Bacchetti, S; Reddel, R R

1995-09-01

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.

Transmission and pathogenesis of vesicular stomatitis viruses

Science.gov (United States)

Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

Influence of repeated infusion of capsaicin-contained red pepper sauce on esophageal secondary peristalsis in humans.

Science.gov (United States)

Liu, T T; Yi, C H; Lei, W Y; Hung, X S; Yu, H C; Chen, C L

2014-10-01

The transient receptor potential vanilloid 1 has been implicated as a target mediator for heartburn perception and modulation of esophageal secondary peristalsis. Our aim was to determine the effect of repeated esophageal infusion of capsaicin-contained red pepper sauce on heartburn perception and secondary peristalsis in healthy adults. Secondary peristalsis was performed with mid-esophageal injections of air in 15 healthy adults. Two separate protocols including esophageal infusion with saline and capsaicin-contained red pepper sauce and 2 consecutive sessions of capsaicin-contained red pepper sauce were randomly performed. After repeated infusion of capsaicin-contained red pepper sauce, the threshold volume to activate secondary peristalsis was significantly increased during slow (p sauce enhanced heartburn perception (p sauce infusion (p = 0.007). Acute infusion of capsaicin-contained red pepper sauce significantly increased pressure wave amplitudes of distal esophagus during slow (p = 0.003) and rapid air injections (p = 0.01), but repeated infusion of capsaicin-contained red pepper sauce significantly decreased pressure wave amplitude of distal esophagus during slow (p = 0.0005) and rapid air injections (p = 0.003). Repeated esophageal infusion of capsaicin appears to attenuate heartburn perception and inhibit distension-induced secondary peristalsis in healthy adults. These results suggest capsaicin-sensitive afferents in modulating sensorimotor function of secondary peristalsis in human esophagus. © 2014 John Wiley & Sons Ltd.

FDA Peanut-Containing Product Recall

Data.gov (United States)

U.S. Department of Health & Human Services — The FDA Peanut-Containing Product Recall widget allows you to browse the Food and Drug Administration (FDA) database of peanut butter and peanut-containing products...

Human neuronal cell protein responses to Nipah virus infection

Directory of Open Access Journals (Sweden)

Hassan Sharifah

2007-06-01

Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

Heterogeneous nuclear ribonucleoprotein K upregulates the kinetochore complex component NUF2 and promotes the tumorigenicity of colon cancer cells

International Nuclear Information System (INIS)

Sugimasa, Hironobu; Taniue, Kenzui; Kurimoto, Akiko; Takeda, Yasuko; Kawasaki, Yoshihiro; Akiyama, Tetsu

2015-01-01

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer. - Highlights: • hnRNP K is required for the tumorigenicity of colon cancer cells. • hnRNP K binds to the promoter region of NUF2 and activates its transcription. • NUF2 expression is correlated with hnRNP K expression in colorectal cancer tissue. • hnRNP K and NUF2 are required for metaphase chromosome alignment. • The hnRNP K-NUF2 axis is important for the proliferation of colon cancer cells

Heterogeneous nuclear ribonucleoprotein K upregulates the kinetochore complex component NUF2 and promotes the tumorigenicity of colon cancer cells

Energy Technology Data Exchange (ETDEWEB)

Sugimasa, Hironobu; Taniue, Kenzui [Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032 (Japan); Kurimoto, Akiko [Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032 (Japan); Oncology Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo, 140-8710 (Japan); Takeda, Yasuko; Kawasaki, Yoshihiro [Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032 (Japan); Akiyama, Tetsu, E-mail: akiyama@iam.u-tokyo.ac.jp [Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032 (Japan)

2015-03-27

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein involved in transcription, mRNA splicing, mRNA stabilization and translation. Although hnRNP K has been suggested to play a role in the development of many cancers, its molecular function in colorectal cancer has remained elusive. Here we show that hnRNP K plays an important role in the mitotic process in HCT116 colon cancer cells. Furthermore, we demonstrate that hnRNP K directly transactivates the NUF2 gene, the product of which is a component of the NDC80 kinetochore complex and which is known to be critical for a stable spindle microtubule-kinetochore attachment. In addition, knockdown of both hnRNP K and NUF2 caused failure in metaphase chromosome alignment and drastic decrease in the growth of colon cancer cells. These results suggest that the hnRNP K-NUF2 axis is important for the mitotic process and proliferation of colon cancer cells and that this axis could be a target for the therapy of colon cancer. - Highlights: • hnRNP K is required for the tumorigenicity of colon cancer cells. • hnRNP K binds to the promoter region of NUF2 and activates its transcription. • NUF2 expression is correlated with hnRNP K expression in colorectal cancer tissue. • hnRNP K and NUF2 are required for metaphase chromosome alignment. • The hnRNP K-NUF2 axis is important for the proliferation of colon cancer cells.

Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA.

Science.gov (United States)

Iakhiaeva, Elena; Iakhiaev, Alexei; Zwieb, Christian

2010-11-13

Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain. We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA. The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

In-vitro study investigating influence of toothpaste containing green tea extract on the microhardness of demineralized human enamel

Science.gov (United States)

Febrian, K.; Triaminingsih, S.; Indrani, DJ

2017-08-01

The aim of this study was to analyze the influence of toothpaste containing green tea extract on the microhardness of demineralized enamel. Human tooth, which was demineralised in citric acid solution, had a toothpaste containing green tea extract of concentration of 5, 10 or 15% application. Microhardness measurement was carried out on each enamel surface of the teeth for initial, after the demineralization and after application of the tooth pastes. It showed that there was significant decrease between enamel microhardness of the teeth at the initial condition and after demineralization. After application of the toothpaste containing green tea extract of each concentration the microhardnss increased significantly. However, there the microhardness was insignificant between the applications of each green tea concentration.

21 CFR 310.530 - Topically applied hormone-containing drug products for over-the-counter (OTC) human use.

Science.gov (United States)

2010-04-01

... labeling or in the ingredient statement is an implied drug claim. The claim implied by the use of this term... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Topically applied hormone-containing drug products for over-the-counter (OTC) human use. 310.530 Section 310.530 Food and Drugs FOOD AND DRUG...

Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18

NARCIS (Netherlands)

Calafat, J.; Kuijpers, T. W.; Janssen, H.; Borregaard, N.; Verhoeven, A. J.; Roos, D.

1993-01-01

Human neutrophils contain a rapidly mobilizable pool of so-called secretory vesicles distinct from the azurophil granules and specific granules. Using human albumin as a marker for these intracellular vesicles in immuno-electron microscopy, we found that part of the cytochrome b558 in non-purified

Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains.

Science.gov (United States)

Miotto, Olivo; Heiny, A T; Albrecht, Randy; García-Sastre, Adolfo; Tan, Tin Wee; August, J Thomas; Brusic, Vladimir

2010-02-03

There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear

Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces.

Science.gov (United States)

Samra, H K; Ganguly, N K; Mahajan, R C

1991-06-01

The effects of human milk, containing specific secretory IgA, on the adherence of Giardia lamblia trophozoites in the presence and in the absence of intestinal mucus in vitro were studied. It was found that the trophozoites treated with breast milk, containing specific secretory IgA to G. lamblia, showed a significant decrease (p less than 0.01) in adherence to nylon fibre columns and glass surfaces than did trophozoites treated with milk containing no SIgA antibodies. The adherence to glass surfaces was significantly more (p less than 0.01) in the presence of intestinal mucus than when the mucus was absent. Milk that did not contain specific secretory SIgA to G. lamblia did not decrease the adherence to glass surfaces either in the presence or in the absence of mucus. The fluorescence study revealed the binding of specific secretory IgA on the trophozoite surface. The results suggest that binding of SIgA antibodies in milk to G. lamblia trophozoites inhibits parasite adherence, thus protecting against this infection in breast-fed babies.

Pituitary tumors containing cholecystokinin

DEFF Research Database (Denmark)

Rehfeld, J F; Lindholm, J; Andersen, B N

1987-01-01

We found small amounts of cholecystokinin in the normal human adenohypophysis and therefore examined pituitary tumors from 87 patients with acromegaly, Cushing's disease, Nelson's syndrome, prolactinoma, or inactive pituitary adenomas. Five adenomas associated with Nelson's syndrome contained......'s disease and 7 acromegaly with adenomas containing ACTH. The cholecystokinin peptides from the tumors were smaller and less sulfated than cholecystokinin from normal pituitary glands. We conclude that ACTH-producing pituitary cells may also produce an altered form of cholecystokinin....

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

Science.gov (United States)

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-01-01

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

Science.gov (United States)

Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

2012-06-01

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Human systemic exposure to [14C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment

NARCIS (Netherlands)

Nohynek, G.J.; Skare, J.A.; Meuling, W.J.A.; Wehmeyer, K.R.; Bie, A.T.H.J. de; Vaes, W.H.J.; Dufour, E.K.; Fautz, R.; Steiling, W.; Bramante, M.; Toutain, H.

2015-01-01

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [14C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [14C] was measured in all

Structural analysis of respiratory syncytial virus reveals the position of M2-1 between the matrix protein and the ribonucleoprotein complex.

Science.gov (United States)

Kiss, Gabriella; Holl, Jens M; Williams, Grant M; Alonas, Eric; Vanover, Daryll; Lifland, Aaron W; Gudheti, Manasa; Guerrero-Ferreira, Ricardo C; Nair, Vinod; Yi, Hong; Graham, Barney S; Santangelo, Philip J; Wright, Elizabeth R

2014-07-01

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family of nonsegmented, negative-sense, single-stranded RNA genome viruses, is a leading cause of lower respiratory tract infections in infants, young children, and the elderly or immunocompromised. There are many open questions regarding the processes that regulate human RSV (hRSV) assembly and budding. Here, using cryo-electron tomography, we identified virus particles that were spherical, filamentous, and asymmetric in structure, all within the same virus preparation. The three particle morphologies maintained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). RNP filaments were traced in three dimensions (3D), and their total length was calculated. The measurements revealed the inclusion of multiple full-length genome copies per particle. RNP was associated with the membrane whenever the M layer was present. The amount of M coverage ranged from 24% to 86% in the different morphologies. Using fluorescence light microscopy (fLM), direct stochastic optical reconstruction microscopy (dSTORM), and a proximity ligation assay (PLA), we provide evidence illustrating that M2-1 is located between RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when hRSV viruses were imaged using Zernike phase contrast (ZPC) cryo-electron tomography. Our studies provide a more complete characterization of the hRSV virion structure and substantiation that M and M2-1 regulate virus organization. hRSV is a leading cause of lower respiratory tract infections in infants and young children as well as elderly or immunocompromised individuals. We used cryo-electron tomography and Zernike phase contrast cryo-electron tomography to visualize populations of purified hRSV in 3D. We observed the three distinct morphologies, spherical, filamentous, and asymmetric, which maintained comparable organizational profiles

Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

Science.gov (United States)

Goldfarb, Katherine C; Cech, Thomas R

2017-01-01

MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

Identification of mathematical model of human breathing in system “Artificial lungs – self-contained breathing apparatus”

Science.gov (United States)

Onevsky, P. M.; Onevsky, M. P.; Pogonin, V. A.

2018-03-01

The structure and mathematical models of the main subsystems of the control system of the “Artificial Lungs” are presented. This structure implements the process of imitation of human external respiration in the system “Artificial lungs - self-contained breathing apparatus”. A presented algorithm for parametric identification of the model is based on spectral operators, which allows using it in real time.

The 3.2 Angstrom Resolution Structure of the Polymorphic Cowpea Chlorotic Mottle Virus Ribonucleoprotein Particle

Science.gov (United States)

Speir, Jeffrey Alan

Structural studies of the polymorphic cowpea chlorotic mottle virus have resulted in high resolution structures for two distinct icosahedral ribonucleoprotein particle conformations dependent upon whether acidic or basic pH conditions prevail. CCMV is stable below pH 6.5, however metal-free particles maintain a 10% increase in hydrodynamic volume at pH >=q 7.5. Identification of this swollen' form of CCMV, which can easily be disrupted with 1M NaCl, led to the first reassembly of an icosahedral virus in vitro from purified viral protein and RNA to form infectious particles, and its assembly has been the subject of biochemical and biophysical investigations for over twenty-five years. Under well defined conditions of pH, ionic strength and divalent metal ion concentration, CCMV capsid protein or capsid protein and RNA will reassemble to form icosahedral particles of various sizes, sheets, tubes, rosettes, and a variety of laminar structures which resemble virion structures from non-related virus families. Analysis of native particles at 3.2A resolution and swollen particles at 28A resolution has suggested that the chemical basis for the formation of polymorphic icosahedral and anisometric structures is: (i) hexamers formed of beta-barrel subunits stabilized by an unusual hexameric parallel beta structure made up of their N-termini, (ii) the location of protein-RNA interactions, (iii) divalent metal cation binding sites that regulate quasi-symmetrical subunit associations, (iv) charge repulsion across the same interfaces when lacking divalent metal ions at basic pH, which induces the formation of sixty 20A diameter portals for RNA release, and (v) a novel, C-terminal-based, subunit dimer assembly unit. The use of C- and N-terminal arms in CCMV has not been observed in other icosahedral RNA virus structures determined at near atomic resolution, however, their detailed interactions and roles in stabilizing the quaternary organization of CCMV are related to that found

Diversity, expression and mRNA targeting abilities of Argonaute-targeting miRNAs among selected vascular plants.

Science.gov (United States)

Jagtap, Soham; Shivaprasad, Padubidri V

2014-12-02

Micro (mi)RNAs are important regulators of plant development. Across plant lineages, Dicer-like 1 (DCL1) proteins process long ds-like structures to produce micro (mi) RNA duplexes in a stepwise manner. These miRNAs are incorporated into Argonaute (AGO) proteins and influence expression of RNAs that have sequence complementarity with miRNAs. Expression levels of AGOs are greatly regulated by plants in order to minimize unwarranted perturbations using miRNAs to target mRNAs coding for AGOs. AGOs may also have high promoter specificity-sometimes expression of AGO can be limited to just a few cells in a plant. Viral pathogens utilize various means to counter antiviral roles of AGOs including hijacking the host encoded miRNAs to target AGOs. Two host encoded miRNAs namely miR168 and miR403 that target AGOs have been described in the model plant Arabidopsis and such a mechanism is thought to be well conserved across plants because AGO sequences are well conserved. We show that the interaction between AGO mRNAs and miRNAs is species-specific due to the diversity in sequences of two miRNAs that target AGOs, sequence diversity among corresponding target regions in AGO mRNAs and variable expression levels of these miRNAs among vascular plants. We used miRNA sequences from 68 plant species representing 31 plant families for this analysis. Sequences of miR168 and miR403 are not conserved among plant lineages, but surprisingly they differ drastically in their sequence diversity and expression levels even among closely related plants. Variation in miR168 expression among plants correlates well with secondary structures/length of loop sequences of their precursors. Our data indicates a complex AGO targeting interaction among plant lineages due to miRNA sequence diversity and sequences of miRNA targeting regions among AGO mRNAs, thus leading to the assumption that the perturbations by viruses that use host miRNAs to target antiviral AGOs can only be species-specific. We also show

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

Energy Technology Data Exchange (ETDEWEB)

Nishio, Sachiyo [Division of Oral and Maxillofacial Biopathological Surgery, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503 (Japan); Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Ohira, Takahito; Sunamura, Naohiro [Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Oshimura, Mitsuo [Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, 683-8503 (Japan); Ryoke, Kazuo [Division of Oral and Maxillofacial Biopathological Surgery, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503 (Japan); Kugoh, Hiroyuki, E-mail: kugoh@med.tottori-u.ac.jp [Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, 683-8503 (Japan); Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, 683-8503 (Japan)

2015-10-30

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.

Repression of hTERT transcription by the introduction of chromosome 3 into human oral squamous cell carcinoma

International Nuclear Information System (INIS)

Nishio, Sachiyo; Ohira, Takahito; Sunamura, Naohiro; Oshimura, Mitsuo; Ryoke, Kazuo; Kugoh, Hiroyuki

2015-01-01

Telomerase is a ribonucleoprotein enzyme that maintains telomere length. Telomerase activity is primarily attributed to the expression of telomerase reverse transcriptase (TERT). It has been reported that introduction of an intact human chromosome 3 into the human oral squamous cell carcinoma cell line HSC3 suppresses the tumorigenicity of these cells. However, the mechanisms that regulate tumorigenicity have not been elucidated. To determine whether this reduction in tumorigenicity was accompanied by a reduction in telomerase activity, we investigated the transcriptional activation of TERT in HSC3 microcell hybrid clones with an introduced human chromosome 3 (HSC3#3). HSC#3 cells showed inhibition of hTERT transcription compared to that of the parental HSC3 cells. Furthermore, cell fusion experiments showed that hybrids of HSC3 cells and cells of the RCC23 renal carcinoma cell line, which also exhibits suppression of TERT transcription by the introduction of human chromosome 3, also displayed suppressed TERT transcription. These results suggested that human chromosome 3 may carry functionally distinct, additional TERT repressor genes. - Highlights: • hTERT mRNA expression level decreased in the chromosome 3 introduced HSC3 clones. • hTERT mRNA expression level was tend to suppressed in HSC3 and RCC23 hybrid cells. • We provide evidence that human chromosome 3 carries at least two distinct hTERT regulatory factors.

How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

Science.gov (United States)

Piecyk, Karolina; Niedzwiecka, Anna; Ferenc-Mrozek, Aleksandra; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena

2015-08-01

Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

Science.gov (United States)

Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

2014-08-08

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

Science.gov (United States)

Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2003-04-01

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

International Nuclear Information System (INIS)

Noisakran, Sansanee; Sengsai, Suchada; Thongboonkerd, Visith; Kanlaya, Rattiyaporn; Sinchaikul, Supachok; Chen, Shui-Tein; Puttikhunt, Chunya

2008-01-01

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells

Overexpression of an Arabidopsis heterogeneous nuclear ribonucleoprotein gene, AtRNP1, affects plant growth and reduces plant tolerance to drought and salt stresses

International Nuclear Information System (INIS)

Wang, Zhenyu; Zhao, Xiuyang; Wang, Bing; Liu, Erlong; Chen, Ni; Zhang, Wei; Liu, Heng

2016-01-01

Heterogeneous nuclear ribonucleoproteins (hnRNPs) participate in diverse regulations of plant growth and environmental stress responses. In this work, an Arabidopsis hnRNP of unknown function, AtRNP1, was investigated. We found that AtRNP1 gene is highly expressed in rosette and cauline leaves, and slightly induced under drought, salt, osmotic and ABA stresses. AtRNP1 protein is localized to both the nucleus and cytoplasm. We performed homologous overexpression of AtRNP1 and found that the transgenic plants showed shortened root length and plant height, and accelerated flowering. In addition, the transgenic plants also showed reduced tolerance to drought, salt, osmotic and ABA stresses. Further studies revealed that under both normal and stress conditions, the proline contents in the transgenic plants are markedly decreased, associated with reduced expression levels of a proline synthase gene and several stress-responsive genes. These results suggested that the overexpression of AtRNP1 negatively affects plant growth and abiotic stress tolerance. - Highlights: • AtRNP1 is a widely expressed gene and its expression is slightly induced under abiotic stresses. • AtRNP1 protein is localized to both the nucleus and cytoplasm. • Overexpression of AtRNP1 affects plant growth. • Overexpression of AtRNP1 reduces plant tolerance to drought and salt stresses. • AtRNP1 overexpression plants show decreased proline accumulation and stress-responsive gene expressions.

Overexpression of an Arabidopsis heterogeneous nuclear ribonucleoprotein gene, AtRNP1, affects plant growth and reduces plant tolerance to drought and salt stresses

Energy Technology Data Exchange (ETDEWEB)

Wang, Zhenyu, E-mail: wzy72609@163.com [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Zhao, Xiuyang, E-mail: xiuzh@psb.vib-ugent.be [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Wang, Bing, E-mail: wangbing@ibcas.ac.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Liu, Erlong, E-mail: liuel14@lzu.edu.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Chen, Ni, E-mail: 63710156@qq.com [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China); Zhang, Wei, E-mail: wzhang1216@yahoo.com [Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444 (China); Liu, Heng, E-mail: hengliu@lzu.edu.cn [Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730030 (China)

2016-04-01

Heterogeneous nuclear ribonucleoproteins (hnRNPs) participate in diverse regulations of plant growth and environmental stress responses. In this work, an Arabidopsis hnRNP of unknown function, AtRNP1, was investigated. We found that AtRNP1 gene is highly expressed in rosette and cauline leaves, and slightly induced under drought, salt, osmotic and ABA stresses. AtRNP1 protein is localized to both the nucleus and cytoplasm. We performed homologous overexpression of AtRNP1 and found that the transgenic plants showed shortened root length and plant height, and accelerated flowering. In addition, the transgenic plants also showed reduced tolerance to drought, salt, osmotic and ABA stresses. Further studies revealed that under both normal and stress conditions, the proline contents in the transgenic plants are markedly decreased, associated with reduced expression levels of a proline synthase gene and several stress-responsive genes. These results suggested that the overexpression of AtRNP1 negatively affects plant growth and abiotic stress tolerance. - Highlights: • AtRNP1 is a widely expressed gene and its expression is slightly induced under abiotic stresses. • AtRNP1 protein is localized to both the nucleus and cytoplasm. • Overexpression of AtRNP1 affects plant growth. • Overexpression of AtRNP1 reduces plant tolerance to drought and salt stresses. • AtRNP1 overexpression plants show decreased proline accumulation and stress-responsive gene expressions.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

Science.gov (United States)

Mout, Rubul; Rotello, Vincent M

2017-10-20

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

Unimpaired dendritic cell functions in MVP/LRP knockout mice.

NARCIS (Netherlands)

Mossink, MH; Groot, de J.; Zon, van A; Franzel-Luiten, E; Schoester, M.; Scheffer, G.L.; Sonneveld, P.; Scheper, R.J.; Wiemer, EA

2003-01-01

Dendritic cells (DCs) act as mobile sentinels of the immune system. By stimulating T lymphocytes, DCs are pivotal for the initiation of both T- and B-cell-mediated immune responses. Recently, ribonucleoprotein particles (vaults) were found to be involved in the development and/or function of human

Fluorine uptake into human enamel around fluoride-containing dental materials during cariogenic pH cycling

Energy Technology Data Exchange (ETDEWEB)

Komatsu, H. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan)], E-mail: kom@den.hokudai.ac.jp; Yamamoto, H. [Graduate School of Dentistry, Osaka University, 1-8 Yamada-Oka, Suita 565-0871 (Japan); Nomachi, M. [Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043 (Japan); Yasuda, K. [The Wakasa wan Energy Research Center, 64-52-1 Hase, Tsuruga 914-0192 (Japan); Matsuda, Y.; Kinugawa, M.; Kijimura, T.; Sano, H. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Satou, T.; Oikawa, S.; Kamiya, T. [Advanced Radiation Technology, TARRI, JAEA, 1233 Watanuki-machi, Takasaki 370-1292 (Japan)

2009-06-15

Using PIGE (Proton Induced Gamma Emission) technique at TARRI (Takasaki Advanced Radiation Research Institute), Japan, we measured fluorine (F) uptake into the tooth enamel around two fluoride-containing materials during caries progression using pH cycling. Class V cavities in extracted human teeth were drilled and filled with fluoride-containing materials (i.e. 'Fuji IX' (FN) and 'UniFil flow with MEGA bond' (UF)) and a non-fluoride-containing material (i.e. 'SOLARE with MEGA bond' (SO)). Three 120 {mu}m longitudinal sections including the filling material were obtained from each tooth. In order to simulate daily acid attack occurring in the oral cavity, the pH cycling (pH 6.8-4.5) was carried out for 1, 3 and 5 weeks, separately. After pH cycling, the caries progression in all specimens was observed using transverse microradiography (TMR). The F and calcium distributions of the specimens were evaluated using PIGE and PIXE techniques. The F distribution of the specimens clearly showed the F uptake from FN into enamel adjacent to the filling material, while the F uptakes from UF and SO were not detected. For UF, the MEGA bond (non-fluoride-containing) between the tooth and UniFil flow interfered with the F absorption into the tooth. For FN, the amount of F uptake into the subsurface enamel increased during pH cycling. The amount of F uptake in 5-week pH cycling had significantly higher value compared to those in 1- and 3-week pH cycling. For UF and SO, there were no significant differences between the different durations of pH cycling. Among fluoride-containing materials, there were some differences in the F uptake with increased pH cycling, which could possibly lead to obtaining difference in clinical performance. The data obtained using PIGE and PIXE techniques were useful in understanding the benefit of fluorine by means of fluoride-containing material for preventing caries.

Fluorine uptake into human enamel around fluoride-containing dental materials during cariogenic pH cycling

International Nuclear Information System (INIS)

Komatsu, H.; Yamamoto, H.; Nomachi, M.; Yasuda, K.; Matsuda, Y.; Kinugawa, M.; Kijimura, T.; Sano, H.; Satou, T.; Oikawa, S.; Kamiya, T.

2009-01-01

Using PIGE (Proton Induced Gamma Emission) technique at TARRI (Takasaki Advanced Radiation Research Institute), Japan, we measured fluorine (F) uptake into the tooth enamel around two fluoride-containing materials during caries progression using pH cycling. Class V cavities in extracted human teeth were drilled and filled with fluoride-containing materials (i.e. 'Fuji IX' (FN) and 'UniFil flow with MEGA bond' (UF)) and a non-fluoride-containing material (i.e. 'SOLARE with MEGA bond' (SO)). Three 120 μm longitudinal sections including the filling material were obtained from each tooth. In order to simulate daily acid attack occurring in the oral cavity, the pH cycling (pH 6.8-4.5) was carried out for 1, 3 and 5 weeks, separately. After pH cycling, the caries progression in all specimens was observed using transverse microradiography (TMR). The F and calcium distributions of the specimens were evaluated using PIGE and PIXE techniques. The F distribution of the specimens clearly showed the F uptake from FN into enamel adjacent to the filling material, while the F uptakes from UF and SO were not detected. For UF, the MEGA bond (non-fluoride-containing) between the tooth and UniFil flow interfered with the F absorption into the tooth. For FN, the amount of F uptake into the subsurface enamel increased during pH cycling. The amount of F uptake in 5-week pH cycling had significantly higher value compared to those in 1- and 3-week pH cycling. For UF and SO, there were no significant differences between the different durations of pH cycling. Among fluoride-containing materials, there were some differences in the F uptake with increased pH cycling, which could possibly lead to obtaining difference in clinical performance. The data obtained using PIGE and PIXE techniques were useful in understanding the benefit of fluorine by means of fluoride-containing material for preventing caries.

Degenerated human intervertebral discs contain autoantibodies against extracellular matrix proteins

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S Capossela

2014-04-01

Full Text Available Degeneration of intervertebral discs (IVDs is associated with back pain and elevated levels of inflammatory cells. It has been hypothesised that discogenic pain is a direct result of vascular and neural ingrowth along annulus fissures, which may expose the avascular nucleus pulposus (NP to the systemic circulation and induce an autoimmune reaction. In this study, we confirmed our previous observation of antibodies in human degenerated and post-traumatic IVDs cultured in vitro. We hypothesised that the presence of antibodies was due to an autoimmune reaction against specific proteins of the disc. Furthermore we identified antigens which possibly trigger an autoimmune response in degenerative disc diseases. We demonstrated that degenerated and post-traumatic IVDs contain IgG antibodies against typical extracellular proteins of the disc, particularly proteins of the NP. We identified IgGs against collagen type II and aggrecan, confirming an autoimmune reaction against the normally immune privileged NP. We also found specific IgGs against collagens types I and V, but not against collagen type III. In conclusion, this study confirmed the association between disc degeneration and autoimmunity, and may open the avenue for future studies on developing prognostic, diagnostic and therapy-monitoring markers for degenerative disc diseases.

Conserved region at the COOH terminus of human immunodeficiency virus gp120 envelope protein contains an immunodominant epitope

International Nuclear Information System (INIS)

Palker, T.J.; Matthews, T.J.; Clark, M.E.

1987-01-01

A highly immunogenic epitope from a conserved COOH-terminal region of the human immunodeficiency virus (HIV) gp120 envelope protein has been identified with antisera from HIV-seropositive subjects and a synthetic peptide (SP-22) containing 15 amino acids from this region (Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg). Peptide SP-22 absorbed up to 100% of anti-gp120 antibody reactivity from select HIV + patient sera in immunoblot assays and up to 79% of serum anti-gp120 antibody reactivity in competition RIA. In RIA, 45% of HIV-seropositive subjects had antibodies that bound to peptide SP-22. Human anti-SP-22 antibodies that bound to and were eluted from an SP-22 affinity column reacted with gp120 in RIA and immunoblot assays but did not neutralize HIV or inhibit HIV-induced syncytium formation in vitro, even though these antibodies comprised 70% of all anti-gp120 antibodies in the test serum. In contrast, the remaining 30% of SP-22 nonreactive anti-gp120 antibodies did not react with gp120 in immunoblot assays but did react in RIA and neutralized HIV in vitro. Thus, ≅ 50% of HIV-seropositive patients make high titers of nonneutralizing antibodies to an immunodominant antigen on gp120 defined by SP-22. Moreover, the COOH terminus of gp120 contains the major antigen or antigens identified by human anti-gp120 antibodies in immunoblot assays

Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.

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Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J

2011-03-01

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.

ANTI-HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN B1 (ANTI-RA33 ANTIBODIES IN RHEUMATOID ARTHRITIS AND SYSTEMIC SCLEROSIS

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P. A. Kuznetsova

2017-01-01

Full Text Available Anti-heterogeneous nuclear ribonucleoprotein (RNP autoantibodies (AAbs are encountered in many autoimmune rheumatic diseases (ARDs. The potential diagnostic value of the RA33 AAb complex consisting of RNP A2 and alternative domains of the splicing proteins RNP B1 and RNP B2 is now of interest to rheumatologists. Subjects and methods. The authors studied the frequency of anti-RNP B1 AAbs in 300 patients with systemic ARDs, including those with rheumatoid arthritis (RA, ankylosing spondylitis (AS, systemic lupus erythematosus (SLE, systemic sclerosis (SSc, and Sjö gren's syndrome (SS and in 53 people without ARDs, who constituted a control group. Serum anti-RNP B1 AAbs were assessed by enzyme immunoassay. Results and discussion. The frequency of anti-RNP B1 AAbs in patients with ARDs was much higher than that in the control group: 170/300 (56.6% and 8/53 (13% patients, respectively. Anti-RNP B1 AAbs were detected in 78.5% (113/144 of the patients with RA; 40.3% (23/57 of those with AS, in 67.5% (27/40 of those with SSc, in 36.4% (16/44 of those with SLE, and in 13.3% (2/15 of those with SS. The diagnostic sensitivity of the marker for RA was 78.5%, its diagnostic specificity was 84.9%; the likelihood ratio of positive and negative results was 5.24 and 0.24, respectively. In the patients with RA, the level of anti-RNP B1 AAbs significantly correlated with that of C-reactive protein and erythrocyte sedimentation rate, while in those with SSc the detection of anti-RNP B1 AAbs was related to the rigidity of the vascular wall and the presence of hypertension. The frequency of anti-RNP B1 AAbs among the RA patients seronegative for rheumatoid factor and anti-cyclic citrullinated peptide antibodies was 15.4%. Conclusion. Anti-RNP B1 AAs are a useful laboratory marker (with the upper limit of the normal range being 3.3 U/ml, but are of limited value in the diagnosis of RA. Anti-RNP B1 AAbs may be regarded as an additional diagnostic marker for RA.

GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

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Guillaume Jannot

2016-12-01

Full Text Available MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.

Trypanosoma brucei gambiense: HMI-9 medium containing methylcellulose and human serum supports the continuous axenic in vitro propagation of the bloodstream form.

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Van Reet, N; Pyana, P P; Deborggraeve, S; Büscher, P; Claes, F

2011-07-01

Trypanosoma brucei (T.b.) gambiense causes the chronic form of human African trypanosomiasis or sleeping sickness. One of the major problems with studying T.b. gambiense is the difficulty to isolate it from its original host and the difficult adaptation to in vivo and in vitro mass propagation. The objective of this study was to evaluate if an established method for axenic culture of pleomorphic bloodstream form T.b. brucei strains, based on methylcellulose containing HMI-9 medium, also facilitated the continuous in vitro propagation of other bloodstream form Trypanozoon strains, in particular of T.b. gambiense. Bloodstream form trypanosomes from one T.b. brucei, two T.b. rhodesiense, one T. evansi and seven T.b. gambiense strains were isolated from mouse blood and each was concurrently cultivated in liquid and methylcellulose-containing HMI-9 based medium, either with or without additional human serum supplementation, for over 10 consecutive sub passages. Although HMI-9 based medium supplemented with 1.1% (w/v) methylcellulose supported the continuous cultivation of all non-gambiense strains better than liquid media could, the in vitro cultivation of all gambiense strains was only achieved in HMI-9 based medium containing 1.1% (w/v) methylcellulose, 15% (v/v) fetal calf serum and 5% (v/v) heat-inactivated human serum. Copyright © 2011 Elsevier Inc. All rights reserved.

Molecular hierarchy in neurons differentiated from mouse ES cells containing a single human chromosome 21.

Science.gov (United States)

Wang, Chi Chiu; Kadota, Mitsutaka; Nishigaki, Ryuichi; Kazuki, Yasuhiro; Shirayoshi, Yasuaki; Rogers, Michael Scott; Gojobori, Takashi; Ikeo, Kazuho; Oshimura, Mitsuo

2004-02-06

Defects in neurogenesis and neuronal differentiation in the fetal brain of Down syndrome (DS) patients lead to the apparent neuropathological abnormalities and contribute to the phenotypic characters of mental retardation, and premature development of Alzheimer's disease, those being the most common phenotype in DS. In order to understand the molecular mechanism underlying the cause of phenotypic abnormalities in the DS brain, we have utilized an in vitro model of TT2F mouse embryonic stem cells containing a single human chromosome 21 (hChr21) to study neuron development and neuronal differentiation by microarray containing 15K developmentally expressed cDNAs. Defective neuronal differentiation in the presence of extra hChr21 manifested primarily the post-transcriptional and translational modification, such as Mrpl10, SNAPC3, Srprb, SF3a60 in the early neuronal stem cell stage, and Mrps18a, Eef1g, and Ubce8 in the late differentiated stage. Hierarchical clustering patterned specific expression of hChr21 gene dosage effects on neuron outgrowth, migration, and differentiation, such as Syngr2, Dncic2, Eif3sf, and Peg3.

Purification of the spliced leader ribonucleoprotein particle from Leptomonas collosoma revealed the existence of an Sm protein in trypanosomes. Cloning the SmE homologue.

Science.gov (United States)

Goncharov, I; Palfi, Z; Bindereif, A; Michaeli, S

1999-04-30

Trans-splicing in trypanosomes involves the addition of a common spliced leader (SL) sequence, which is derived from a small RNA, the SL RNA, to all mRNA precursors. The SL RNA is present in the cell in the form of a ribonucleoprotein, the SL RNP. Using conventional chromatography and affinity selection with 2'-O-methylated RNA oligonucleotides at high ionic strength, five proteins of 70, 16, 13, 12, and 8 kDa were co-selected with the SL RNA from Leptomonas collosoma, representing the SL RNP core particle. Under conditions of lower ionic strength, additional proteins of 28 and 20 kDa were revealed. On the basis of peptide sequences, the gene coding for a protein with a predicted molecular weight of 11.9 kDa was cloned and identified as homologue of the cis-spliceosomal SmE. The protein carries the Sm motifs 1 and 2 characteristic of Sm antigens that bind to all known cis-spliceosomal uridylic acid-rich small nuclear RNAs (U snRNAs), suggesting the existence of Sm proteins in trypanosomes. This finding is of special interest because trypanosome snRNPs are the only snRNPs examined to date that are not recognized by anti-Sm antibodies. Because of the early divergence of trypanosomes from the eukaryotic lineage, the trypanosome SmE protein represents one of the primordial Sm proteins in nature.

A 90-day safety study in Sprague-Dawley rats fed milk powder containing recombinant human lactoferrin (rhLF) derived from transgenic cloned cattle.

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Zhou, Cui; Wang, Jian Wu; Huang, Kun Lun; He, XiaoYun; Chen, Xiu Ping; Sun, Hong; Yu, Tian; Che, Hui Lian

2011-10-01

Transgenic cloned animals expressing beneficial human nutritional traits offer a new strategy for large-scale production of some kinds of functional substances. In some cases, the required safety testing for genetically modified (GM) foods do not seem appropriate for human food safety, though regulations do not seem to provide alternatives. A 90-day rat feeding study is the core study for the safety assessment of GM foods. The test material in this 90-day study was prepared nonfat milk powder containing recombinant human lactoferrin (rhLF), which was expressed in transgenic cloned cattle. Groups of 10 male and female Sprague-Dawley rats were given a nutritionally balanced purified diet containing 7.5, 15, or 30% transgenic or conventional milk powder for 90 days. A commercial AIN93G diet was used as an additional control group. Clinical, biological, and pathological parameters were compared between groups. The only significant effect of treatment was higher mean ferritin and Fe(+) concentrations for both male and female rats fed the transgenic milk powder diets, as compared to rats fed nontransgenic milk diets or the commercial diet. The results of the present study are consistent with previous research, which indicates that milk powder containing rhLF derived from healthy transgenic cloned cattle is as safe as conventional milk powder.

Human hair follicle benzo(a)pyrene and benzo(a)pyrene 7, 8-diol metabolism: effect of exposure to a coal tar-containing shampoo

Energy Technology Data Exchange (ETDEWEB)

Merk, H.F.; Mukhtar, H.; Kaufmann, I.; Das, M.; Bickers, D.R.

1987-01-01

Hair follicles are a readily available source of human epithelial tissue and offer an excellent system with which to study carcinogen metabolism in human populations. In this study hair follicles were employed to measure the metabolism of benzo(a)pyrene (BP), benzo(a)pyrene - 7,8-diol (BP 7,8-diol) and the enzyme mediated binding of /sup 3/H-BP to DNA. The effect of human exposure to a crude coal tar (CCT) - containing shampoo, a preparation rich in polycyclic aromatic hydrocarbons (PAHs on these parameters was also evaluated. It was found that aryl hydrocarbon hydroxylase (AHH) activity increased after use of the shampoo and enhancement of enzyme-mediated binding of BP to DNA was detected in most subjects. Hair follicles were shown to convert BP to several metabolic species and BP, 7,8-diol was also metabolised. Clotrimazole, a known inhibitor of the metabolism of BP was found to inhibit AHH and the metabolism of BP and BP 7,8-diol in human hair follicles, as were other imidazole compounds. The studies show that hair follicles represent an accessible tissue suitable for assessing the extent of PAH carcinogen metabolism in human subjects. Furthermore enzyme activity critical to cancer induction by PAHs was shown to be inducible following the use of a CCT-containing shampoo. Imidazole compounds were shown to be possible effective anti-carcinogens in human populations. 29 refs.

Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite.

Science.gov (United States)

Hills, Mark; Jeyapalan, Jennie N; Foxon, Jennifer L; Royle, Nicola J

2007-04-01

Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

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B Cisterna

2009-08-01

Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

Science.gov (United States)

Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

2008-01-01

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Native gel analysis for RISC assembly.

Science.gov (United States)

Kawamata, Tomoko; Tomari, Yukihide

2011-01-01

Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) regulate expression of their target mRNAs via the RNA-induced silencing complex (RISC). A core component of RISC is the Argonaute (Ago) protein, which dictates the RISC function. In Drosophila, miRNAs and siRNAs are generally loaded into Ago1-containing RISC (Ago1-RISC) and Ago2-containing RISC (Ago2-RISC), respectively. We developed a native agarose gel system to directly detect Ago1-RISC, Ago2-RISC, and their precursor complexes. Methods presented here will provide powerful tools to biochemically dissect the RISC assembly pathways.

Synergistic antitumor cytotoxic actions of ascorbate and menadione on human prostate (DU145) cancer cells in vitro: nucleus and other injuries preceding cell death by autoschizis.

Science.gov (United States)

Gilloteaux, Jacques; Jamison, James M; Neal, Deborah; Summers, Jack L

2014-04-01

Scanning (SEM) and transmission electron microscopy (TEM) were used to characterize the cytotoxic effects of ascorbate (VC), menadione (VK3), or a VC:VK3 combination on a human prostate carcinoma cell line (DU145) following a 1-h vitamin treatment and a subsequent 24-h incubation in culture medium. Cell alterations examined by light and electron microscopy were treatment-dependent with VC + VK3 >VK3 > VC > Sham. Oxidative stress-induced damage was found in most organelles. This report describes injuries in the tumor cell nucleus (chromatin and nucleolus), mitochondria, endomembranes, lysosomal bodies (autophagocytoses) and inclusions. Morphologic alterations suggest that cytoskeleton damage is likely responsible for the superficial cytoplasmic changes, including major changes in cell shape and size and the self-excising phenomena. Unlike apoptotic bodies, the excised pieces contain ribonucleoproteins, but not organelles. These deleterious events cause a progressive, significant reduction in the tumor cell size. During nuclear alterations, the nuclei maintain their envelope during chromatolysis and karyolysis until cell death, while nucleoli undergo a characteristic segregation of their components. In addition, changes in fat and glycogen storage are consistent the cytotoxic and metabolic alterations caused by the respective treatments. All cellular ultrastructural changes are consistent with cell death by autoschizis and not apoptosis or other kinds of cell death.

Acyl CoA Binding Domain Containing 3 (ACBD3) Protein in Huntington’s Disease 
Human Skin Fibroblasts

Czech Academy of Sciences Publication Activity Database

Kratochvílová, H.; Rodinová, M.; Sládková, J.; Klempíř, J.; Lišková, Irena; Motlík, Jan; Zeman, J.; Hansíková, H.; Tesařová, M.

2015-01-01

Roč. 78, Suppl. 2 (2015), s. 34-38 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. Liblice, 08.11.2015-10.11.2015] R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : Huntington’s disease * Acyl-CoA binding domain containing 3 protein * human skin fibroblasts Subject RIV: FH - Neurology Impact factor: 0.209, year: 2015

Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

Science.gov (United States)

Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

2010-10-15

Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma

International Nuclear Information System (INIS)

Chung, I-Che; Li, Hsin-Pai; Chang, Yu-Sun; Chen, Lih-Chyang; Chung, An-Ko; Chao, Mei; Huang, Hsin-Yi; Hsueh, Chuen; Tsang, Ngan-Ming; Chang, Kai-Ping; Liang, Ying

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC. We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231. MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning −42 to −33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells. Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but

Isolation and characterization of the heterogeneous nuclear RNA-ribonucleoprotein complex

International Nuclear Information System (INIS)

Choi, Y.D.

1985-01-01

Exposure of cells to UV light of sufficient intensity brings about crosslinking of RNA to proteins which are in direct contact with it in vivo. The major [ 35 S]methionine-labeled proteins which become crosslinked to poly(A) + hnRNA in HeLa cells are of 120K, 68K, 53K, 43K, 41K, 38K, and 36K (K = kilodaltons). By immunizing mice with UV crosslinked complexes two monoclonal antibodies (2B12 and 4F4) against the C proteins (41K and 43K) and one (3G6) against the 120K protein of the hnRNP complex were obtained. Immunofluorescence microscopy demonstrates that the C proteins and 120K are segregated to the nucleus and are not associated with nucleoli or chromatin. The two C proteins are highly related to each other antigenically. Monoclonal antibody 4F4 identifies the C proteins of the hnRNP complex in widely divergent species from human to lizard. The C proteins are phosphorylated and are in contact with hnRNA in vivo. The hnRNP complex was isolated from vertebrate cell nuclei by immunoprecipitation with these monoclonal antibodies. This complex contains proteins and hnRNA of up to ∼10 kb. The major steady state labeled [ 35 S]methionine labeled proteins of the isolated complex from HeLa cells are of 34K, 36K, 36K (A1 and A2), 37K, 38K (B1 and B2), 41K, 43K (C1 and C2) and doublets at 68K and at 120K. These proteins are organized into a 30S particle. Large hnRNP complexes are composed of multiples of 30S particles which are connected by highly nuclease sensitive stretches of hnRNA. It it concluded that the hnRNP structure is an integral component of the mRNA formation pathway in the eukaryotic cell

Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Momand, J.; Clarke, S.

1987-12-01

The authors have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Try-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater that the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by (/sup 3/H) diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.

SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

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Xiao-Peng Xiong

2015-08-01

Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

A transcriptome-wide study on the microRNA- and the Argonaute 1-enriched small RNA-mediated regulatory networks involved in plant leaf senescence.

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Qin, J; Ma, X; Yi, Z; Tang, Z; Meng, Y

2016-03-01

Leaf senescence is an important physiological process during the plant life cycle. However, systemic studies on the impact of microRNAs (miRNAs) on the expression of senescence-associated genes (SAGs) are lacking. Besides, whether other Argonaute 1 (AGO1)-enriched small RNAs (sRNAs) play regulatory roles in leaf senescence remains unclear. In this study, a total of 5,123 and 1,399 AGO1-enriched sRNAs, excluding miRNAs, were identified in Arabidopsis thaliana and rice (Oryza sativa), respectively. After retrieving SAGs from the Leaf Senescence Database, all of the AGO1-enriched sRNAs and the miRBase-registered miRNAs of these two plants were included for target identification. Supported by degradome signatures, 200 regulatory pairs involving 120 AGO1-enriched sRNAs and 40 SAGs, and 266 regulatory pairs involving 64 miRNAs and 42 SAGs were discovered in Arabidopsis. Moreover, 13 genes predicted to interact with some of the above-identified target genes at protein level were validated as regulated by 17 AGO1-enriched sRNAs and ten miRNAs in Arabidopsis. In rice, only one SAG was targeted by three AGO1-enriched sRNAs, and one SAG was targeted by miR395. However, five AGO1-enriched sRNAs were conserved between Arabidopsis and rice. Target genes conserved between the two plants were identified for three of the above five sRNAs, pointing to the conserved roles of these regulatory pairs in leaf senescence or other developmental procedures. Novel targets were discovered for three of the five AGO1-enriched sRNAs in rice, indicating species-specific functions of these sRNA-target pairs. These results could advance our understanding of the sRNA-involved molecular processes modulating leaf senescence. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA

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Zwieb Christian

2010-11-01

Full Text Available Abstract Background Human cells depend critically on the signal recognition particle (SRP for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain. Results We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA. Conclusions The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

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Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

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Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

2012-01-01

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA

NARCIS (Netherlands)

Fontijn, R.; Hop, C.; Brinkman, H. J.; Slater, R.; Westerveld, A.; van Mourik, J. A.; Pannekoek, H.

1995-01-01

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more

Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle.

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Smith, Johanna A; Daniel, Rene

2016-11-13

Vaginal transmission is crucial to the spread of HIV-1 around the world. It is not yet clear what type (s) of innate defenses against HIV-1 infection are present in the vagina. Here, we aimed to determine whether human vaginal fluid contains exosomes that may possess anti-HIV-1 activity. The exosomal fraction was isolated from samples of vaginal fluids. The presence of exosomes was confirmed by flow cytometry and western blotting. The newly discovered exosomes were tested for their ability to block early steps of HIV-1 infection in vitro using established cell culture systems and real time PCR-based methods. Vaginal fluid contains exosomes expressing CD9, CD63, and CD81 exosomal markers. The exosomal fraction of the fluid-reduced transmission of HIV-1 vectors by 60%, the efficiency of reverse transcription step by 58.4%, and the efficiency of integration by 47%. Exosomes had no effect on the entry of HIV-1 vectors. Human vaginal fluid exosomes are newly discovered female innate defenses that may protect women against HIV-1 infection.

The Argonaute protein TbAGO1 contributes to large and mini-chromosome segregation and is required for control of RIME retroposons and RHS pseudogene-associated transcripts.

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Durand-Dubief, Mickaël; Absalon, Sabrina; Menzer, Linda; Ngwabyt, Sandra; Ersfeld, Klaus; Bastin, Philippe

2007-12-01

The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.

A microneedle patch containing measles vaccine is immunogenic in non-human primates.

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Edens, Chris; Collins, Marcus L; Goodson, James L; Rota, Paul A; Prausnitz, Mark R

2015-09-08

Very high vaccination coverage is required to eliminate measles, but achieving high coverage can be constrained by the logistical challenges associated with subcutaneous injection. To simplify the logistics of vaccine delivery, a patch containing micron-scale polymeric needles was formulated to encapsulate the standard dose of measles vaccine (1000 TCID₅₀) and the immunogenicity of the microneedle patch was compared with subcutaneous injection in rhesus macaques. The microneedle patch was administered without reconstitution with diluent, dissolved in skin within 10 min, and caused only mild, transient skin erythema. Both groups of rhesus macaques generated neutralizing antibody responses to measles that were consistent with protection and the neutralizing antibody titers were equivalent. In addition, the microneedle patches maintained an acceptable level of potency after storage at elevated temperature suggesting improved thermostability compared to standard lyophilized vaccine. In conclusion, a measles microneedle patch vaccine was immunogenic in non-human primates, and this approach offers a promising delivery method that could help increase vaccination coverage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Choline acetyltransferase-containing neurons in the human parietal neocortex

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V Benagiano

2009-06-01

Full Text Available A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT, the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.

Intratracheal injection of adenovirus containing the human MNSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis

International Nuclear Information System (INIS)

Epperly, Michael W.; Bray, Jenifer A.; Krager, Stephen; Berry, Luann M.; Gooding, William; Engelhardt, John F.; Zwacka, Ralf; Travis, Elizabeth L.; Greenberger, Joel S.

1999-01-01

Purpose: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. Methods and Materials: Athymic Nude (Nu/J) mice were intratracheally injected with 10 9 plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-α, TGF-β, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H and E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. Results: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry

Generation of a panel of somatic cell hybrids containing unselected fragments of human chromosome 10 by X-ray irradiation and cell fusion: Application to isolating the MEN2A region in hybrid cells

International Nuclear Information System (INIS)

Goodfellow, P.J.; Povey, S.; Nevanlinna, H.A.; Goodfellow, P.N.

1990-01-01

We have used X-ray irradiation and cell fusion to generate somatic cell hybrids containing fragments of human chromosome 10. Our experiments were directed towards isolating the region of the MEN2A gene in hybrids and to use those as the source of DNA for cloning and mapping new markers from near the MEN2A locus. A number of hybrid clones containing human sequences that are tightly linked to the MEN2A gene were identified. Some 25% of our hybrids, however, proved to contain more than one human chromosome 10-derived fragment or showed evidence of deletions and/or rearrangements. A detailed analysis of the human content of X-ray irradiation hybrids is required to assess the integrity and number of human fragments retained. Despite retention of multiple human-derived fragments, these hybrids will prove useful as cloning and mapping resources

Immunocapture of CD133-positive cells from human cancer cell lines by using monodisperse magnetic poly(glycidyl methacrylate) microspheres containing amino groups

Energy Technology Data Exchange (ETDEWEB)

Kuan, Wei-Chih [Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhisung 621, Taiwan (China); Horák, Daniel, E-mail: horak@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6 (Czech Republic); Plichta, Zdeněk [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6 (Czech Republic); Lee, Wen-Chien [Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhisung 621, Taiwan (China)

2014-01-01

Magnetic poly(glycidyl methacrylate)-based macroporous microspheres with an average particle size of 4.2 μm were prepared using a modified multi-step swelling polymerization method and by introducing amino functionality on their surfaces. Antibody molecules were oxidized on their carbohydrate moieties and bound to the amino-containing magnetic microspheres via a site-directed procedure. CD133-positive cells could be effectively captured from human cancer cell lines (HepG2, HCT116, MCF7, and IMR-32) by using magnetic microspheres conjugated to an anti-human CD133 antibody. After further culture, the immunocaptured CD133-expressing cells from IMR-32 proliferated and gradually detached from the magnetic microspheres. Flow-cytometric analysis confirmed the enrichment of CD133-expressing cells by using the antibody-bound magnetic microspheres. Such microspheres suitable for immunocapture are very promising for cancer diagnosis because the CD133-expressing cells in cancer cell lines have been suggested to be cancer stem cells. - Highlights: • Multi-step swelling polymerization produced poly(glycidyl methacrylate) microspheres. • Anti-human CD133 antibodies were bound to the amino-containing magnetic microspheres. • CD133-positive cells were effectively captured from human cancer cell lines. • Immunocaptured CD133-expressing cells proliferated and were detached from microspheres. • Enrichment of CD133-expressing cells was confirmed by flow-cytometric analysis.

An In Vitro RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity.

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Morin, Benjamin; Liang, Bo; Gardner, Erica; Ross, Robin A; Whelan, Sean P J

2017-01-01

We report an in vitro RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression. The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L

Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing

Energy Technology Data Exchange (ETDEWEB)

Dumortier, Jerome; Streblow, Daniel N.; Moses, Ashlee V.; Jacobs, Jon M.; Kreklywich, Craig N.; Camp, David G.; Smith, Richard D.; Orloff, Susan L.; Nelson, Jay

2008-07-01

Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

International Nuclear Information System (INIS)

Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

1990-01-01

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

Mercury-Containing Devices and Demolition

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Some items inside residential buildings contain mercury, which poses a persistent and toxic human health and environmental threat. These materials should be carefully salvaged for proper recycling to prevent mercury contamination prior to demolition.

Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

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Poulsen, Ebbe Toftgaard; Pedersen, Kata Wolff; Marzeda, Anna Maria; Enghild, Jan J

2017-02-14

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca 2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

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Aktaş, Tuğçe; Avşar Ilık, İbrahim; Maticzka, Daniel; Bhardwaj, Vivek; Pessoa Rodrigues, Cecilia; Mittler, Gerhard; Manke, Thomas; Backofen, Rolf; Akhtar, Asifa

2017-04-06

Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post

The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

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Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

2014-07-18

In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

Science.gov (United States)

Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

2011-12-20

Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Science.gov (United States)

Moser, Joanna J; Fritzler, Marvin J

2010-10-18

GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

The microRNA and messengerRNA profile of the RNA-induced silencing complex in human primary astrocyte and astrocytoma cells.

Directory of Open Access Journals (Sweden)

Joanna J Moser

2010-10-01

Full Text Available GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA-mediated messenger RNA (mRNA silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC. To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1 miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2 astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3 miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4 the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Potential anticancer activity of curcumin analogs containing sulfone on human cancer cells

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Zhang Qiuyan

2016-01-01

Full Text Available Three curcumin analogs(S1-S3 containing sulfone were investigated for their effects on human prostate cancer PC-3, colon cancer HT-29, lung cancer H1299 and pancreatic cancer BxPC-3 cells. The three compounds were approximately 16-to 96-fold more active than curcumin in these cell lines as determined by the MTT assay. The effects of these compounds on cell growth were further studied in prostate cancer PC-3 cells in both two dimensional (2D and three dimensional (3D cultures. S1-S3strongly inhibited the growth and induced cell death in PC-3 cells, and the effects of these compounds were associated with suppression of nuclear factor kappa B (NF-κB transcriptional activity. Moreover, treatment of PC-3 cells with all three compounds caused a decrease in the level of phosphorylated signal transducer and activator of transcription-3 (p-STAT3 (Tyr705,but not p-STAT3(Ser727. Only S1and S2decreased the presence of phosphorylated Akt (p-Akt in PC-3 cells. These curcumin analogs warrant further in vivo studies for anticancer activities in suitable animal models.

p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.

Science.gov (United States)

Ihry, Robert J; Worringer, Kathleen A; Salick, Max R; Frias, Elizabeth; Ho, Daniel; Theriault, Kraig; Kommineni, Sravya; Chen, Julie; Sondey, Marie; Ye, Chaoyang; Randhawa, Ranjit; Kulkarni, Tripti; Yang, Zinger; McAllister, Gregory; Russ, Carsten; Reece-Hoyes, John; Forrester, William; Hoffman, Gregory R; Dolmetsch, Ricardo; Kaykas, Ajamete

2018-06-11

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells 1-3 . Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells 3-13 . Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

Immunization of early adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant.

NARCIS (Netherlands)

Pedersen, C.; Petaja, T.; Strauss, G.; Rumke, H.C.; Poder, A.; Richardus, J.H.; Spiessens, B.; Descamps, D.; Hardt, K.; Lehtinen, M.; Dubin, G.

2007-01-01

PURPOSE: In female individuals 15-25-years of age, the AS04-containing human papillomavirus (HPV)-16/18 vaccine is highly immunogenic and provides up to 100% protection against HPV-16/18 persistent infection and associated cervical lesions up to 4.5 years. Optimal cervical cancer prevention will

21 CFR 700.16 - Use of aerosol cosmetic products containing zirconium.

Science.gov (United States)

2010-04-01

... zirconium. 700.16 Section 700.16 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... cosmetic products containing zirconium. (a) Zirconium-containing complexes have been used as an ingredient... indicates that certain zirconium compounds have caused human skin granulomas and toxic effects in the lungs...

IMP3 RNP Safe Houses Prevent miRNA-Directed HMGA2 mRNA Decay in Cancer and Development

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Lars Jønson

2014-04-01

Full Text Available The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.

NSun2-Mediated Cytosine-5 Methylation of Vault Noncoding RNA Determines Its Processing into Regulatory Small RNAs

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Shobbir Hussain

2013-07-01

Full Text Available Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs, yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs. Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.

The proliferative human monocyte subpopulation contains osteoclast precursors

Science.gov (United States)

Lari, Roya; Kitchener, Peter D; Hamilton, John A

2009-01-01

Introduction Immediate precursors of bone-resorbing osteoclasts are cells of the monocyte/macrophage lineage. Particularly during clinical conditions showing bone loss, it would appear that osteoclast precursors are mobilized from bone marrow into the circulation prior to entering tissues undergoing such loss. The observed heterogeneity of peripheral blood monocytes has led to the notion that different monocyte subpopulations may have special or restricted functions, including as osteoclast precursors. Methods Human peripheral blood monocytes were sorted based upon their degree of proliferation and cultured in macrophage colony-stimulating factor (M-CSF or CSF-1) and receptor activator of nuclear factor-kappa-B ligand (RANKL). Results The monocyte subpopulation that is capable of proliferation gave rise to significantly more multinucleated, bone-resorbing osteoclasts than the bulk of the monocytes. Conclusions Human peripheral blood osteoclast precursors reside in the proliferative monocyte subpopulation. PMID:19222861

APOBEC3DE Inhibits LINE-1 Retrotransposition by Interacting with ORF1p and Influencing LINE Reverse Transcriptase Activity.

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Weizi Liang

Full Text Available Human long interspersed elements 1 (LINE-1 or L1 is the only autonomous non-LTR retroelement in humans and has been associated with genome instability, inherited genetic diseases, and the development of cancer. Certain human APOBEC3 family proteins are known to have LINE-1 restriction activity. The mechanisms by which APOBEC3 affects LINE-1 retrotransposition are not all well characterized; here, we confirm that both A3B and A3DE have a strong ability to inhibit LINE-1 retrotransposition. A3DE interacts with LINE-1 ORF1p to target LINE-1 ribonucleoprotein particles in an RNA-dependent manner. Moreover, A3DE binds to LINE-1 RNA and ORF1 protein in cell culture system. Fluorescence microscopy demonstrated that A3DE co-localizes with ORF1p in cytoplasm. Furthermore, A3DE inhibits LINE-1 reverse transcriptase activity in LINE-1 ribonucleoprotein particles in a cytidine deaminase-independent manner. In contrast, A3B has less inhibitory effects on LINE-1 reverse transcriptase activity despite its strong inhibition of LINE-1 retrotransposition. This study demonstrates that different A3 proteins have been evolved to inhibit LINE-1 activity through distinct mechanisms.

Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities.

Science.gov (United States)

Didychuk, Allison L; Montemayor, Eric J; Carrocci, Tucker J; DeLaitsch, Andrew T; Lucarelli, Stefani E; Westler, William M; Brow, David A; Hoskins, Aaron A; Butcher, Samuel E

2017-09-08

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3' phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2-8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome.The mechanism of U6 small nuclear ribonucleoprotein (snRNP) biogenesis is not well understood. Here the authors characterize the enzymatic activities and structures of yeast and human U6 RNA processing enzyme Usb1, reconstitute post-transcriptional assembly of yeast U6 snRNP in vitro, and propose a model for U6 snRNP assembly.

Inhibition of growth of human breast cancer cells in culture by neutron capture using liposomes containing 10B.

Science.gov (United States)

Yanagië, H; Kobayashi, H; Takeda, Y; Yoshizaki, I; Nonaka, Y; Naka, S; Nojiri, A; Shinnkawa, H; Furuya, Y; Niwa, H; Ariki, K; Yasuhara, H; Eriguchi, M

2002-03-01

Cell destruction in boron neutron capture therapy is effected by nuclear reaction between 10B and thermal neutrons with the release of alpha-particles (4He) and lithium-7 ions (7Li). 4He kills cells within 10 microm of the site of 4He generation, therefore it is theoretically possible to destroy tumour cells without affecting adjacent healthy tissue, given selective delivery of compounds containing 10B. Liposomes wore prepared by vortex dispersion of solutions containing 10B compounds with dried lipid films and the effects of those compounds on human breast cancer cells in culture were examined after thermal neutral irradiation. [3H]-TdR incorporation by MRKnu/nu-1 cells treated with 10B-containing liposomes showed 40% suppression compared with liposomes without 10B, at 2 x 1012 n/cm2 thermal neutron fluence. Inhibition of tumour cell growth with liposomes prepared with 100 mm 10B-compound was as significant as with those made with 500 ppm 10B solution. The concentration of 10B in liposomes was 76.5 +/- 3.4 microg/mL. Boronated liposomes can thus deliver sufficient 10B atoms to this line of breast cancer cells in culture to effect cytotoxicity and suppression of growth after thermal neutron irradiation.

Detection of cyclobutane thymine dimers in DNA of human cells with monoclonal antibodies raised against a thymine dimer-containing tetranucleotide

Energy Technology Data Exchange (ETDEWEB)

Roza, L; Wulp, K.J.M. van der; MacFarlane, S J; Lohman, P H.M.; Baan, R A

1988-11-01

A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)/sub 5/. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m/sup 2/). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.

Susceptibility of Mice to Trypanosoma evansi Treated with Human Plasma Containing Different Concentrations of Apolipoprotein L-1

Science.gov (United States)

Fanfa, Vinicius R.; Otto, Mateus A.; Gressler, Lucas T.; Tavares, Kaio C.S.; Lazzarotto, Cícera R.; Tonin, Alexandre A.; Miletti, Luiz C.; Duarte, Marta M.M.F.; Monteiro, Silvia G.

2011-01-01

The aim of this study was to test the susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1 (APOL1). For this experiment, a strain of T. evansi and human plasma (plasmas 1, 2, and 3) from 3 adult males clinically healthy were used. In vivo test used 50 mice divided in 5 groups (A to E) with 10 animals in each group. Animals of groups B to E were infected, and then treated with 0.2 ml of human plasma in the following outline: negative control (A), positive control (B), treatment with plasma 1 (C), treatment with plasma 2 (D), and treatment with plasma 3 (E). Mice treated with human plasma showed an increase in longevity of 40.9±0.3 (C), 20±9.0 (D) and 35.6±9.3 (E) days compared to the control group (B) which was 4.3±0.5 days. The number of surviving mice and free of the parasite (blood smear and PCR negative) at the end of the experiment was 90%, 0%, and 60% for groups C, D, and E, respectively. The quantification of APOL1 was performed due to the large difference in the treatments that differed in the source plasma. In plasmas 1, 2, and 3 was detected the concentration of 194, 99, and 115 mg/dl of APOL1, respectively. However, we believe that this difference in the treatment efficiency is related to the level of APOL1 in plasmas. PMID:22355213

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

Science.gov (United States)

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

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Aaron P. Landry

2014-01-01

Full Text Available Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0. The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss and double-stranded (ds DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes.

Science.gov (United States)

Steinhagen, Folkert; Zillinger, Thomas; Peukert, Konrad; Fox, Mario; Thudium, Marcus; Barchet, Winfried; Putensen, Christian; Klinman, Dennis; Latz, Eicke; Bode, Christian

2018-04-01

Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi-Goutières Syndrome (AGS). The recently discovered cyclic-GMP-AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1-deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self-DNA. Collectively, these findings might lead to the development of new therapeutics against IFN-driven pathologies due to cGAS activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

75 FR 33744 - Pesticide Management and Disposal; Standards for Pesticide Containers and Containment; Proposed...

Science.gov (United States)

2010-06-15

... the likelihood of unreasonable adverse effects on human health and the environment. The container and... submitted soon. This is based on an evaluation of the resources and the time it would take for EPA to..., and the FIFRA Scientific Advisory Panel (SAP). The SAP and the Secretary of Agriculture waived review...

Structural basis for the inhibition of human alkyladenine DNA glycosylase (AAG) by 3,N4-ethenocytosine-containing DNA.

Science.gov (United States)

Lingaraju, Gondichatnahalli M; Davis, C Ainsley; Setser, Jeremy W; Samson, Leona D; Drennan, Catherine L

2011-04-15

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

Human retroviruses and AIDS, 1991. [CONTAINS GLOSSARY

Energy Technology Data Exchange (ETDEWEB)

Myers, G.; Korber, B. (eds.) (Los Alamos National Lab., NM (USA)); Berzofsky, J.A.; Pavlakis, G.N. (eds.) (National Cancer Inst., Bethesda, MD (USA)); Smith, R.F. (ed.) (Harvard Univ. (USA))

1991-05-01

This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses.The scope of the compendium and database is best summarized by the five parts that it comprises: (1) HIV and SIV Nucleotide Sequences; (2) Amino Acid Sequences; (3) Analyses; (4) Related Sequences; and (5) Database Communications. Information within all the parts is updated at least twice in each year, which accounts for the modes of binding and pagination in the compendium.

Sensitivity of human peripheral lymphocyte chromosomes to various X-ray doses and subsequent storage in Plexiglass or glass containers

International Nuclear Information System (INIS)

Ivanov, B.; Bulanova, M.; Geogieva, I.

1979-01-01

A study was performed to determine whether chromosomal aberrations produced in vitro by various X-ray doses in human lymphocytes were affected by post-irradiation storage of the blood in plastic or glass containers. Following X-ray doses of up to 400 R, the yields of cells with aberrations and the incidence of dicentrics, rings, interstitial deletions, symmetrical changes and chromosome fragments increased with dose. After storage of the irradiated lymphocytes in either Plexiglass or glass, the values for exchange aberrations, deletions and aberrant cells were compared. The only statistically significant difference was a slight increase in the percentage of aberrant cells stored in the plastic containers at the 400 R dose level. It was concluded that plastics appear to have a sensitizing effect on the genetic structure of the peripheral lymphocyte and thus the use of this material to store blood in biological dosimetry studies should be discouraged. (U.K.)

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

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Hsiao Chiu-Bin

2006-11-01

Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

KAUST Repository

Vishal, Chaturvedi

2011-06-07

Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/ MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity. the author(s), publisher and licensee Libertas Academica Ltd.

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans.

Science.gov (United States)

Opländer, Christian; Volkmar, Christine M; Paunel-Görgülü, Adnana; Fritsch, Thomas; van Faassen, Ernst E; Mürtz, Manfred; Grieb, Gerrit; Bozkurt, Ahmet; Hemmrich, Karsten; Windolf, Joachim; Suschek, Christoph V

2012-02-15

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively. Copyright © 2012 Elsevier Inc. All rights reserved.

Human systemic exposure to [¹⁴C]-paraphenylenediamine-containing oxidative hair dyes: Absorption, kinetics, metabolism, excretion and safety assessment.

Science.gov (United States)

Nohynek, Gerhard J; Skare, Julie A; Meuling, Wim J A; Wehmeyer, Kenneth R; de Bie, Albertus Th H J; Vaes, Wouter H J; Dufour, Eric K; Fautz, Rolf; Steiling, Winfried; Bramante, Mario; Toutain, Herve

2015-07-01

Systemic exposure was measured in humans after hair dyeing with oxidative hair dyes containing 2.0% (A) or 1.0% (B) [(14)C]-p-phenylenediamine (PPD). Hair was dyed, rinsed, dried, clipped and shaved; blood and urine samples were collected for 48 hours after application. [(14)C] was measured in all materials, rinsing water, hair, plasma, urine and skin strips. Plasma and urine were also analysed by HLPC/MS/MS for PPD and its metabolites (B). Total mean recovery of radioactivity was 94.30% (A) or 96.21% (B). Mean plasma Cmax values were 132.6 or 97.4 ng [(14)C]-PPDeq/mL, mean AUC(0-∞) values 1415 or 966 ng [(14)C]-PPDeq/mL*hr in studies A or B, respectively. Urinary excretion of [(14)C] mainly occurred within 24 hrs after hair colouring with a total excretion of 0.72 or 0.88% of applied radioactivity in studies A or B, respectively. Only N,N'-diacetylated-PPD was detected in plasma and the urine. A TK-based human safety assessment estimated margins of safety of 23.3- or 65-fold relative to respective plasma AUC or Cmax values in rats at the NOAEL of a toxicity study. Overall, hair dyes containing PPD are unlikely to pose a health risk since they are used intermittently and systemic exposure is limited to the detoxified metabolite N,N'-diacetyl-PPD. Copyright © 2015 Elsevier Ltd. All rights reserved.

MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

Science.gov (United States)

Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

2010-04-08

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

MicroRNA–Directed siRNA Biogenesis in Caenorhabditis elegans

Science.gov (United States)

Corrêa, Régis L.; Steiner, Florian A.; Berezikov, Eugene; Ketting, René F.

2010-01-01

RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi–related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer. PMID:20386745

21 CFR 310.509 - Parenteral drug products in plastic containers.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Parenteral drug products in plastic containers... Parenteral drug products in plastic containers. (a) Any parenteral drug product packaged in a plastic... parenteral drug product for intravenous use in humans that is packaged in a plastic immediate container on or...

Halophytic plants as a component of a bioregenerative life support system for recycling of NaCl contained in human liquid waste.

Science.gov (United States)

Balnokin, Yurii; Balnokin, Yurii; Myasoedov, Nikolay; Popova, Larissa; Tikhomirov, Alexander A.; Ushakova, Sofya; Tikhomirova, Natalia; Lasseur, Christophe; Gros, Jean-Bernard

Currently, the closure of matter turnover is one of the urgent problems of bioregenerative life support system (BLSS) designing. The important aspect of the problem is involving of substances contained in liquid and solid exometabolites of humans inhabiting BLSS into intrasystem matter turnover. Recycling of Na+ and Cl- contained in human liquid exometabolites, i.e. urine is acknowledged to be among the main tasks of the matter turnover in BLSS. The ions excreted with urine may be returned to human organism with food. A way to allow this is including edible halophytic plants into the phototrophic compartment of BLSS. Halophytes are defined as plants which can grow on saline soils and produce high biomass under these conditions. Some halophytes can take up high quantities of Na+ and Cl- and accumulate the ions in the shoots or extrude them to leaf surface by means of salt glands. To allow Na+ and Cl- recycling through halophyte utilization, the following principal steps should be accomplished: (i) mineralization of the exometabolites by physicochemical methods; (ii) oxidation of ammonia formed during the exometabolite mineralization to nitrate by nitrifying bacteria, (iii) growing the halophyte on the nutrient solution prepared on the basis of the mineralized exometabolites, (iv) introducing the halophyte green biomass into human food. The present work is devoted to the following problems: (i) selection of a salt-accumulating/extruding halophytic plant suitable for Na+ and Cl- recycling in BLSS and (ii) parameter evaluation of a plant conveyor containing the halophytic plants at various ages. Halophytic plants selected for BLSS should meet the following criteria: (i) ability to grow under 24-hour-illumination, (ii) high productivity, (iii) ability to accumulate Na+ and Cl- in high quantities in shoots or to excrete salts to leaf surface, (iv) edibility, and (v) high nutritive value of the biomass. Relying on these criteria, salt-accumulating halophyte Salicornia

Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes.

Science.gov (United States)

Iwasaki, Shintaro; Kobayashi, Maki; Yoda, Mayuko; Sakaguchi, Yuriko; Katsuma, Susumu; Suzuki, Tsutomu; Tomari, Yukihide

2010-07-30

Small silencing RNAs--small interfering RNAs (siRNAs) or microRNAs (miRNAs)--direct posttranscriptional gene silencing of their mRNA targets as guides for the RNA-induced silencing complex (RISC). Both siRNAs and miRNAs are born double stranded. Surprisingly, loading these small RNA duplexes into Argonaute proteins, the core components of RISC, requires ATP, whereas separating the two small RNA strands within Argonaute does not. Here we show that the Hsc70/Hsp90 chaperone machinery is required to load small RNA duplexes into Argonaute proteins, but not for subsequent strand separation or target cleavage. We envision that the chaperone machinery uses ATP and mediates a conformational opening of Ago proteins so that they can receive bulky small RNA duplexes. Our data suggest that the chaperone machinery may serve as the driving force for the RISC assembly pathway. Copyright 2010 Elsevier Inc. All rights reserved.

Expression of human Piwi-like genes is associated with prognosis for soft tissue sarcoma patients

International Nuclear Information System (INIS)

Greither, Thomas; Taubert, Helge; Koser, Franziska; Kappler, Matthias; Bache, Matthias; Lautenschläger, Christine; Göbel, Steffen; Holzhausen, Hans-Jürgen; Wach, Sven; Würl, Peter

2012-01-01

Argonaute genes are essential for RNA interference, stem cell maintenance and differentiation. The Piwi-like genes, a subclass of the Argonaute genes, are expressed mainly in the germline. These genes may be re-expressed in tumors, and expression of the Piwi-like genes is associated with prognosis in several types of tumors. We measured the expression of Piwi-like mRNAs (Piwi-like 2–4) in 125 soft tissue sarcoma (STS) samples by qPCRs. Statistical tests were applied to study the correlation of expression levels with tumor-specific survival for STS patients. In multivariate Cox’s regression analyses, we showed that low Piwi-like 2 and Piwi-like 4 mRNA expression were significantly associated with a worse prognosis (RR = 1.87; p = 0.032 and RR = 1.82; p = 0.039). Low expression of both genes was associated with a 2.58-fold increased risk of tumor-related death (p = 0.01). Piwi-like 4 and combined Piwi-like 2 and 4 mRNA levels correlated significantly with prognosis (RR = 3.53; p = 0.002 and RR = 5.23; p = 0.004) only for female but not for male patients. However, combined low Piwi-like 2 and 3 transcript levels were associated with worse survival (RR = 5.90; p = 0.02) for male patients. In this study, we identified a significant association between the expression of Piwi-like 2 and 4 mRNAs and the tumor-specific survival of soft tissue sarcoma patients. Furthermore, a connection between sex and the impact of Piwi-like mRNA expressions on STS patients’ prognosis was shown for the first time

Differentiated human midbrain-derived neural progenitor cells express excitatory strychnine-sensitive glycine receptors containing α2β subunits.

Directory of Open Access Journals (Sweden)

Florian Wegner

Full Text Available BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+-K(+-Cl(- co-transporter 1 (NKCC1-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro.

Crystal Structure of the Human, FIC-Domain Containing Protein HYPE and Implications for Its Functions

Science.gov (United States)

Bunney, Tom D.; Cole, Ambrose R.; Broncel, Malgorzata; Esposito, Diego; Tate, Edward W.; Katan, Matilda

2014-01-01

Summary Protein AMPylation, the transfer of AMP from ATP to protein targets, has been recognized as a new mechanism of host-cell disruption by some bacterial effectors that typically contain a FIC-domain. Eukaryotic genomes also encode one FIC-domain protein, HYPE, which has remained poorly characterized. Here we describe the structure of human HYPE, solved by X-ray crystallography, representing the first structure of a eukaryotic FIC-domain protein. We demonstrate that HYPE forms stable dimers with structurally and functionally integrated FIC-domains and with TPR-motifs exposed for protein-protein interactions. As HYPE also uniquely possesses a transmembrane helix, dimerization is likely to affect its positioning and function in the membrane vicinity. The low rate of autoAMPylation of the wild-type HYPE could be due to autoinhibition, consistent with the mechanism proposed for a number of putative FIC AMPylators. Our findings also provide a basis to further consider possible alternative cofactors of HYPE and distinct modes of target-recognition. PMID:25435325

21 CFR 211.94 - Drug product containers and closures.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Drug product containers and closures. 211.94 Section 211.94 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... and use that can cause deterioration or contamination of the drug product. (c) Drug product containers...

Self-contained induction of neurons from human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Tsuyoshi Okuno

Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

Human skeletal muscle contains no detectable guanidinoacetic acid

DEFF Research Database (Denmark)

Ostojic, Sergej M; Ostojic, Jelena

2018-01-01

We analyzed data from previously completed trials to determine the effects of supplemental guanidinoacetic acid (GAA) on markers of muscle bioenergetics in healthy men using 1.5 T magnetic resonance spectroscopy. No detectable GAA (<0.1 μmol/L) was found in the vastus medialis muscle at baseline ...... nor at follow-up. This implies deficient GAA availability in the human skeletal muscle, suggesting absent or negligible potential for creatine synthesis from GAA inside this tissue, even after GAA loading....

Quantitative determination of BAF312, a S1P-R modulator, in human urine by LC-MS/MS: prevention and recovery of lost analyte due to container surface adsorption.

Science.gov (United States)

Li, Wenkui; Luo, Suyi; Smith, Harold T; Tse, Francis L S

2010-02-15

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC-MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the 'non-specific loss' of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC-MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the 'non-specific binding' of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. beta-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of beta-cyclodextrin to untreated urine samples does not recover the 'lost' analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200-20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from -3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid-liquid extraction in combination with an isocratic LC separation was employed. The

Engineering of blood vessel patterns by angio-morphogens [angiotropins]: non-mitogenic copper-ribonucleoprotein cytokins [CuRNP ribokines] with their metalloregulated constituents of RAGE-binding S100-EF-hand proteins and extracellular RNA bioaptamers in vascular remodeling of tissue and angiogenesis in vitro

Energy Technology Data Exchange (ETDEWEB)

Wissler, J.H. [ARCONS Applied Research, Bad Nauheim (Germany)

2001-12-01

Tissue vascularization is requisite to successful cell-based therapies, biomaterial design and implant integration. Thus, known problems in ossointegration of avascular implants in connection with the generation of bone tissue reflect arrays of general problems of socio-economic relevance existing in reparative medicine still waiting for to be solved. For this purpose, morphogenesis and remodeling of endothelial angio-architectures in tissue and in vitro by isolated non-mitogenic angio-morphogens [angiotropins] are considered in terms of their structure, function and action mechanisms. Extracellular angiotropins are secreted by activated leukocytes/monocytes/macrophages. They are a family of cytokines with morphogen bioactivity selectively directed to endothelial cells. Their structure was deciphered as metalloregulated copper-ribonucleoproteins [CuRNP ribokines]. They are built up of angiotropin-related S100-EF-hand protein [ARP] and highly modified and edited 5'end-phosphorylated RNA [ARNA], complexed together by copper ions. Oxidant-sensitive ARNA and their precursors represent novel types in a RNA world: They are the first isolated and sequenced forms of extracellular RNA [eRNA], may act as cytokine and bioaptamer, contain isoguanosine [crotonoside] as modified nucleoside and show up copper as RNA-structuring transition metal ion. By metalloregulated bioaptamer functions, ARNA impart novel biofunctions to RAGE-binding S100-EF-hand proteins. Angiotropin morphogens were shown suitable for neointiation and remodeling of blood vessel patterns in different, adult, embryonal and artificial tissues. These neovascular patterns manifest regulated hemodynamics for preventing tissue necrosis, supporting tissue functions and promoting wound healing. As evaluated in skin and muscle vascularization, the neovascular patterns are integrated into homeostatic control mechanisms of tissue. Thus, the morphogens show up beneficial perspectives and are suggested useful tools

Prednisolone phosphate-containing TRX-20 liposomes inhibit cytokine and chemokine production in human fibroblast-like synovial cells: a novel approach to rheumatoid arthritis therapy.

Science.gov (United States)

Harigai, Takashi; Hagiwara, Hitomi; Ogawa, Yumi; Ishizuka, Takanobu; Kaneda, Shinichi; Kimura, Junji

2007-01-01

To evaluate the potential of using prednisolone phosphate (PSLP)-containing 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) liposomes to treat rheumatoid arthritis (RA), we examined their ability to bind human fibroblast-like synovial (HFLS) cells and their effects in these cells. To test for binding, Lissamine rhodamine B-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine)-labelled PSLP-containing TRX-20 liposomes were added to HFLS cells, and the fluorescence intensity of the rhodamine bound to the cells was evaluated. Rhodamine-labelled PSLP-containing liposomes without TRX-20 were used as a negative control. To evaluate the uptake of liposomes by the HFLS cells, we used TRX-20 liposomes containing 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and p-xylene-bis-pyridinium bromide (DPX), and observed the cells by fluorescence microscopy. The effects of the PSLP in TRX-20 liposomes on HFLS cells were assessed by the inhibition of the production of two inflammatory cytokines (interleukin 6 and granulocyte macrophage colony-stimulating factor) and one inflammatory chemokine (interleukin 8). The interaction of the PSLP-containing TRX-20 liposomes with HFLS cells was approximately 40 times greater than that of PSLP-containing liposomes without TRX-20. PSLP-containing TRX-20 liposomes bound to HFLS cells primarily via chondroitin sulfate. TRX-20 liposomes taken up by the cell were localized to acidic compartments. Furthermore, the PSLP-containing TRX-20 liposomes inhibited the production of the inflammatory cytokines and the chemokine more effectively than did the PSLP-containing liposomes without TRX-20. These results indicate that PSLP-containing TRX-20 liposomes show promise as a novel drug delivery system that could enhance the clinical use of glucocorticoids for treating RA.

Trends in the development of reactor containments

International Nuclear Information System (INIS)

Turricchia, A.

1977-01-01

The paper presents a review of the technical developments which the containment systems of BWRs, PWRs and HWRs have undergone in the USA, Europe and Canada. The great variety of existing containment systems have been classified for each type of reactor, according to: principle of operation (i.e. dry containment; pressure suppression containment; containment with vacuum building; sub-atmospheric containment; etc.); type and number of the physical barriers provided against the dispersion of fission products to the environment (single containment, double containment, single containment with local collection of the leaks from the most probable leakage paths, or with pressurization of the potential leakage paths, etc.); and structural characteristics of the containment barriers (reinforced concrete, prestressed concrete, metal containment, liners). In the framework of this classification the main features of the various containment systems are illustrated, with special emphasis on the most modern designs. The difference in trend among various countries and various designers are highlighted. A review is also made of the various containment auxiliary systems which would operate in accident conditions (isolation systems, heat removal system, atmosphere control system, etc.). Also for these systems the main design criteria are set forth and present trends are described. Finally, a brief survey is also made of the present trends with regards to the design of containment structures against external events be they of natural origin (earthquake, tornadoes, etc.) or connected with human activities (airplane crash, explosions, etc.) [fr

Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

OpenAIRE

Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

2003-01-01

For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

Human Capital Accumulation: The Role of Human Resource Development.

Science.gov (United States)

Garavan, Thomas N.; Morley, Michael; Gunnigle, Patrick; Collins, Eammon

2001-01-01

Presents definitions of intellectual and human capital. Examines human capital from the individual perspective (employability, performance, career development) and organization perspective (investment, ownership, knowledge management). Reviews papers in the theme issue. (Contains 117 references.) (SK)

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein

Science.gov (United States)

Marsh, Elizabeth K.; Delury, Craig P.; Davies, Nicholas J.; Weston, Christopher J.; Miah, Mohammed A.L.; Banks, Lawrence; Parish, Joanna L.

2017-01-01

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification. PMID:28061478

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

Science.gov (United States)

Marsh, Elizabeth K; Delury, Craig P; Davies, Nicholas J; Weston, Christopher J; Miah, Mohammed A L; Banks, Lawrence; Parish, Joanna L; Higgs, Martin R; Roberts, Sally

2017-03-21

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

Identification of the functional domains of ANT-1, a novel coactivator of the androgen receptor

International Nuclear Information System (INIS)

Fan Shuli; Goto, Kiminobu; Chen Guangchun; Morinaga, Hidetaka; Nomura, Masatoshi; Okabe, Taijiro; Nawata, Hajime; Yanase, Toshihiko

2006-01-01

Previously, we identified a transcriptional coactivator for the activation function-1 (AF-1) domain of the human androgen receptor (AR) and designated it androgen receptor N-terminal domain transactivating protein-1 (ANT-1). This coactivator, which contains multiple tetratricopeptide repeat (TPR) motifs from amino acid (aa) 294, is identical to a component of U5 small nuclear ribonucleoprotein particles and binds specifically to the AR or glucocorticoid receptor. Here, we identified four distinct functional domains. The AR-AF-1-binding domain, which bound to either aa 180-360 or 360-532 in AR-AF-1, clearly overlapped with TAU-1 and TAU-5. This domain and the subnuclear speckle formation domain in ANT-1 were assigned within the TPR motifs, while the transactivating and nuclear localization signal domains resided within the N-terminal sequence. The existence of these functional domains may further support the idea that ANT-1 can function as an AR-AF-1-specific coactivator while mediating a transcription-splicing coupling

RNA-Interference Components Are Dispensable for Transcriptional Silencing of the Drosophila Bithorax-Complex

KAUST Repository

Cernilogar, Filippo M.

2013-06-13

Background:Beyond their role in post-transcriptional gene silencing, Dicer and Argonaute, two components of the RNA interference (RNAi) machinery, were shown to be involved in epigenetic regulation of centromeric heterochromatin and transcriptional gene silencing. In particular, RNAi mechanisms appear to play a role in repeat induced silencing and some aspects of Polycomb-mediated gene silencing. However, the functional interplay of RNAi mechanisms and Polycomb group (PcG) pathways at endogenous loci remains to be elucidated.Principal Findings:Here we show that the endogenous Dicer-2/Argonaute-2 RNAi pathway is dispensable for the PcG mediated silencing of the homeotic Bithorax Complex (BX-C). Although Dicer-2 depletion triggers mild transcriptional activation at Polycomb Response Elements (PREs), this does not induce transcriptional changes at PcG-repressed genes. Moreover, Dicer-2 is not needed to maintain global levels of methylation of lysine 27 of histone H3 and does not affect PRE-mediated higher order chromatin structures within the BX-C. Finally bioinformatic analysis, comparing published data sets of PcG targets with Argonaute-2-bound small RNAs reveals no enrichment of these small RNAs at promoter regions associated with PcG proteins.Conclusions:We conclude that the Dicer-2/Argonaute-2 RNAi pathway, despite its role in pairing sensitive gene silencing of transgenes, does not have a role in PcG dependent silencing of major homeotic gene cluster loci in Drosophila. © 2013 Cernilogar et al.

Fluorine uptake into the human enamel surface from fluoride-containing sealing materials during cariogenic pH cycling

Science.gov (United States)

Yasuhiro, Matsuda; Katsushi, Okuyama; Hiroko, Yamamoto; Hisanori, Komatsu; Masashi, Koka; Takahiro, Sato; Naoki, Hashimoto; Saiko, Oki; Chiharu, Kawamoto; Hidehiko, Sano

2015-04-01

To prevent the formation of caries and reduce dentin hypersensitivity, sealing materials, either with or without fluoride, are generally applied on the tooth in clinical practice. Application of fluoride-free sealing materials results in the formation of an acid-resistant layer on the tooth surface. On the other hand, fluoride-containing sealing materials might not only form an acid-resistant layer but could possibly further provide fluoride to enhance remineralization and reduce demineralization. In this study, the demineralization prevention ability and fluorine uptake rate in human enamel of fluoride-containing sealing materials ["MS coats F" (MSF)] and fluoride-free sealing materials ("hybrid coats 2" [HI]) were evaluated using an automatic pH cycling system. Each material was applied to the original tooth surface, the cut surfaces were covered with sticky wax, and the automatic pH-cycling system simulated daily acid changes (pH 6.8-4.5) occurring in the oral cavity for 4 weeks. Caries progression was analyzed using transverse microradiography (TMR) taken pre and post the 4 weeks of pH cycling. The fluorine and calcium distributions in the carious lesion in each specimen were evaluated using the proton-induced gamma emission (PIGE) and proton-induced X-ray (PIXE) techniques, respectively. TMR analysis showed that both MSF and HI had a caries-preventing effect after 4 weeks of pH cycling. PIGE/PIXE analysis demonstrated that only MSF resulted in fluoride uptake in the enamel surface. Therefore, MSF can help to form an acid-resistant layer and provide fluoride to the enamel surface. The presence of fluoride on the enamel surface suggested that MSF could prevent demineralization, even if the acid-resistant layer was removed, in clinical settings. The data obtained using the PIGE and PIXE techniques are useful for understanding the benefits of the use of a fluoride-containing sealing material for preventing caries.

The human Ago2 MC region does not contain an eIF4E-like mRNA cap binding motif

Directory of Open Access Journals (Sweden)

Grishin Nick V

2009-01-01

Full Text Available Abstract Background Argonaute (Ago proteins interact with small regulatory RNAs to mediate gene regulatory pathways. A recent report by Kiriakidou et al. 1 describes an MC sequence region identified in Ago2 that displays similarity to the cap-binding motif in translation initiation factor 4E (eIF4E. In a cap-bound eIF4E structure, two important aromatic residues of the motif stack on either side of a 7-methylguanosine 5'-triphosphate (m7Gppp base. The corresponding Ago2 aromatic residues (F450 and F505 were hypothesized to perform the same cap-binding function. However, the detected similarity between the MC sequence and the eIF4E cap-binding motif was questionable. Results A number of sequence-based and structure-based bioinformatics methods reveal the reported similarity between the Ago2 MC sequence region and the eIF4E cap-binding motif to be spurious. Alternatively, the MC sequence region is confidently assigned to the N-terminus of the Ago piwi module, within the mid domain of experimentally determined prokaryotic Ago structures. Confident mapping of the Ago2 MC sequence region to the piwi mid domain results in a homology-based structure model that positions the identified aromatic residues over 20 Å apart, with one of the aromatic side chains (F450 contributing instead to the hydrophobic core of the domain. Conclusion Correct functional prediction based on weak sequence similarity requires substantial evolutionary and structural support. The evolutionary context of the Ago mid domain suggested by multiple sequence alignment is limited to a conserved hydrophobicity profile required for the fold and a motif following the MC region that binds guide RNA. Mapping of the MC sequence to the mid domain structure reveals Ago2 aromatics that are incompatible with eIF4E-like mRNA cap-binding, yet display some limited local structure similarities that cause the chance sequence match to eIF4E. Reviewers This article was reviewed by Arcady Mushegian

Effects of Genetically Modified Milk Containing Human Beta-Defensin-3 on Gastrointestinal Health of Mice.

Directory of Open Access Journals (Sweden)

Xin Chen

Full Text Available This study was performed to investigate the effects of genetically modified (GM milk containing human beta-defensin-3 (HBD3 on mice by a 90-day feeding study. The examined parameters included the digestibility of GM milk, general physical examination, gastric emptying function, intestinal permeability, intestinal microflora composition of mice, and the possibility of horizontal gene transfer (HGT. The emphasis was placed on the effects on gastrointestinal (GI tract due to the fact that GI tract was the first site contacting with food and played crucial roles in metabolic reactions, nutrition absorption and immunity regulation in the host. However, the traditional methods for analyzing the potential toxicological risk of GM product pay little attention on GI health. In this study, the results showed GM milk was easy to be digested in simulated gastric fluid, and it did not have adverse effects on general and GI health compared to conventional milk. And there is little possibility of HGT. This study may enrich the safety assessment of GM product on GI health.

Research reactors in Austria - Present situation

International Nuclear Information System (INIS)

Boeck, H.; Musilek, A.; Villa, M.

2005-01-01

In the past decades Austria operated three research reactors, the 10 MW ASTRA reactor at Seibersdorf, the 250 kW TRIGA reactor at the Atominstitut and the 1 kW Argonaut reactor at the Technical University in Graz. Since the shut down of the ASTRA on July 31th, 1999 and its immediate decommissioning reactor and the shut down of the Argonaut reactor in Graz on August 31st, 2004 only one reactor remains operational for keeping nuclear competence in Austria which is the 250 kW TRIGA Mark II reactor. (author)

The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

Science.gov (United States)

Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

2016-08-15

Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

Compendium of botanicals reported to contain naturally occuring substances of possible concern for human health when used in food and food supplements

DEFF Research Database (Denmark)

Pilegaard, Kirsten

In April 2009, EFSA published on its website a Compendium of botanicals reported to contain toxic, addictive, psychotropic or other substances of concern. The purpose of the Compendium is to assist risk assessors responsible for the evaluation of specific ingredients in food supplements, in more...... one lists botanicals for which, although some data were available, the Scientific Committee could not identify substances of concern, or other reasons for the inclusion in the compendium. This new “Compendium of botanicals reported to contain naturally occuring substances of possible concern for human...... health” replaces the first version published in 2009; it lists in alphabetical order botanicals without any judgment on whether they are suitable or not suitable for food applications in Europe; it has no legal or regulatory force pertaining to the legal classification of products or substances....

Human Respiratory Syncytial Virus and Human Metapneumovirus

Directory of Open Access Journals (Sweden)

Luciana Helena Antoniassi da Silva

2009-08-01

Full Text Available The human respiratory syncytial virus (hRSV and the human metapneumovírus (hMPV are main etiological agents of acute respiratory infections (ARI. The ARI is an important cause of childhood morbidity and mortality worldwide.  hRSV and hMPV are members of the Paramyxoviridae. They are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV is the best characterized agent viral of this group, associated with respiratory diseases in lower respiratory tract. Recently, a new human pathogen belonging to the subfamily Pneumovirinae was identified, the human metapneumovirus (hMPV, which is structurally similar to the hRSV, in genomic organization, viral structure, antigenicity and clinical symptoms.  The subfamily Pneumovirinae contains two genera: genus Pneumovirus contains hRSV, the bovine (bRSV, as well as the ovine and caprine respiratory syncytial virus and pneumonia virus of mice, the second genus Metapneumovirus, consists of avian metapneumovirus (aMPV and human metapneumovirus (hMPV. In this work, we present a brief narrative review of the literature on important aspects of the biology, epidemiology and clinical manifestations of infections by two respiratory viruses.

Repeatability of two-dimensional chemical shift imaging multivoxel proton magnetic resonance spectroscopy for measuring human cerebral choline-containing compounds.

Science.gov (United States)

Puri, Basant K; Egan, Mary; Wallis, Fintan; Jakeman, Philip

2018-03-22

To investigate the repeatability of proton magnetic resonance spectroscopy in the in vivo measurement of human cerebral levels of choline-containing compounds (Cho). Two consecutive scans were carried out in six healthy resting subjects at a magnetic field strength of 1.5 T. On each occasion, neurospectroscopy data were collected from 64 voxels using the same 2D chemical shift imaging (CSI) sequence. The data were analyzed in the same way, using the same software, to obtain the values for each voxel of the ratio of Cho to creatine. The Wilcoxon related-samples signed-rank test, coefficient of variation (CV), repeatability coefficient (RC), and intraclass correlation coefficient (ICC) were used to assess the repeatability. The CV ranged from 2.75% to 33.99%, while the minimum RC was 5.68%. There was excellent reproducibility, as judged by significant ICC values, in 26 voxels. Just three voxels showed significant differences according to the Wilcoxon related-samples signed-rank test. It is therefore concluded that when CSI multivoxel proton neurospectroscopy is used to measure cerebral choline-containing compounds at 1.5 T, the reproducibility is highly acceptable.

21 CFR 1230.41 - Delivery of containers.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Delivery of containers. 1230.41 Section 1230.41 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) REGULATIONS UNDER CERTAIN OTHER ACTS ADMINISTERED BY THE FOOD AND DRUG ADMINISTRATION REGULATIONS UNDER THE...

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

OpenAIRE

Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

2015-01-01

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

Fluorine uptake into human enamel around a fluoride-containing dental material during cariogenic pH cycling

International Nuclear Information System (INIS)

Komatsu, H.; Yamamoto, H.; Nomachi, M.; Yasuda, K.; Matsuda, Y.; Murata, Y.; Kijimura, T.; Sano, H.; Sakai, T.; Kamiya, T.

2007-01-01

Using PIGE (TIARA, JAPAN) technique, we measured fluorine (F) uptake into the tooth enamel around a fluoride-containing material during caries progression using pH cycling. Class I cavities in the buccal surfaces of 6 extracted human teeth were drilled and filled with fluoride-containing material; a glass ionomer cement (Fuji IX(GC)). Three 300 μm sections through the material were obtained from each tooth. Two of these specimens were utilized to measure the F distribution in enamel adjacent to the material. A 1.7 MeV proton beam accelerated by the TIARA single-ended accelerator was delivered to a micro-beam apparatus. The beam spot size was about 1 μm with a beam current of about 100 pA. A nuclear reaction, 19 F(p, αγ) 16 0, was used to measure the F concentration and the gamma-rays from this reaction were detected with a 4' NaI detector. X-rays induced by proton were detected with a Si(Li) detector to measure calcium concentration and the beam intensity was monitored with the X-ray yield from a copper foil for quantitative analysis. After measurement of F uptake, all specimens were polished to a thickness of 120 μm. In order to simulate daily acid challenges occurring in the oral cavity, the pH cycling (pH6.8-pH4.5) was carried out for 1, 3 and 5 weeks, separately. The duration that the solution remained below pH 5.5 was 37 min per cycle. The cycles were repeated 6 times per day with 2 h interval between cycles, and the specimens were kept in remineralizing solution for the rest of pH cycle. After pH cycling, F and calcium distribution of each specimen was evaluated using PIGE technique. The F distribution of the specimens before pH cycling clearly showed the F uptake from fluoride-containing material into enamel adjacent to the material. After pH cycling, the caries progression in all specimens was observed by the image of transverse microradiography (TMR). The depth of caries and mineral loss progressed with increasing the duration of pH cycling, although

Fluorine uptake into human enamel around a fluoride-containing dental material during cariogenic pH cycling

Energy Technology Data Exchange (ETDEWEB)

Komatsu, H. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan)]. E-mail: kom@den.hokudai.ac.jp; Yamamoto, H. [Graduate School of Dentistry, Osaka University, 1-8 Yamada-Oka, Suita 565-0871 (Japan); Nomachi, M. [Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043 (Japan); Yasuda, K. [Wakasa wan Energy Research Center, 64-52-1 Hase, Tsuruga 914-0192 (Japan); Matsuda, Y. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Murata, Y. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Kijimura, T. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Sano, H. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Sakai, T. [Takasaki Advanced Radiation Research Institute, JAEA, 1233 Watanuki-machi, Takasaki 370-1292 (Japan); Kamiya, T. [Takasaki Advanced Radiation Research Institute, JAEA, 1233 Watanuki-machi, Takasaki 370-1292 (Japan)

2007-07-15

Using PIGE (TIARA, JAPAN) technique, we measured fluorine (F) uptake into the tooth enamel around a fluoride-containing material during caries progression using pH cycling. Class I cavities in the buccal surfaces of 6 extracted human teeth were drilled and filled with fluoride-containing material; a glass ionomer cement (Fuji IX(GC)). Three 300 {mu}m sections through the material were obtained from each tooth. Two of these specimens were utilized to measure the F distribution in enamel adjacent to the material. A 1.7 MeV proton beam accelerated by the TIARA single-ended accelerator was delivered to a micro-beam apparatus. The beam spot size was about 1 {mu}m with a beam current of about 100 pA. A nuclear reaction, {sup 19}F(p, {alpha}{gamma}){sup 16}0, was used to measure the F concentration and the gamma-rays from this reaction were detected with a 4' NaI detector. X-rays induced by proton were detected with a Si(Li) detector to measure calcium concentration and the beam intensity was monitored with the X-ray yield from a copper foil for quantitative analysis. After measurement of F uptake, all specimens were polished to a thickness of 120 {mu}m. In order to simulate daily acid challenges occurring in the oral cavity, the pH cycling (pH6.8-pH4.5) was carried out for 1, 3 and 5 weeks, separately. The duration that the solution remained below pH 5.5 was 37 min per cycle. The cycles were repeated 6 times per day with 2 h interval between cycles, and the specimens were kept in remineralizing solution for the rest of pH cycle. After pH cycling, F and calcium distribution of each specimen was evaluated using PIGE technique. The F distribution of the specimens before pH cycling clearly showed the F uptake from fluoride-containing material into enamel adjacent to the material. After pH cycling, the caries progression in all specimens was observed by the image of transverse microradiography (TMR). The depth of caries and mineral loss progressed with increasing the

The RNA synthesis machinery of negative-stranded RNA viruses

International Nuclear Information System (INIS)

Ortín, Juan; Martín-Benito, Jaime

2015-01-01

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes

The RNA synthesis machinery of negative-stranded RNA viruses

Energy Technology Data Exchange (ETDEWEB)

Ortín, Juan, E-mail: jortin@cnb.csic.es [Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CSIC) and CIBER de Enfermedades Respiratorias (ISCIII), Madrid (Spain); Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es [Department of Macromolecular Structures, Centro Nacional de Biotecnología (CSIC), Madrid (Spain)

2015-05-15

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

Fluorine uptake into the human enamel surface from fluoride-containing sealing materials during cariogenic pH cycling

Energy Technology Data Exchange (ETDEWEB)

Yasuhiro, Matsuda, E-mail: matsuda@den.hokudai.ac.jp [Department of Restorative Dentistry, Graduate School of Dental Medicine Hokkaido University (Japan); Katsushi, Okuyama [Department of Restorative Dentistry, Graduate School of Dental Medicine Hokkaido University (Japan); Hiroko, Yamamoto [Graduate School of Dentistry, Osaka University (Japan); Hisanori, Komatsu [Department of Restorative Dentistry, Graduate School of Dental Medicine Hokkaido University (Japan); Masashi, Koka; Takahiro, Sato [Takasaki Advanced Radiation Research Institute, JAEA (Japan); Naoki, Hashimoto; Saiko, Oki; Chiharu, Kawamoto; Hidehiko, Sano [Department of Restorative Dentistry, Graduate School of Dental Medicine Hokkaido University (Japan)

2015-04-01

To prevent the formation of caries and reduce dentin hypersensitivity, sealing materials, either with or without fluoride, are generally applied on the tooth in clinical practice. Application of fluoride-free sealing materials results in the formation of an acid-resistant layer on the tooth surface. On the other hand, fluoride-containing sealing materials might not only form an acid-resistant layer but could possibly further provide fluoride to enhance remineralization and reduce demineralization. In this study, the demineralization prevention ability and fluorine uptake rate in human enamel of fluoride-containing sealing materials [“MS coats F” (MSF)] and fluoride-free sealing materials (“hybrid coats 2” [HI]) were evaluated using an automatic pH cycling system. Each material was applied to the original tooth surface, the cut surfaces were covered with sticky wax, and the automatic pH-cycling system simulated daily acid changes (pH 6.8–4.5) occurring in the oral cavity for 4 weeks. Caries progression was analyzed using transverse microradiography (TMR) taken pre and post the 4 weeks of pH cycling. The fluorine and calcium distributions in the carious lesion in each specimen were evaluated using the proton-induced gamma emission (PIGE) and proton-induced X-ray (PIXE) techniques, respectively. TMR analysis showed that both MSF and HI had a caries-preventing effect after 4 weeks of pH cycling. PIGE/PIXE analysis demonstrated that only MSF resulted in fluoride uptake in the enamel surface. Therefore, MSF can help to form an acid-resistant layer and provide fluoride to the enamel surface. The presence of fluoride on the enamel surface suggested that MSF could prevent demineralization, even if the acid-resistant layer was removed, in clinical settings. The data obtained using the PIGE and PIXE techniques are useful for understanding the benefits of the use of a fluoride-containing sealing material for preventing caries.

Fluorine uptake into the human enamel surface from fluoride-containing sealing materials during cariogenic pH cycling

International Nuclear Information System (INIS)

Yasuhiro, Matsuda; Katsushi, Okuyama; Hiroko, Yamamoto; Hisanori, Komatsu; Masashi, Koka; Takahiro, Sato; Naoki, Hashimoto; Saiko, Oki; Chiharu, Kawamoto; Hidehiko, Sano

2015-01-01

To prevent the formation of caries and reduce dentin hypersensitivity, sealing materials, either with or without fluoride, are generally applied on the tooth in clinical practice. Application of fluoride-free sealing materials results in the formation of an acid-resistant layer on the tooth surface. On the other hand, fluoride-containing sealing materials might not only form an acid-resistant layer but could possibly further provide fluoride to enhance remineralization and reduce demineralization. In this study, the demineralization prevention ability and fluorine uptake rate in human enamel of fluoride-containing sealing materials [“MS coats F” (MSF)] and fluoride-free sealing materials (“hybrid coats 2” [HI]) were evaluated using an automatic pH cycling system. Each material was applied to the original tooth surface, the cut surfaces were covered with sticky wax, and the automatic pH-cycling system simulated daily acid changes (pH 6.8–4.5) occurring in the oral cavity for 4 weeks. Caries progression was analyzed using transverse microradiography (TMR) taken pre and post the 4 weeks of pH cycling. The fluorine and calcium distributions in the carious lesion in each specimen were evaluated using the proton-induced gamma emission (PIGE) and proton-induced X-ray (PIXE) techniques, respectively. TMR analysis showed that both MSF and HI had a caries-preventing effect after 4 weeks of pH cycling. PIGE/PIXE analysis demonstrated that only MSF resulted in fluoride uptake in the enamel surface. Therefore, MSF can help to form an acid-resistant layer and provide fluoride to the enamel surface. The presence of fluoride on the enamel surface suggested that MSF could prevent demineralization, even if the acid-resistant layer was removed, in clinical settings. The data obtained using the PIGE and PIXE techniques are useful for understanding the benefits of the use of a fluoride-containing sealing material for preventing caries

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

International Nuclear Information System (INIS)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

2008-01-01

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

Critical human-factors issues in nuclear-power regulation and a recommended comprehensive human-factors long-range plan. Critical discussion of human factors areas of concern

International Nuclear Information System (INIS)

Hopkins, C.O.; Snyder, H.L.; Price, H.E.; Hornick, R.J.; Mackie, R.R.; Smillie, R.J.; Sugarman, R.C.

1982-08-01

This comprehensive long-range human factors plan for nuclear reactor regulation was developed by a Study Group of the Human Factors Society, Inc. This Study Group was selected by the Executive Council of the Society to provide a balanced, experienced human factors perspective to the applications of human factors scientific and engineering knowledge to nuclear power generation. The report is presented in three volumes. Volume 1 contains an Executive Summary of the 18-month effort and its conclusions. Volume 2 summarizes all known nuclear-related human factors activities, evaluates these activities wherever adequate information is available, and describes the recommended long-range (10-year) plan for human factors in regulation. Volume 3 elaborates upon each of the human factors issues and areas of recommended human factors involvement contained in the plan, and discusses the logic that led to the recommendations

Fatal human poisoning with Padan: a cartap-containing pesticide.

Science.gov (United States)

Kurisaki, Emiko; Kato, Naho; Ishida, Tokiya; Matsumoto, Akinori; Shinohara, Kazuaki; Hiraiwa, Kouichi

2010-02-01

We report a patient who ingested about 13 g of Padan SG, a cartap-containing pesticide. After ingestion, the patient developed multiple seizures and dyspnea and lost consciousness. The patient did not recover and died on the fifth hospital day despite treatment at the early stage of poisoning. The cause of death was multisystem organ failure. Results of toxicological analysis were as follows: concentrations of nereistoxin (cartap metabolite) were 10.6 microg/mL in plasma, 18.2 microg/mL in urine, and 2.6 mg/mL in gastric fluid. Results of drug screening of urine by Triage DOA Panels and using an organophosphate detection kit were negative.

Human major histocompatibility complex contains a minimum of 19 genes between the complement cluster and HLA-B

International Nuclear Information System (INIS)

Spies, T.; Bresnahan, M.; Strominger, J.L.

1989-01-01

A 600-kilobase (kb) DNA segment from the human major histocompatibility complex (MHC) class III region was isolated by extension of a previous 435-kb chromosome walk. The contiguous series of cloned overlapping cosmids contains the entire 555-kb interval between C2 in the complement gene cluster and HLA-B. This region is known to encode the tumor necrosis factors (TNFs) α and β, B144, and the major heat shock protein HSP70. Moreover, a cluster of genes, BAT1-BAT5 (HLA-B-associated transcripts) have been localized in the vicinity of the genes for TNFα and TNFβ. An additional four genes were identified by isolation of corresponding cDNA clones with cosmid DNA probes. These genes for BAT6-BAT9 were mapped near the gene for C2 within a 120-kb region that includes a HSP70 gene pair. These results, together with complementary data from a similar recent study, indicated the presence of a minimum of 19 genes within the C2-HLA-B interval of the MHC class III region. Although the functional properties of most of these genes are yet unknown, they may be involved in some aspects of immunity. This idea is supported by the genetic mapping of the hematopoietic histocompatibility locus-1 (Hh-1) in recombinant mice between TNFα and H-2S, which is homologous to the complement gene cluster in humans

Fructose containing sugars do not raise blood pressure or uric acid at normal levels of human consumption.

Science.gov (United States)

Angelopoulos, Theodore J; Lowndes, Joshua; Sinnett, Stephanie; Rippe, James M

2015-02-01

The impact of fructose, commonly consumed with sugars by humans, on blood pressure and uric acid has yet to be defined. A total of 267 weight-stable participants drank sugar-sweetened milk every day for 10 weeks as part of their usual, mixed-nutrient diet. Groups 1 and 2 had 9% estimated caloric intake from fructose or glucose, respectively, added to milk. Groups 3 and 4 had 18% of estimated caloric intake from high fructose corn syrup or sucrose, respectively, added to the milk. Blood pressure and uric acid were determined prior to and after the 10-week intervention. There was no effect of sugar type on either blood pressure or uric acid (interaction P>.05), and a significant time effect for blood pressure was noted (Pfructose at the 50th percentile level, whether consumed as pure fructose or with fructose-glucose-containing sugars, does not promote hyperuricemia or increase blood pressure. © 2014 Wiley Periodicals, Inc.

Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase.

Science.gov (United States)

Liu, Xiao; Pichulik, Tica; Wolz, Olaf-Oliver; Dang, Truong-Minh; Stutz, Andrea; Dillen, Carly; Delmiro Garcia, Magno; Kraus, Helene; Dickhöfer, Sabine; Daiber, Ellen; Münzenmayer, Lisa; Wahl, Silke; Rieber, Nikolaus; Kümmerle-Deschner, Jasmin; Yazdi, Amir; Franz-Wachtel, Mirita; Macek, Boris; Radsak, Markus; Vogel, Sebastian; Schulte, Berit; Walz, Juliane Sarah; Hartl, Dominik; Latz, Eicke; Stilgenbauer, Stephan; Grimbacher, Bodo; Miller, Lloyd; Brunner, Cornelia; Wolz, Christiane; Weber, Alexander N R

2017-10-01

The Nod-like receptor NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK) are protagonists in innate and adaptive immunity, respectively. NLRP3 senses exogenous and endogenous insults, leading to inflammasome activation, which occurs spontaneously in patients with Muckle-Wells syndrome; BTK mutations cause the genetic immunodeficiency X-linked agammaglobulinemia (XLA). However, to date, few proteins that regulate NLRP3 inflammasome activity in human primary immune cells have been identified, and clinically promising pharmacologic targeting strategies remain elusive. We sought to identify novel regulators of the NLRP3 inflammasome in human cells with a view to exploring interference with inflammasome activity at the level of such regulators. After proteome-wide phosphoproteomics, the identified novel regulator BTK was studied in human and murine cells by using pharmacologic and genetic BTK ablation. Here we show that BTK is a critical regulator of NLRP3 inflammasome activation: pharmacologic (using the US Food and Drug Administration-approved inhibitor ibrutinib) and genetic (in patients with XLA and Btk knockout mice) BTK ablation in primary immune cells led to reduced IL-1β processing and secretion in response to nigericin and the Staphylococcus aureus toxin leukocidin AB (LukAB). BTK affected apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and caspase-1 cleavage and interacted with NLRP3 and ASC. S aureus infection control in vivo and IL-1β release from cells of patients with Muckle-Wells syndrome were impaired by ibrutinib. Notably, IL-1β processing and release from immune cells isolated from patients with cancer receiving ibrutinib therapy were reduced. Our data suggest that XLA might result in part from genetic inflammasome deficiency and that NLRP3 inflammasome-linked inflammation could potentially be targeted pharmacologically through BTK. Copyright © 2017 American Academy of Allergy

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif.

Science.gov (United States)

Goldie, Belinda J; Fitzsimmons, Chantel; Weidenhofer, Judith; Atkins, Joshua R; Wang, Dan O; Cairns, Murray J

2017-01-01

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

Dicer is dispensable for asymmetric RISC loading in mammals.

Science.gov (United States)

Betancur, Juan G; Tomari, Yukihide

2012-01-01

In flies, asymmetric loading of small RNA duplexes into Argonaute2-containing RNA-induced silencing complex (Ago2-RISC) requires Dicer-2/R2D2 heterodimer, which acts as a protein sensor for the thermodynamic stabilities of the ends of small RNA duplexes. However, the mechanism of small RNA asymmetry sensing in mammalian RISC assembly remains obscure. Here, we quantitatively examined RISC assembly and target silencing activity in the presence or absence of Dicer in mammals. Our data show that, unlike the well-characterized fly Ago2-RISC assembly pathway, mammalian Dicer is dispensable for asymmetric RISC loading in vivo and in vitro.

Multilayer checkpoints for microRNA authenticity during RISC assembly.

Science.gov (United States)

Kawamata, Tomoko; Yoda, Mayuko; Tomari, Yukihide

2011-09-01

MicroRNAs (miRNAs) function through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) protein at the core. RISC assembly follows a two-step pathway: miRNA/miRNA* duplex loading into Ago, and separation of the two strands within Ago. Here we show that the 5' phosphate of the miRNA strand is essential for duplex loading into Ago, whereas the preferred 5' nucleotide of the miRNA strand and the base-pairing status in the seed region and the middle of the 3' region function as additive anchors to Ago. Consequently, the miRNA authenticity is inspected at multiple steps during RISC assembly.

Waste container and method for containing waste

International Nuclear Information System (INIS)

Ono, Akira; Matsushita, Mitsuhiro; Doi, Makoto; Nakatani, Seiichi.

1990-01-01

In a waste container, water-proof membranes and rare earth element layers are formed on the inner surface of a steel plate concrete container in which steel plates are embedded. Further, rear earth element detectors are disposed each from the inner side of the steel plate concrete container by way of a pressure pipe to the outer side of the container. As a method for actually containing wastes, when a plurality of vessels in which wastes are fixed are collectively enhoused to the waste container, cussioning materials are attached to the inner surface of the container and wastes fixing containers are stacked successively in a plurality of rows in a bag made of elastic materials. Subsequently, fixing materials are filled and tightly sealed in the waste container. When the waste container thus constituted is buried underground, even if it should be deformed to cause intrusion of rain water to the inside of the container, the rare earth elements in the container dissolved in the rain water can be detected by the detectors, the containers are exchanged before the rain water intruding to the inner side is leached to the surrounding ground, to previously prevent the leakage of radioactive nuclides. (K.M.)

High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases

Science.gov (United States)

Qin, Yidan; Yao, Jun; Wu, Douglas C.; Nottingham, Ryan M.; Mohr, Sabine; Hunicke-Smith, Scott; Lambowitz, Alan M.

2016-01-01

Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from RNA in RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling. PMID:26554030

Disease containment strategies based on mobility and information dissemination

OpenAIRE

Pejović, Veljko; Lima, Antonio; De Domenico, Manlio; Musolesi, Mirco

2016-01-01

Human mobility and social structure are at the basis of disease spreading. Disease containment strategies are usually devised from coarse-grained assumptions about human mobility. Cellular networks data, however, provides finer-grained information, not only about how people move, but also about how they communicate. In this paper we analyze the behavior of a large number of individuals in Ivory Coast using cellular network data. We model mobility and communication between individuals by means...

The antimicrobial resistance containment and surveillance approach--a public health tool.

Science.gov (United States)

Simonsen, Gunnar S.; Tapsall, John W.; Allegranzi, Benedetta; Talbot, Elizabeth A.; Lazzari, Stefano

2004-01-01

Antimicrobial drug resistance (AMR) is widely recognized as a global public health threat because it endangers the effectiveness of treatment of infectious diseases. In 2001 WHO issued the Global Strategy for Containment of Antimicrobial Resistance, but it has proved difficult to translate the recommendations of the Global Strategy into effective public health actions. The purpose of the Antimicrobial Resistance Containment and Surveillance (ARCS) approach is to facilitate the formulation of public health programmes and the mobilization of human and financial resources for the containment of AMR. The ARCS approach highlights the fundamental link between rational drug use and containment of AMR. Clinical management of human and animal infections should be improved through better disease control and prevention, high quality diagnostic testing, appropriate treatment regimens and consumer health education. At the same time, systems for supplying antimicrobial drugs should include appropriate regulations, lists of essential drugs, and functional mechanisms for the approval and delivery of drugs. Containment of AMR is defined in the ARCS approach as the continuous application of this package of core interventions. Surveillance of the extent and trends of antimicrobial resistance as well as the supply, selection and use of antimicrobial drugs should be established to monitor the process and outcome of containment of AMR. The ARCS approach is represented in the ARCS diagram (Fig. 2) which provides a simplified, but comprehensive illustration of the complex problem of containment and monitoring of AMR. PMID:15654407

Activation of autoreactive B cells by endogenous TLR7 and TLR3 RNA ligands.

Science.gov (United States)

Green, Nathaniel M; Moody, Krishna-Sulayman; Debatis, Michelle; Marshak-Rothstein, Ann

2012-11-16

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.

Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

Directory of Open Access Journals (Sweden)

Daniel Menezes-Souza

2015-01-01

Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

Science.gov (United States)

Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

2015-03-01

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Secondary Containers and Service Containers for Pesticides

Science.gov (United States)

Secondary containers and service containers are used by pesticide applicators in the process of applying a pesticide. EPA does not require secondary containers or service containers to be labeled or to meet particular construction standards. Learn more.

Global Argonauts: Returnees and Diaspora as Sources of Innovation in China and Israel

Science.gov (United States)

Welch, Anthony; Hao, Jie

2016-01-01

The paper focuses on returnees and knowledge diaspora as important sources for human resources development, identifying push and pull factors that also contribute significantly to innovation. For both China and Israel, their high-skilled diaspora are a major policy priority: each has a substantial, high-skilled diaspora and policies and programmes…

Fluorine analysis of human enamel around fluoride-containing materials under different pH-cycling by {mu}-PIGE/PIXE system

Energy Technology Data Exchange (ETDEWEB)

Komatsu, H., E-mail: kom@den.hokudai.ac.jp [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Yamamoto, H. [Graduate School of Dentistry, Osaka University, 1-8 Yamada-Oka, Suita 565-0871 (Japan); Matsuda, Y.; Kijimura, T.; Kinugawa, M.; Okuyama, K. [Graduate School of Dental Medicine, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo 060-8586 (Japan); Nomachi, M. [Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka 560-0043 (Japan); Yasuda, K. [Wakasa Wan Energy Research Center, 64-52-1 Hase, Tsuruga 914-0192 (Japan); Satoh, T. [Advanced Radiation Technology, TARRI, JAEA, 1233 Watanuki-Machi, Takasaki 370-1292 (Japan); Oikawa, S. [National Institute of Radiological Science, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555 (Japan)

2011-10-15

The caries preventive effect of fluoride-containing materials (FCMs) might depend on the caries risk of the individuals. Two pairs of demineralizing and remineralizing solutions of pH-cycling were prepared for simulating low and high caries risk. The purpose of this study was to determine fluorine (F) uptake into human enamel around FCMs under different pH-cycling using the in-air {mu}-PIGE/PIXE system. Fluoride-containing glass ionomer cement (Fuji IX{sub GP} FAST CAPSULE (FN)), and composite resin (BEAUTIFIL II with FLUORO BOND SHAKE ONE (BS)) were used in this study. The pH-cycling (pH 6.8-4.5) was carried out for 5 weeks. After pH-cycling, the caries progression was analyzed using transverse micro-radiography (TMR). The fluorine and calcium distributions in the carious lesion in each specimen were evaluated using the PIGE/PIXE system. From TMR analysis, there was a difference in caries risk between the two kinds of pH-cycling. Although the caries preventive effect of BS and FN was confirmed at low risk, the effect at high risk was confirmed for FN only. From the analysis of the fluorine uptake in the outer 200 {mu}m of the lesion we concluded that there was no significant difference between the pH-cycling solutions. However, we found different fluorine concentrations in the enamel for the two FCMs. The decreased caries progression under high risk for FN indicated that an adequate amount of fluorine supplied from the material is required at higher caries risk. It was confirmed that the caries preventive effect of FCM depends on the caries risk. The fluorine analysis of teeth under various pH-cycling conditions gives information to evaluate the caries preventive effect of fluoride-containing materials according to the caries risk.

Fluorine analysis of human enamel around fluoride-containing materials under different pH-cycling by μ-PIGE/PIXE system

International Nuclear Information System (INIS)

Komatsu, H.; Yamamoto, H.; Matsuda, Y.; Kijimura, T.; Kinugawa, M.; Okuyama, K.; Nomachi, M.; Yasuda, K.; Satoh, T.; Oikawa, S.

2011-01-01

The caries preventive effect of fluoride-containing materials (FCMs) might depend on the caries risk of the individuals. Two pairs of demineralizing and remineralizing solutions of pH-cycling were prepared for simulating low and high caries risk. The purpose of this study was to determine fluorine (F) uptake into human enamel around FCMs under different pH-cycling using the in-air μ-PIGE/PIXE system. Fluoride-containing glass ionomer cement (Fuji IX GP FAST CAPSULE (FN)), and composite resin (BEAUTIFIL II with FLUORO BOND SHAKE ONE (BS)) were used in this study. The pH-cycling (pH 6.8-4.5) was carried out for 5 weeks. After pH-cycling, the caries progression was analyzed using transverse micro-radiography (TMR). The fluorine and calcium distributions in the carious lesion in each specimen were evaluated using the PIGE/PIXE system. From TMR analysis, there was a difference in caries risk between the two kinds of pH-cycling. Although the caries preventive effect of BS and FN was confirmed at low risk, the effect at high risk was confirmed for FN only. From the analysis of the fluorine uptake in the outer 200 μm of the lesion we concluded that there was no significant difference between the pH-cycling solutions. However, we found different fluorine concentrations in the enamel for the two FCMs. The decreased caries progression under high risk for FN indicated that an adequate amount of fluorine supplied from the material is required at higher caries risk. It was confirmed that the caries preventive effect of FCM depends on the caries risk. The fluorine analysis of teeth under various pH-cycling conditions gives information to evaluate the caries preventive effect of fluoride-containing materials according to the caries risk.

Human Resource Management in Virtual Organizations. Research in Human Resource Management Series.

Science.gov (United States)

Heneman, Robert L., Ed.; Greenberger, David B., Ed.

This document contains 14 papers on human resources (HR) and human resource management (HRM) in virtual organizations. The following papers are included: "Series Preface" (Rodger Griffeth); "Volume Preface" (Robert L. Heneman, David B. Greenberger); "The Virtual Organization: Definition, Description, and…

Characterization of RNA interference in rat PC12 cells

DEFF Research Database (Denmark)

Thonberg, Håkan; Schéele, Camilla C; Dahlgren, Cecilia

2004-01-01

strand of the siRNA guides a multi-protein complex, RNA-induced silencing complex (RISC), to cleave target mRNA. Although the exact function and composition of RISC is still unclear, it has been shown to include several proteins of the Argonaute protein family. Here we report of a robust system...... of the rat Golgi-ER protein 95 kDa (GERp95), an Argonaute family protein, by siRNA methodology. After GERp95-ablation, sequential knockdown of NPY by siRNA was shown to be impaired. Thus, we report that the GERp95 protein is functionally required for RNAi targeting NPY in rat PC12 cells....

Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

International Nuclear Information System (INIS)

Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

1986-01-01

Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

Characterization of MRP RNA-protein interactions within the perinucleolar compartment.

Science.gov (United States)

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-03-15

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA-processing (MRP) RNA, pyrimidine tract-binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA-containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)-PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA-protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif

Directory of Open Access Journals (Sweden)

Belinda J. Goldie

2017-08-01

Full Text Available While the cytoplasmic function of microRNA (miRNA as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

Greater mortality and morbidity in extremely preterm infants fed a diet containing cow milk protein products.

Science.gov (United States)

Abrams, Steven A; Schanler, Richard J; Lee, Martin L; Rechtman, David J

2014-01-01

Provision of human milk has important implications for the health and outcomes of extremely preterm (EP) infants. This study evaluated the effects of an exclusive human milk diet on the health of EP infants during their stay in the neonatal intensive care unit. EP infants milk fortified with a human milk protein-based fortifier (HM) (n=167) or a diet containing variable amounts of milk containing cow milk-based protein (CM) (n=93). Principal outcomes were mortality, necrotizing enterocolitis (NEC), growth, and duration of parenteral nutrition (PN). Mortality (2% versus 8%, p=0.004) and NEC (5% versus 17%, p=0.002) differed significantly between the HM and CM groups, respectively. For every 10% increase in the volume of milk containing CM, the risk of sepsis increased by 17.9% (pmilk diet, devoid of CM-containing products, was associated with lower mortality and morbidity in EP infants without compromising growth and should be considered as an approach to nutritional care of these infants.

Container crane for sea freight containers

NARCIS (Netherlands)

Luttekes, E.; Rijsenbrij, J.C.

2001-01-01

The invention relates to a container crane for loading and unloading seaborne containers. The container crane comprises a bridge girder (7), a jib (8), at least two crabs (11, 12) which can travel along the said bridge girder and/or jib and are provided with hoist means for lifting and lowering the

In vivo anticancer evaluation of the hyperthermic efficacy of anti-human epidermal growth factor receptor-targeted PEG-based nanocarrier containing magnetic nanoparticles

Directory of Open Access Journals (Sweden)

Baldi G

2014-06-01

Full Text Available Giovanni Baldi,1 Costanza Ravagli,1 Filippo Mazzantini,1 George Loudos,2 Jaume Adan,3 Marc Masa,3 Dimitrios Psimadas,2 Eirini A Fragogeorgi,2 Erica Locatelli,4 Claudia Innocenti,5,6 Claudio Sangregorio,5,7 Mauro Comes Franchini4 1CERICOL, Sovigliana-Vinci, Italy; 2Technological Educational Institute of Athens, Athens, Greece; 3Leitat Technological Center, Barcelona, Spain; 4Department of Industrial Chemistry Toso Montanari, University of Bologna, Bologna, 5Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM, 6Dipartimento di Chimica U Schiff, Università di Firenze, Firenze, 7Centro Nazionale delle Ricerche (ICCOM – CNR, Firenze, Italy Abstract: Polymeric nanoparticles with targeting moieties containing magnetic nanoparticles as theranostic agents have considerable potential for the treatment of cancer. Here we report the chemical synthesis and characterization of a poly(D,L-lactide-co-glycolide-b-poly(ethylene glycol-based nanocarrier containing iron oxide nanoparticles and human epithelial growth factor receptor on the outer shell. The nanocarrier was also radiolabeled with 99mTc and tested as a theranostic nanomedicine, ie, it was investigated for both its diagnostic ability in vivo and its therapeutic hyperthermic effects in a standard A431 human tumor cell line. Following radiolabeling with 99mTc, the biodistribution and therapeutic hyperthermic effects of the nanosystem were studied noninvasively in vivo in tumor-bearing mice. A substantial decrease in tumor size correlated with an increase in both nanoparticle concentration and local temperature was achieved, confirming the possibility of using this multifunctional nanosystem as a therapeutic tool for epidermoid carcinoma. Keywords: magnetic nanoparticles, polymeric nanocarriers, skin cancer, hyperthermia, single-photon emission computed tomography, imaging

FAUST/CONTAIN; FAUST/CONTAIN

Energy Technology Data Exchange (ETDEWEB)

Cherdron, W.; Minges, J.; Sauter, H.; Schuetz, W.

1995-08-01

The FAUNA facility has been restructured after completion of the sodium fire experiments. It is now serving LWR research, cf. report II on program no. 32.21.02 concerning steam explosions. The CONTAIN code system for computing the thermodynamic, aerosol and radiological phenomena in a containment under severe accident conditions is being developed with a new to fission product release and transport. (orig.)

Effectiveness of containment sprays in containment management

International Nuclear Information System (INIS)

Nourbakhsh, H.P.; Perez, S.E.; Lehner, J.R.

1993-05-01

A limited study has been performed assessing the effectiveness of containment sprays-to mitigate particular challenges which may occur during a severe accident. Certain aspects of three specific topics related to using sprays under severe accident conditions were investigated. The first was the effectiveness of sprays connected to an alternate water supple and pumping source because the actual containment spray pumps are inoperable. This situation could occur during a station blackout. The second topic concerned the adverse as well as beneficial effects of using containment sprays during severe accident scenario where the containment atmosphere contains substantial quantities of hydrogen along with steam. The third topic was the feasibility of using containment sprays to moderate the consequences of DCH

APR1400 Containment Simulation with CONTAIN code

Energy Technology Data Exchange (ETDEWEB)

Hwang, Moon Kyu; Chung, Bub Dong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2010-05-15

The more realistic containment pressure variation predicted by the CONTAIN code through the coupled analysis during a large break loss of coolant accident in the nuclear power plant is expected to provide more accurate prediction for the plant behavior than a standalone MARS-KS calculation. The input deck has been generated based on the already available ARP- 1400 input for CONTEMPT code. Similarly to the CONTEMPT input deck, a simple two-cell model was adopted to model the containment behavior, one cell for the containment inner volume and another cell for the environment condition. The developed input for the CONTAIN code is to be eventually applied for the coupled code calculation of MARS-KS/CONTAIN

APR1400 Containment Simulation with CONTAIN code

International Nuclear Information System (INIS)

Hwang, Moon Kyu; Chung, Bub Dong

2010-01-01

The more realistic containment pressure variation predicted by the CONTAIN code through the coupled analysis during a large break loss of coolant accident in the nuclear power plant is expected to provide more accurate prediction for the plant behavior than a standalone MARS-KS calculation. The input deck has been generated based on the already available ARP- 1400 input for CONTEMPT code. Similarly to the CONTEMPT input deck, a simple two-cell model was adopted to model the containment behavior, one cell for the containment inner volume and another cell for the environment condition. The developed input for the CONTAIN code is to be eventually applied for the coupled code calculation of MARS-KS/CONTAIN

Prenatal Experiences of Containment in the Light of Bion's Model of Container/Contained

Science.gov (United States)

Maiello, Suzanne

2012-01-01

This paper explores the idea of possible proto-experiences of the prenatal child in the context of Bion's model of container/contained. The physical configuration of the embryo/foetus contained in the maternal uterus represents the starting point for an enquiry into the unborn child's possible experiences of its state of being contained in a…

Human milk metagenome: a functional capacity analysis

Science.gov (United States)

2013-01-01

Background Human milk contains a diverse population of bacteria that likely influences colonization of the infant gastrointestinal tract. Recent studies, however, have been limited to characterization of this microbial community by 16S rRNA analysis. In the present study, a metagenomic approach using Illumina sequencing of a pooled milk sample (ten donors) was employed to determine the genera of bacteria and the types of bacterial open reading frames in human milk that may influence bacterial establishment and stability in this primal food matrix. The human milk metagenome was also compared to that of breast-fed and formula-fed infants’ feces (n = 5, each) and mothers’ feces (n = 3) at the phylum level and at a functional level using open reading frame abundance. Additionally, immune-modulatory bacterial-DNA motifs were also searched for within human milk. Results The bacterial community in human milk contained over 360 prokaryotic genera, with sequences aligning predominantly to the phyla of Proteobacteria (65%) and Firmicutes (34%), and the genera of Pseudomonas (61.1%), Staphylococcus (33.4%) and Streptococcus (0.5%). From assembled human milk-derived contigs, 30,128 open reading frames were annotated and assigned to functional categories. When compared to the metagenome of infants’ and mothers’ feces, the human milk metagenome was less diverse at the phylum level, and contained more open reading frames associated with nitrogen metabolism, membrane transport and stress response (P milk metagenome also contained a similar occurrence of immune-modulatory DNA motifs to that of infants’ and mothers’ fecal metagenomes. Conclusions Our results further expand the complexity of the human milk metagenome and enforce the benefits of human milk ingestion on the microbial colonization of the infant gut and immunity. Discovery of immune-modulatory motifs in the metagenome of human milk indicates more exhaustive analyses of the functionality of the human

Continuous containment monitoring with containment pressure fluctuation

International Nuclear Information System (INIS)

Dick, J.E.

1996-01-01

The monitoring of the integrity of containments particularly but not exclusively for nuclear plants is dealt with in this invention. While this application is primarily concerned with containment monitoring in the context of the single unit design, it is expected that the concepts presented will be universally applicable to any containment design, including containments for non-nuclear applications such as biological laboratories. The nuclear industry has long been interested in a means of monitoring containment integrity on a continuous basis, that is, while the reactor is operating normally. 12 refs., 2 figs

Cross- and Co-Packaging of Retroviral RNAs and Their Consequences

Directory of Open Access Journals (Sweden)

Lizna M. Ali

2016-10-01

Full Text Available Retroviruses belong to the family Retroviridae and are ribonucleoprotein (RNP particles that contain a dimeric RNA genome. Retroviral particle assembly is a complex process, and how the virus is able to recognize and specifically capture the genomic RNA (gRNA among millions of other cellular and spliced retroviral RNAs has been the subject of extensive investigation over the last two decades. The specificity towards RNA packaging requires higher order interactions of the retroviral gRNA with the structural Gag proteins. Moreover, several retroviruses have been shown to have the ability to cross-/co-package gRNA from other retroviruses, despite little sequence homology. This review will compare the determinants of gRNA encapsidation among different retroviruses, followed by an examination of our current understanding of the interaction between diverse viral genomes and heterologous proteins, leading to their cross-/co-packaging. Retroviruses are well-known serious animal and human pathogens, and such a cross-/co-packaging phenomenon could result in the generation of novel viral variants with unknown pathogenic potential. At the same time, however, an enhanced understanding of the molecular mechanisms involved in these specific interactions makes retroviruses an attractive target for anti-viral drugs, vaccines, and vectors for human gene therapy.

A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

Energy Technology Data Exchange (ETDEWEB)

Wu, Di, E-mail: DiWu@mail.nankai.edu.cn; Wu, Shian

2013-04-19

Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

The effect of calcium phosphate-containing desensitizing agent on ...

African Journals Online (AJOL)

Objective: The aim of this study was to investigate the effect of calcium phosphate containing desensitizing pretreatments on the microtensile bond strength (MTBS) and microleakage of the multimode adhesive agent to dentin. Materials and Methods: In this study, twelve noncarious, freshly extracted human third molar teeth ...

The pollen of metaphor: Box, cage, and trap as containment in the eighteenth century.

Science.gov (United States)

Milne, Anne

2016-06-01

This article uses the concept of "the pollen of metaphor" to discuss three forms of non-human animal containment in the eighteenth century: François Huber's Leaf or Book Hive bee box first described in his Nouvelles Observations sur les Abeilles (1792, English translation 1806), Sarah Trimmer's bird cages in her didactic children's book, Fabulous Histories; Or, The Story of the Robins (1786), and a mouse trap in Anna Letitia Barbauld's 1773 poem, "The Mouse's Petition, found in the trap where he had been confined all night by Dr. Priestley, for the sake of making experiments with different kinds of air." All three works highlight the eighteenth-century art of observation. The inherent commitment to relationships in the observation process suggests that interpreting ocular evidence involves "plausible relations," metaphor and/or "productive analogy." The article teases out subtle differences between the ways that each author uses containments and concludes that while Huber seeks to circumscribe non-human animal behavior within the bounds of 'reasonable' animal husbandry to better serve human needs, Trimmer goes further to connect 'appropriate' non-human animal containment to moral strictures governing humans. Barbauld's intervention using a literate, speaking animal subject confronts such moral governance to argue for equal rights based on principles of true equality rather than what is observed to be 'reasonable' and/or 'moral.' Copyright © 2016 Elsevier Ltd. All rights reserved.

Aedes aegypti Global Suitability Maps Using a Water Container Energy Balance Model for Dengue Risk Applications

Science.gov (United States)

Steinhoff, D.

2015-12-01

Dengue infections are estimated to total nearly 400 million per year worldwide, with both the geographic range and the magnitude of infections having increased in the past 50 years. The primary dengue vector mosquito Aedes aegypti is closely associated with humans. It lives exclusively in urban and semi-urban areas, preferentially bites humans, and spends its developmental stages in artificial water containers. Climate regulates the development of Ae. aegypti immature mosquitoes in artificial containers. Potential containers for Ae. aegypti immature development include, but are not limited to, small sundry items (e.g., bottles, cans, plastic containers), buckets, tires, barrels, tanks, and cisterns. Successful development of immature mosquitoes from eggs to larvae, pupae, and eventually adults is largely dependent on the availability of water and the thermal properties of the water in the containers. Recent work has shown that physics-based approaches toward modeling container water properties are promising for resolving the complexities of container water dynamics and the effects on immature mosquito development. An energy balance container model developed by the author, termed the Water Height And Temperature in Container Habitats Energy Model (WHATCH'EM), solves for water temperature and height for user-specified containers with readily available weather data. Here we use WHATCH'EM with NASA Earth Science products used as input to construct global suitability maps based on established water temperature ranges for immature Ae. aegypti mosquitoes. A proxy for dengue risk is provided from habitat suitability, but also population estimates, as Ae. aegypti is closely associated with human activity. NASA gridded Global Population of the World data is used to mask out rural areas with low dengue risk. Suitability maps are illustrated for a variety of containers (size, material, color) and shading scenarios.

Red wine contains a potent inhibitor of phenolsulphotransferase.

Science.gov (United States)

Littlewood, J T; Glover, V; Sandler, M

1985-01-01

Many ethanolic drinks, especially red wine, contain potent inhibitors of phenolsulphotransferase. At a dilution of 1/75 from the original beverage, extracts from six types of red wine inhibited human platelet phenolsulphotransferase P by a mean of 99% and human platelet phenolsulphotransferase M by 12%. Such extracts had no significant effect on rat liver monoamine oxidase A or human platelet monoamine oxidase B. The inhibitors, which have not yet been identified, can be extracted into ethyl acetate at acid or neutral pH. Thus, they are not monoamines. Flavonoid phenols are plausible candidates. As phenolsulphotransferase M and P are involved in the metabolism of many phenols, including drugs, the inhibition of these enzymes could result in the enhancement of pharmacological potency and have important clinical consequences. PMID:3857069

Use of an in vitro human skin permeation assay to assess bioequivalence of two topical cream formulations containing butenafine hydrochloride (1%, w/w).

Science.gov (United States)

Mitra, Amitava; Kim, Nanhye; Spark, Darren; Toner, Frank; Craig, Susan; Roper, Clive; Meyer, Thomas A

2016-12-01

The primary objective of this work was to investigate, using an in vitro human skin permeation study, whether changes in the excipients of butenafine hydrochloride cream would have any effect on bioperformance of the formulation. Such in vitro data would be a surrogate for any requirement of a bioequivalence (BE) study to demonstrate formulation similarity. A LC-MS/MS method for quantitation of butenafine in various matrices was developed and validated. A pilot study was performed to validate the in vitro skin permeation methodology using three cream formulations containing butenafine hydrochloride at concentrations of 0.5, 1.0 and 1.5% (w/w). Finally, a definitive in vitro human skin permeation study was conducted, comparing the extent of butenafine hydrochloride permeation from the new formulation to that from the current formulation. The results of the study comparing the two formulations showed that there was no statistically significant difference in the extent of butenafine permeation into human skin. In conclusion, these in vitro data demonstrated that the formulation change is likely to have no significant impact on the bioperformance of 1% (w/w) butenafine hydrochloride cream. Copyright © 2016 Elsevier Inc. All rights reserved.

21 CFR 584.200 - Ethyl alcohol containing ethyl acetate.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Ethyl alcohol containing ethyl acetate. 584.200 Section 584.200 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS...

21 CFR 864.3250 - Specimen transport and storage container.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Specimen transport and storage container. 864.3250 Section 864.3250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Pathology Instrumentation and Accessories § 864...

Effect of Apacox, a feed additive containing herb extracts and ...

African Journals Online (AJOL)

PC

basal diet but contained an additional 200 mg α-tocopheryl acetate/kg (TOC), ... of this resistance to humans through the food chain, all antibacterial feed additives will be ... main biologically active constituents of saffron are crocins, a family of ...

Critical human-factors issues in nuclear-power regulation and a recommended comprehensive human-factors long-range plan. Executive summary

International Nuclear Information System (INIS)

Hopkins, C.O.; Snyder, H.L.; Price, H.E.; Hornick, R.J.; Mackie, R.R.; Smillie, R.J.; Sugarman, R.C.

1982-08-01

This comprehensive long-range human factors plan for nuclear reactor regulation was developed by a Study Group of the Human Factors Society, Inc. This Study Group was selected by the Executive Council of the Society to provide a balanced, experienced human factors perspective to the applications of human factors scientific and engineering knowledge to nuclear power generation. The report is presented in three volumes. Volume 1 contains an Executive Summary of the 18-month effort and its conclusions. Volume 2 summarizes all known nuclear-related human factors activities, evaluates these activities wherever adequate information is available, and describes the recommended long-range (10-year) plan for human factors in regulation. Volume 3 elaborates upon each of the human factors issues and areas of recommended human factors involvement contained in the plan, and discusses the logic that led to the recommendations

NATO Human View Architecture and Human Networks

Science.gov (United States)

Handley, Holly A. H.; Houston, Nancy P.

2010-01-01

The NATO Human View is a system architectural viewpoint that focuses on the human as part of a system. Its purpose is to capture the human requirements and to inform on how the human impacts the system design. The viewpoint contains seven static models that include different aspects of the human element, such as roles, tasks, constraints, training and metrics. It also includes a Human Dynamics component to perform simulations of the human system under design. One of the static models, termed Human Networks, focuses on the human-to-human communication patterns that occur as a result of ad hoc or deliberate team formation, especially teams distributed across space and time. Parameters of human teams that effect system performance can be captured in this model. Human centered aspects of networks, such as differences in operational tempo (sense of urgency), priorities (common goal), and team history (knowledge of the other team members), can be incorporated. The information captured in the Human Network static model can then be included in the Human Dynamics component so that the impact of distributed teams is represented in the simulation. As the NATO militaries transform to a more networked force, the Human View architecture is an important tool that can be used to make recommendations on the proper mix of technological innovations and human interactions.

The biological activities of (1,3)-(1,6)-{beta}-d-glucan and porous electrospun PLGA membranes containing {beta}-glucan in human dermal fibroblasts and adipose tissue-derived stem cells

Energy Technology Data Exchange (ETDEWEB)

Woo, Yeon I; Park, Bong Joo; Kim, Hye-Lee; Lee, Mi Hee; Kim, Jungsung; Park, Jong-Chul [Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Yang, Young-Il [Department of Pathology, School of Medicine, Paik Institute for Clinical Research, Inje University, 633-165 Gae-dong, Busan-jin-gu, Busan 614-735 (Korea, Republic of); Kim, Jung Koo [Department of Biomedical Engineering, College of Biomedical Science and Engineering, Inje University, Kimhae 621-749 (Korea, Republic of); Tsubaki, Kazufumi [R and D division, Asahi Denka Co. Ltd, 7-2-35 Higashi-ogu, Arakawa-ku, Tokyo 116-8554 (Japan); Han, Dong-Wook, E-mail: parkjc@yuhs.a [Department of Nanomedical Engineering, College of Nanoscience and Nanotechnology, Pusan National University, geumjeong-gu, Busan 609-735 (Korea, Republic of)

2010-08-01

In this study, we investigated the possible roles of (1,3)-(1,6)-{beta}-d-glucan ({beta}-glucan) and porous electrospun poly-lactide-co-glycolide (PLGA) membranes containing {beta}-glucan for skin wound healing, especially their effect on adult human dermal fibroblast (aHDF) and adipose tissue-derived stem cell (ADSC) activation, proliferation, migration, collagen gel contraction and biological safety tests of the prepared membrane. This study demonstrated that {beta}-glucan and porous PLGA membranes containing {beta}-glucan have enhanced the cellular responses, proliferation and migration, of aHDFs and ADSCs and the result of a collagen gel contraction assay also revealed that collagen gels contract strongly after 4 h post-gelation incubation with {beta}-glucan. Furthermore, we confirmed that porous PLGA membranes containing {beta}-glucan are biologically safe for wound healing study. These results indicate that the porous PLGA membranes containing {beta}-glucan interacted favorably with the membrane and the topical administration of {beta}-glucan was useful in promoting wound healing. Therefore, our study suggests that {beta}-glucan and porous PLGA membranes containing {beta}-glucan may be useful as a material for enhancing wound healing.

Containment

International Nuclear Information System (INIS)

Anon.

1987-01-01

The primary mission of the Containment Group is to ensure that underground nuclear tests are satisfactorily contained. The main goal is the development of sound technical bases for containment-related methodology. Major areas of activity include siting, geologic description, emplacement hole stemming, and phenomenological predictions. Performance results of sanded gypsum concrete plugs on the Jefferson, Panamint, Cornucopia, Labquark, and Bodie events are given. Activities are also described in the following areas: computational capabilities site description, predictive modeling, and cavity-pressure measurement. Containment publications are listed. 8 references

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...

CONTAIN code analyses of direct containment heating experiments

International Nuclear Information System (INIS)

Williams, D.C.; Griffith, R.O.; Tadios, E.L.; Washington, K.E.

1995-01-01

In some nuclear reactor core-melt accidents, a potential exists for molten core-debris to be dispersed into the containment under high pressure. Resulting energy transfer to the containment atmosphere can pressurize the containment. This process, known as direct containment heating (DCH), has been the subject of extensive experimental and analytical programs sponsored by the U.S. Nuclear Regulatory Commission (NRC). The DCH modeling has been an important focus for the development of the CONTAIN code. Results of a detailed independent peer review of the CONTAIN code were published recently. This paper summarizes work performed in support of the peer review in which the CONTAIN code was applied to analyze DCH experiments. Goals of this work were comparison of calculated and experimental results, CONTAIN DCH model assessment, and development of guidance for code users, including development of a standardized input prescription for DCH analysis

A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine.

Science.gov (United States)

de Waard, Pim W J; Peijnenburg, Ad A C M; Baykus, Hakan; Aarts, Jac M M J G; Hoogenboom, Ron L A P; van Schooten, Frederik J; de Kok, Theo M C M

2008-10-22

Binding and activation of the aryl hydrocarbon receptor (AhR) is thought to be an essential step in the toxicity of the environmental pollutants dioxins and dioxin-like PCBs. However, also a number of natural compounds, referred to as NAhRAs (natural Ah-receptor agonists), which are present in, for example, fruits and vegetables, can bind and activate this receptor. To study their potential effects in humans, we first investigated the effect of the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gene expression in ex vivo exposed freshly isolated human lymphocytes, and compared the resulting gene expression profile with those caused by the well-known NAhRA indolo[3,2-b]carbazole (ICZ), originating from cruciferous vegetables, and by a hexane extract of NAhRA-containing grapefruit juice (GJE). Only ICZ induced a gene expression profile similar to TCDD in the lymphocytes, and both significantly up-regulated CYP1B1 and TIPARP (TCDD-inducible poly (ADP-ribose) polymerase) mRNA. Next, we performed a human intervention study with NAhRA-containing cruciferous vegetables and grapefruit juice. The expression of the prototypical AhR-responsive genes CYP1A1, CYP1B1 and NQO1 in whole blood cells and in freshly isolated lymphocytes was not significantly affected. Also enzyme activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO), as judged by caffeine metabolites in urine, were unaffected, except for a small down-regulation of NAT2 activity by grapefruit juice. Examination of blood plasma with DR CALUX showed a 12% increased AhR agonist activity 3 and 24 h after consumption of cruciferous vegetables, but did not show a significant effect of grapefruit juice consumption. We conclude that intake of NAhRAs from food may result in minor AhR-related effects measurable in human blood and urine.

Direct containment heating models in the CONTAIN code

International Nuclear Information System (INIS)

Washington, K.E.; Williams, D.C.

1995-08-01

The potential exists in a nuclear reactor core melt severe accident for molten core debris to be dispersed under high pressure into the containment building. If this occurs, the set of phenomena that result in the transfer of energy to the containment atmosphere and its surroundings is referred to as direct containment heating (DCH). Because of the potential for DCH to lead to early containment failure, the U.S. Nuclear Regulatory Commission (USNRC) has sponsored an extensive research program consisting of experimental, analytical, and risk integration components. An important element of the analytical research has been the development and assessment of direct containment heating models in the CONTAIN code. This report documents the DCH models in the CONTAIN code. DCH models in CONTAIN for representing debris transport, trapping, chemical reactions, and heat transfer from debris to the containment atmosphere and surroundings are described. The descriptions include the governing equations and input instructions in CONTAIN unique to performing DCH calculations. Modifications made to the combustion models in CONTAIN for representing the combustion of DCH-produced and pre-existing hydrogen under DCH conditions are also described. Input table options for representing the discharge of debris from the RPV and the entrainment phase of the DCH process are also described. A sample calculation is presented to demonstrate the functionality of the models. The results show that reasonable behavior is obtained when the models are used to predict the sixth Zion geometry integral effects test at 1/10th scale

Direct containment heating models in the CONTAIN code

Energy Technology Data Exchange (ETDEWEB)

Washington, K.E.; Williams, D.C.

1995-08-01

The potential exists in a nuclear reactor core melt severe accident for molten core debris to be dispersed under high pressure into the containment building. If this occurs, the set of phenomena that result in the transfer of energy to the containment atmosphere and its surroundings is referred to as direct containment heating (DCH). Because of the potential for DCH to lead to early containment failure, the U.S. Nuclear Regulatory Commission (USNRC) has sponsored an extensive research program consisting of experimental, analytical, and risk integration components. An important element of the analytical research has been the development and assessment of direct containment heating models in the CONTAIN code. This report documents the DCH models in the CONTAIN code. DCH models in CONTAIN for representing debris transport, trapping, chemical reactions, and heat transfer from debris to the containment atmosphere and surroundings are described. The descriptions include the governing equations and input instructions in CONTAIN unique to performing DCH calculations. Modifications made to the combustion models in CONTAIN for representing the combustion of DCH-produced and pre-existing hydrogen under DCH conditions are also described. Input table options for representing the discharge of debris from the RPV and the entrainment phase of the DCH process are also described. A sample calculation is presented to demonstrate the functionality of the models. The results show that reasonable behavior is obtained when the models are used to predict the sixth Zion geometry integral effects test at 1/10th scale.

Glycogen synthase kinase-3 inhibition sensitizes human induced pluripotent stem cells to thiol-containing antioxidants induced apoptosis.

Science.gov (United States)

Tu, Chengyi; Xu, Robert; Koleti, Meghana; Zoldan, Janet

2017-08-01

Inhibition of glycogen synthase kinase 3 (GSK3) is an extensively used strategy to activate Wnt pathway for pluripotent stem cell (PSC) differentiation. However, the effects of such inhibition on PSCs, besides upregulating the Wnt pathway, have rarely been investigated despite that GSK3 is broadly involved in other cellular activities such as insulin signaling and cell growth/survival regulation. Here we describe a previously unknown synergistic effect between GSK3 inhibition (e.g., Chir99021 and LY2090314) and various normally non-toxic thiol-containing antioxidants (e.g., N-acetylcysteine, NAC) on the induction of apoptosis in human induced pluripotent stem cells (iPSCs). Neither Chir99021 nor the antioxidants individually induced significant apoptosis, whereas their combined treatment resulted in rapid and extensive apoptosis, with substantial caspase 3 activity observed within 3h and over 90% decrease in cell viability after 24h. We confirmed the generality of this phenomenon with multiple independent iPSCs lines, various thiol-based antioxidants and distinct GSK3 inhibitors. Mechanistically, we demonstrated that rapamycin treatment could substantially reduce cell death, suggesting the critical role of mammalian target of rapamycin (mTOR). Akt dysregulation was also found to partially contribute to cell apoptosis but was not the primary cause. Further, this coordinated proapoptotic effect was not detected in mouse ESCs but was present in another human cells line: a breast cancer cell line (MDA-MB-231). Given the wide use of GSK3 inhibition in biomedical research: from iPSC differentiation to cancer intervention and the treatment of neuronal diseases, researchers can potentially take advantage of or avoid this synergistic effect for improved experimental or clinical outcome. Copyright © 2017. Published by Elsevier B.V.

Effect of peritoneal dialysis fluid containing osmo-metabolic agents on human endothelial cells

Directory of Open Access Journals (Sweden)

Bonomini M

2016-11-01

Full Text Available Mario Bonomini,1,2 Sara Di Silvestre,3,4 Pamela Di Tomo,3,4 Natalia Di Pietro,2,4 Domitilla Mandatori,3,4 Lorenzo Di Liberato,1 Vittorio Sirolli,1,2 Francesco Chiarelli,2,4 Cesare Indiveri,5 Assunta Pandolfi,3,4 Arduino Arduini6 1Unit of Nephrology and Dialysis, 2Department of Medicine and Aging Sciences, 3Department of Medical, Oral and Biotechnological Sciences, 4Aging Research Center and Translational Medicine, CeSI-MeT, University “G. d’Annunzio”, Chieti-Pescara, 5Department DiBEST (Biologia, Ecologia, Scienze della Terra, Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende CS, Italy; 6CoreQuest, Manno, Switzerland Background: The use of glucose as the only osmotic agent in peritoneal dialysis (PD solutions (PDSs is believed to exert local (peritoneal and systemic detrimental actions, particularly in diabetic PD patients. To improve peritoneal biocompatibility, we have developed more biocompatible PDSs containing xylitol and carnitine along with significantly less amounts of glucose and have tested them in cultured Human Vein Endothelial Cells (HUVECs obtained from the umbilical cords of healthy (C and gestational diabetic (GD mothers. Methods: Primary C- and GD-HUVECs were treated for 72 hours with our PDSs (xylitol 0.7% and 1.5%, whereas carnitine and glucose were fixed at 0.02% and 0.5%, respectively and two glucose-based PDSs (glucose 1.36% or 2.27%. We examined their effects on endothelial cell proliferation (cell count, viability (3-(4,5-dimethylthiazolyl-2-2,5-diphenyltetrazolium bromide assay, intracellular nitro-oxidative stress (peroxynitrite levels, Vascular Cell Adhesion Molecule-1 and Intercellular Adhesion Molecule-1 membrane exposure (flow cytometry, and HUVEC-monocyte interactions (U937 adhesion assay. Results: Compared to glucose-based PDSs, our in vitro studies demonstrated that the tested PDSs did not change the proliferative potential both in C- and GD-HUVECs. Moreover, our

Metabolism of phthalates in humans

DEFF Research Database (Denmark)

Frederiksen, Hanne; Skakkebaek, Niels E; Andersson, Anna-Maria

2007-01-01

on the foetal testis and they are similar to those seen in humans with testicular dysgenesis syndrome. Therefore, exposure of the human foetus and infants to phthalates via maternal exposure is a matter of concern. The metabolic pathways of phthalate metabolites excreted in human urine are partly known for some...... phthalates, but our knowledge about metabolic distribution in the body and other biological fluids, including breast milk, is limited. Compared to urine, human breast milk contains relatively more of the hydrophobic phthalates, such as di-n-butyl phthalate and the longer-branched, di(2-ethylhexyl) phthalate...... (DEHP) and di-iso-nonyl phthalate (DiNP); and their monoester metabolites. Urine, however, contains relatively more of the secondary metabolites of DEHP and DiNP, as well as the monoester phthalates of the more short-branched phthalates. This differential distribution is of special concern as...

Beyond the Conventional Argonauts Explanations

DEFF Research Database (Denmark)

Rezaei, Shahamak

professional and cultural experiences and their network in their home and host country to start up new firms in their home country. Following Schumpeter (1942) these firms are pr definition the outcome of innovative activities as the bifocality (Honig et al. 2010) of the transnational entrepreneurs allows...... for novel types of resource combinations and thus new products (i.e. new to the firm and market) and possible new organizational forms (e.g. double location sourcing). The centrality of transnational entrepreneurs within the ICT industry has been thoroughly documented in the US, Taiwan, China and India...

How to combine human resource management systems and human capital portfolios to achieve superior innovation performance

OpenAIRE

Rupietta, Christian; Backes-Gellner, Uschi

2013-01-01

Firms generate new knowledge that leads to innovations by recombining existing knowledge sources. A successful recombination depends on both the availability of a knowledge stock (human capital pool) that contains innovation-relevant knowledge and the regulation of the knowledge flow through the application of human resource management practices. However, while human resource theory expects complementarities between both the human capital pool and the human resource management system it does ...

Shatavarins (containing Shatavarin IV) with anticancer activity from the roots of Asparagus racemosus

Science.gov (United States)

Mitra, Shankar K.; Prakash, Neswi S.; Sundaram, Ramachandran

2012-01-01

Objectives: The anticancer activity of shatavarins (containing shatavarin IV) isolated from the roots of Asparagus racemosus (Wild) was evaluated using in vitro and in vivo experimental models. Material and Methods: The shatavarin IV was isolated from ethyl acetate insoluble fraction (AR-2B) of chloroform:methanol (2:1) (AR-2) extract of A. racemosus roots. The cytotoxicity (in vitro) of shatavarin IV and other shatavarins rich fraction was carried out using of MTT assay using MCF-7 (human breast cancer), HT-29 (human colon adenocarcinoma), and A-498 (human kidney carcinoma) cell lines. The in vivo anticancer activity of shatavarins (containing shatavarin IV) was evaluated against Ehrlich ascites carcinoma (EAC) tumor bearing mice. Results: The isolated shatavarin IV (84.69 %) along with shatavarins rich fraction, coded AR-2B containing 5.05% shatavarin IV showed potent cytotoxicity. Oral administration of AR-2B to tumor bearing mice at doses of 250 and 500 mg/kg body weight for 10 days, showed significant reduction in percent increase in body weight, tumor volume, packed cell volume, viable tumor cell count, and increased non-viable cell count when compared to the untreated mice of the EAC control group. The restoration of hematological parameters towards normalcy was also observed. Conclusion: The result suggests that the shatavarins (containing shatavarin IV) rich fraction (AR-2B) exhibits significant anticancer activity in both in vitro and in vivo experimental models. PMID:23248403

Human serum amyloid genes--molecular characterization

International Nuclear Information System (INIS)

Sack, G.H.; Lease, J.J.

1986-01-01

Three clones containing human genes for serum amyloid A protein (SAA) have been isolated and characterized. Each of two clones, GSAA 1 and 2 (of 12.8 and 15.9 kilobases, respectively), contains two exons, accouting for amino acids 12-58 and 58-103 of mature SAA; the extreme 5' termini and 5' untranslated regions have not yet been defined but are anticipated to be close based on studies of murine SAA genes. Initial amino acid sequence comparisons show 78/89 identical residues. At 4 of the 11 discrepant residues, the amino acid specified by the codon is the same as the corresponding residue in murine SAA. Identification of regions containing coding regions has permitted use of selected subclones for blot hybridization studies of larger human SAA chromosomal gene organization. The third clone, GSAA 3 also contains SAA coding information by DNA sequence analysis but has a different organization which has not yet been fully described. We have reported the isolation of clones of human DNA hybridizing with pRS48 - a plasmid containing a complementary DNA (cDNA) clone for murine serum amyloid A (SAA; 1, 2). We now present more detailed data confirming the identity and defining some of the organizational features of these clones

Progress towards construction of a total restriction fragment map of a human chromosome.

NARCIS (Netherlands)

H. Vissing; F.G. Grosveld (Frank); E. Solomon; G. Moore; N. Lench; N. Shennan; R. Williamson

1987-01-01

textabstractWe present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human

Sulphur-containing Amino Acids: Protective Role Against Free Radicals and Heavy Metals.

Science.gov (United States)

Colovic, Mirjana B; Vasic, Vesna M; Djuric, Dragan M; Krstic, Danijela Z

2018-01-30

Sulphur is an abundant element in biological systems, which plays an important role in processes essential for life as a constituent of proteins, vitamins and other crucial biomolecules. The major source of sulphur for humans is plants being able to use inorganic sulphur in the purpose of sulphur-containing amino acids synthesis. Sulphur-containing amino acids include methionine, cysteine, homocysteine, and taurine. Methionine and cysteine are classified as proteinogenic, canonic amino acids incorporated in protein structure. Sulphur amino acids are involved in the synthesis of intracellular antioxidants such as glutathione and N-acetyl cysteine. Moreover, naturally occurring sulphur-containing ligands are effective and safe detoxifying agents, often used in order to prevent toxic metal ions effects and their accumulation in human body. Literature search for peer-reviewed articles was performed using PubMed and Scopus databases, and utilizing appropriate keywords. This review is focused on sulphur-containing amino acids - methionine, cysteine, taurine, and their derivatives - glutathione and N-acetylcysteine, and their defense effects as antioxidant agents against free radicals. Additionally, the protective effects of sulphur-containing ligands against the toxic effects of heavy and transition metal ions, and their reactivation role towards the enzyme inhibition are described. Sulphur-containing amino acids represent a powerful part of cell antioxidant system. Thus, they are essential in the maintenance of normal cellular functions and health. In addition to their worthy antioxidant action, sulphur-containing amino acids may offer a chelating site for heavy metals. Accordingly, they may be supplemented during chelating therapy, providing beneficial effects in eliminating toxic metals. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Proposed Objective Odor Control Test Methodology for Waste Containment

Science.gov (United States)

Vos, Gordon

2010-01-01

The Orion Cockpit Working Group has requested that an odor control testing methodology be proposed to evaluate the odor containment effectiveness of waste disposal bags to be flown on the Orion Crew Exploration Vehicle. As a standardized "odor containment" test does not appear to be a matter of record for the project, a new test method is being proposed. This method is based on existing test methods used in industrial hygiene for the evaluation of respirator fit in occupational settings, and takes into consideration peer reviewed documentation of human odor thresholds for standardized contaminates, industry stardnard atmostpheric testing methodologies, and established criteria for laboratory analysis. The proposed methodology is quantitative, though it can readily be complimented with a qualitative subjective assessment. Isoamyl acetate (IAA - also known at isopentyl acetate) is commonly used in respirator fit testing, and there are documented methodologies for both measuring its quantitative airborne concentrations. IAA is a clear, colorless liquid with a banana-like odor, documented detectable smell threshold for humans of 0.025 PPM, and a 15 PPB level of quantation limit.

Effectiveness of travel restrictions in the rapid containment of human influenza: a systematic review

Science.gov (United States)

Mateus, Ana LP; Otete, Harmony E; Beck, Charles R; Dolan, Gayle P; Nguyen-Van-Tam, Jonathan S

2014-01-01

Abstract Objective To assess the effectiveness of internal and international travel restrictions in the rapid containment of influenza. Methods We conducted a systematic review according to the requirements of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Health-care databases and grey literature were searched and screened for records published before May 2014. Data extraction and assessments of risk of bias were undertaken by two researchers independently. Results were synthesized in a narrative form. Findings The overall risk of bias in the 23 included studies was low to moderate. Internal travel restrictions and international border restrictions delayed the spread of influenza epidemics by one week and two months, respectively. International travel restrictions delayed the spread and peak of epidemics by periods varying between a few days and four months. Travel restrictions reduced the incidence of new cases by less than 3%. Impact was reduced when restrictions were implemented more than six weeks after the notification of epidemics or when the level of transmissibility was high. Travel restrictions would have minimal impact in urban centres with dense populations and travel networks. We found no evidence that travel restrictions would contain influenza within a defined geographical area. Conclusion Extensive travel restrictions may delay the dissemination of influenza but cannot prevent it. The evidence does not support travel restrictions as an isolated intervention for the rapid containment of influenza. Travel restrictions would make an extremely limited contribution to any policy for rapid containment of influenza at source during the first emergence of a pandemic virus. PMID:25552771

Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

International Nuclear Information System (INIS)

Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R.

1990-01-01

Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV

Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

Energy Technology Data Exchange (ETDEWEB)

Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

1999-02-01

The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

Antitumor Active Protein-containing Glycans from the Body of Ganoderma tsugae

Institute of Scientific and Technical Information of China (English)

LIU Ying; LI Yue-fei; ZHENG Ke-yan; FEI Xiao-fang

2012-01-01

To explore the effects of traditional herbal medicine Ganoderma tsugae(G.tsugae) on immunomodulatory and antitumor activities,the crude polysaccharides ofG.tsugae were purified by filtration,diethylaminoethyl(DEAE)sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography.Two main fractions,protein-containing glycans CSSLP-I and CSSLP-2,were obtained via the gradient elution.The protein content,molecular weight,and monosaccharide composition of the two fractions were analyzed.Furthermore,the influence of the protein-containing glycans from G.tsugae on the activation of human acute monocytic leukemia cell line(THP-1 ) and their antitumor activities to the human hepatocellular liver carcinoma cell(HepG-2) in vitro were evaluated.The results indicate that CSSLP-I and CSSLP-2 could increase the pinocytic activity of THP-1 cells and induce THP-1 cells to produce the cytokines of TNFa and IL-2,significantly.CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell(NepG-2).Moreover,the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the participation of TNFa and 1L-2 or other antitumor factors induced from THP-1 cclls by G.tsugae protein-containing glycan fractions.

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

Science.gov (United States)

Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

2016-07-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. © FEMS 2016.

Storage of Human Breast Milk

Directory of Open Access Journals (Sweden)

Gamze Can

2007-10-01

Full Text Available Storage of human breast milk by freezing or refrigeration of milk has been recommended especially at some social circumstances of most mothers who are regularly separated from their infants because of work. The greatest fear that has hindered the prospects of in - vitro storage of breast milk for any considerable period of time is the possibility of bacterial contamination and growth of infectious pathogens in the stored milk, there by rendering them unsafe for human consumption. The storage container can influence the cell content of milk, as the cells adhere to the walls of a glass container but not to polyethylene or polypropylene containers. Bacteriological examination of refrigerated milks has proven their safety for human consumption for even up to 72 h. For a storage over longer periods up to 1 month, freezing at - 20 0C could be recommended, but the most preferred method, especially for longer storage would be fresh freezing at - 70 0C, if affordable or available. The nutrient value of human milk is essentially unchanged, but the immunological properties are reduced by various storage techniques. Boiling and microwave radiation have not been recommended. [TAF Prev Med Bull 2007; 6(5.000: 375-379

45 CFR 91.18 - Age distinctions contained in HHS regulations.

Science.gov (United States)

2010-10-01

....18 Section 91.18 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION... distinctions contained in a rule or regulation issued by HHS shall be presumed to be necessary to the achievement of a statutory objective of the program or activity to which the rule or regulation applies...

Reactor container

International Nuclear Information System (INIS)

Ichiki, Tadaharu; Saba, Kazuhisa.

1979-01-01

Purpose: To improve the earthquake resistance as well as reduce the size of a container for a nuclear reactor with no adverse effects on the decrease of impact shock to the container and shortening of construction step. Constitution: Reinforcing profile steel materials are welded longitudinally and transversely to the inner surface of a container, and inner steel plates are secured to the above profile steel materials while keeping a gap between the materials and the container. Reactor shielding wall planted to the base concrete of the container is mounted to the pressure vessel, and main steam pipeways secured by the transverse beams and led to the outside of container is connected. This can improve the rigidity earthquake strength and the safetiness against the increase in the inside pressure upon failures of the container. (Yoshino, Y.)

Cloning, sequencing, and expression of cDNA for human β-glucuronidase

International Nuclear Information System (INIS)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-01-01

The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

Linear ion-trap mass spectrometric characterization of human pituitary nitrotyrosine-containing proteins

Science.gov (United States)

Zhan, Xianquan; Desiderio, Dominic M.

2007-01-01

The nitric oxide-mediated Tyr-nitration of endogenous proteins is associated with several pathological and physiological processes. In order to investigate the presence - and potential roles - of Tyr-nitration in the human pituitary, a large-format two-dimensional gel separation plus a Western blot against a specific anti-3-nitrotyrosine antibody were used to separate and detect nitroproteins from a human pituitary proteome. The nitroproteins were subjected to in-gel trypsin digestion, and high-sensitivity vacuum matrix-assisted laser desorption/ionization (vMALDI) linear ion-trap tandem mass spectrometry was used to analyze the tryptic peptides. Those MS/MS data were used to determine the amino acid sequence and the specific nitration site of each tryptic nitropeptide, and were matched to corresponding proteins with Bioworks TuboSEQUEST software. Compared to our previous study, 16 new nitrotyrosine-immunoreactive positive Western blot spots were found within the area pI 3.0-10 and Mr 10-100 kDa. Four new nitroproteins were discovered: the stanniocalcin 1 precursor--involved in calcium and phosphate metabolism; mitochondrial co-chaperone protein HscB, which might act as a co-chaperone in iron-sulfur cluster assembly in mitochrondria; progestin and adipoQ receptor family member III--a seven-transmembrane receptor; proteasome subunit alpha type 2--involved in an ATP/ubiquitin-dependent non-lysosomal proteolytic pathway. Those data demonstrate that nitric oxide-mediated Tyr-nitration might participate in various biochemical, metabolic, and pathological processes in the human pituitary.

LCA comparison of container systems in municipal solid waste management

International Nuclear Information System (INIS)

Rives, Jesus; Rieradevall, Joan; Gabarrell, Xavier

2010-01-01

The planning and design of integrated municipal solid waste management (MSWM) systems requires accurate environmental impact evaluation of the systems and their components. This research assessed, quantified and compared the environmental impact of the first stage of the most used MSW container systems. The comparison was based on factors such as the volume of the containers, from small bins of 60-80 l to containers of 2400 l, and on the manufactured materials, steel and high-density polyethylene (HDPE). Also, some parameters such as frequency of collections, waste generation, filling percentage and waste container contents, were established to obtain comparable systems. The methodological framework of the analysis was the life cycle assessment (LCA), and the impact assessment method was based on CML 2 baseline 2000. Results indicated that, for the same volume, the collection systems that use HDPE waste containers had more of an impact than those using steel waste containers, in terms of abiotic depletion, global warming, ozone layer depletion, acidification, eutrophication, photochemical oxidation, human toxicity and terrestrial ecotoxicity. Besides, the collection systems using small HDPE bins (60 l or 80 l) had most impact while systems using big steel containers (2400 l) had less impact. Subsequent sensitivity analysis about the parameters established demonstrated that they could change the ultimate environmental impact of each waste container collection system, but that the comparative relationship between systems was similar.

Safety evaluation of an IPP tripeptide-containing milk protein hydrolysate

NARCIS (Netherlands)

Ponstein-Simarro Doorten, A.Y.; Wiel, J.A.G. van de; Jonker, D.

2009-01-01

Tensguard™ is a milk protein hydrolysate containing the lactotripeptide IPP. It is derived from cow's milk, which is present in the human diet and has a safe history of consumption. The final Tensguard™ product, a supplement or a functional food ingredient, is intended for use by people who want to

Structural and catalytic characterization of a thermally stable and acid-stable variant of human carbonic anhydrase II containing an engineered disulfide bond

Energy Technology Data Exchange (ETDEWEB)

Boone, Christopher D.; Habibzadegan, Andrew [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States); Tu, Chingkuang; Silverman, David N. [University of Florida, PO Box 100267, Gainesville, FL 32610 (United States); McKenna, Robert, E-mail: rmckenna@ufl.edu [University of Florida, PO Box 100245, Gainesville, FL 32610 (United States)

2013-08-01

The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) has been solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO{sub 2} to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost-inefficient high-maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild-type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X-ray crystallographic structure of the disulfide-containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active-site geometry. Kinetic studies utilizing the measurement of {sup 18}O-labeled CO{sub 2} by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.

Comparison of diet-induced thermogenesis of foods containing medium- versus long-chain triacylglycerols.

Science.gov (United States)

Kasai, Michio; Nosaka, Naohisa; Maki, Hideaki; Suzuki, Yoshie; Takeuchi, Hiroyuki; Aoyama, Toshiaki; Ohra, Atsushi; Harada, Youji; Okazaki, Mitsuko; Kondo, Kazuo

2002-12-01

The purpose of this study was to investigate the effect of 5-10 g of medium-chain triacylglycerols (MCT) on diet-induced thermogenesis in healthy humans. The study compared diet-induced thermogenesis after ingestion of test foods containing MCT and long-chain triacylglycerols (LCT), using a double-blind, crossover design. Eight male and eight female subjects participated in study 1 and study 2, respectively. In both studies, the LCT was a blend of rapeseed oil and soybean oil. In study 1, the liquid meals contained 10 g MCT (10M), a mixture of 5 g MCT and 5 g LCT (5M5L), and 10 g LCT (10L). In study 2, the subjects were given a meal (sandwich and clear soup) with the mayonnaise or margarine containing 5 g of MCT or LCT. Postprandial energy expenditure was measured by indirect calorimetry before and during the 6 h after ingestion of the test meals. Diet-induced thermogenesis was significantly greater after 5M5L and 10M Ingestion as compared to 10L ingestion. Ingestion of the mayonnaise or margarine containing 5 g MCT caused significantly larger diet-induced thermogenesis as compared to that of LCT. These results suggest that, in healthy humans, the intake of 5-10 g of MCT causes larger diet-induced thermogenesis than that of LCT, irrespective of the form of meal containing the MCT.

Characterization of MRP RNA–protein interactions within the perinucleolar compartment

Science.gov (United States)

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-01-01

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA–processing (MRP) RNA, pyrimidine tract–binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA–containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)–PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA–protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC. PMID:21233287

Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

Energy Technology Data Exchange (ETDEWEB)

Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

1994-09-01

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

Directory of Open Access Journals (Sweden)

Venkateswara Reddy Gogulamudi

2015-10-01

Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

7 CFR 906.340 - Container, pack, and container marking regulations.

Science.gov (United States)

2010-01-01

... 7 Agriculture 8 2010-01-01 2010-01-01 false Container, pack, and container marking regulations... AGRICULTURE ORANGES AND GRAPEFRUIT GROWN IN LOWER RIO GRANDE VALLEY IN TEXAS Container and Pack Requirements § 906.340 Container, pack, and container marking regulations. (a) No handler shall handle any variety of...

Development of container failure models

International Nuclear Information System (INIS)

Garisto, N.C.

1990-01-01

In order to produce a complete performance assessment for a Canadian waste vault some prediction of container failure times is required. Data are limited; however, the effects of various possible failure scenarios on the rest of the vault model can be tested. For titanium and copper, the two materials considered in the Canadian program, data are available on the frequency of failures due to manufacturing defects; there is also an estimate on the expected size of such defects. It can be shown that the consequences of such small defects in terms of the dose to humans are acceptable. It is not clear, from a modelling point of view, whether titanium or copper are preferable

Reactor container

International Nuclear Information System (INIS)

Naruse, Yoshihiro.

1990-01-01

The thickness of steel shell plates in a reactor container embedded in sand cussions is monitored to recognize the corrosion of the steel shell plates. That is, the reactor pressure vessel is contained in a reactor container shell and the sand cussions are disposed on the lower outside of the reactor container shell to elastically support the shell. A pit is disposed at a position opposing to the sand cussions for measuring the thickness of the reactor container shell plates. The pit is usually closed by a closing member. In the reactor container thus constituted, the closing member can be removed upon periodical inspection to measure the thickness of the shell plates. Accordingly, the corrosion of the steel shell plates can be recognized by the change of the plate thickness. (I.S.)

A container

DEFF Research Database (Denmark)

2012-01-01

A container assembly for the containment of fluids or solids under a pressure different from the ambient pressure comprising a container (2) comprising an opening and an annular sealing, a lid (3) comprising a central portion (5) and engagement means (7) for engaging the annular flange, and sealing...... means (10) wherein the engagement means (7) is adapted, via the sealing means, to seal the opening when the pressure of the container assembly differs from the ambient pressure in such a way that the central portion (5) flexes in the axial direction which leads to a radial tightening of the engagement...... means (7) to the container, wherein the container further comprises locking means (12) that can be positioned so that the central portion is hindered from flexing in at least one direction....

Human waste

NARCIS (Netherlands)

Amin, Md Nurul; Kroeze, Carolien; Strokal, Maryna

2017-01-01

Many people practice open defecation in south Asia. As a result, lot of human waste containing nutrients such as nitrogen (N) and phosphorus (P) enter rivers. Rivers transport these nutrients to coastal waters, resulting in marine pollution. This source of nutrient pollution is, however, ignored in

Climate change: The need for a human rights agenda within a framework of shared human security

OpenAIRE

Gasper, Des

2012-01-01

textabstractThis edited volume publishes as a special issue of the quarterly social science journal Social Research, a collection of articles on Human Rights and the Global Economy. The topics addressed are Human Rights and Economy Policy; Global Poverty and the Obligations of Rich Countries; Human Rights, Climate Change and Global Justice; and Corporations and Human Rights Obligations. This issue contains the edited proceedings of the November 2011 conference at the New School, where experts...

Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

Science.gov (United States)

Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

2015-06-01

The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The Kinetics Human Action Video Dataset

OpenAIRE

Kay, Will; Carreira, Joao; Simonyan, Karen; Zhang, Brian; Hillier, Chloe; Vijayanarasimhan, Sudheendra; Viola, Fabio; Green, Tim; Back, Trevor; Natsev, Paul; Suleyman, Mustafa; Zisserman, Andrew

2017-01-01

We describe the DeepMind Kinetics human action video dataset. The dataset contains 400 human action classes, with at least 400 video clips for each action. Each clip lasts around 10s and is taken from a different YouTube video. The actions are human focussed and cover a broad range of classes including human-object interactions such as playing instruments, as well as human-human interactions such as shaking hands. We describe the statistics of the dataset, how it was collected, and give some ...

Individual microRNAs (miRNAs) display distinct mRNA targeting "rules".

Science.gov (United States)

Wang, Wang-Xia; Wilfred, Bernard R; Xie, Kevin; Jennings, Mary H; Hu, Yanling Hu; Stromberg, Arnold J; Nelson, Peter T

2010-01-01

MicroRNAs (miRNAs) guide Argonaute (AGO)-containing microribonucleoprotein (miRNP) complexes to target mRNAs.It has been assumed that miRNAs behave similarly to each other with regard to mRNA target recognition. The usual assumptions, which are based on prior studies, are that miRNAs target preferentially sequences in the 3'UTR of mRNAs,guided by the 5' "seed" portion of the miRNAs. Here we isolated AGO- and miRNA-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with anti-AGO antibody. Cells were transfected with miR-107, miR-124,miR-128, miR-320, or a negative control miRNA. Co-IPed RNAs were subjected to downstream high-density Affymetrix Human Gene 1.0 ST microarray analyses using an assay we validated previously-a "RIP-Chip" experimental design. RIP-Chip data provided a list of mRNAs recruited into the AGO-miRNP in correlation to each miRNA. These experimentally identified miRNA targets were analyzed for complementary six nucleotide "seed" sequences within the transfected miRNAs. We found that miR-124 targets tended to have sequences in the 3'UTR that would be recognized by the 5' seed of miR-124, as described in previous studies. By contrast, miR-107 targets tended to have 'seed' sequences in the mRNA open reading frame, but not the 3' UTR. Further, mRNA targets of miR-128 and miR-320 are less enriched for 6-mer seed sequences in comparison to miR-107 and miR-124. In sum, our data support the importance of the 5' seed in determining binding characteristics for some miRNAs; however, the "binding rules" are complex, and individual miRNAs can have distinct sequence determinants that lead to mRNA targeting.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

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Noelia Losino

Full Text Available Embryonic stem cells (ESC need a set of specific factors to be propagated. They can also grow in conditioned medium (CM derived from a bovine granulosa cell line BGC (BGC-CM, a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+. Here, we investigated if the FN EDA(+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-, and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

Containing Ebola at the Source with Ring Vaccination.

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Stefano Merler

2016-11-01

Full Text Available Interim results from the Guinea Ebola ring vaccination trial suggest high efficacy of the rVSV-ZEBOV vaccine. These findings open the door to the use of ring vaccination strategies in which the contacts and contacts of contacts of each index case are promptly vaccinated to contain future Ebola virus disease outbreaks. To provide a numerical estimate of the effectiveness of ring vaccination strategies we introduce a spatially explicit agent-based model to simulate Ebola outbreaks in the Pujehun district, Sierra Leone, structurally similar to previous modelling approaches. We find that ring vaccination can successfully contain an outbreak for values of the effective reproduction number up to 1.6. Through an extensive sensitivity analysis of parameters characterising the readiness and capacity of the health care system, we identify interventions that, alongside ring vaccination, could increase the likelihood of containment. In particular, shortening the time from symptoms onset to hospitalisation to 2-3 days on average through improved contact tracing procedures, adding a 2km spatial component to the vaccination ring, and decreasing human mobility by quarantining affected areas might contribute increase our ability to contain outbreaks with effective reproduction number up to 2.6. These results have implications for future control of Ebola and other emerging infectious disease threats.

Empty Container Logistics

Directory of Open Access Journals (Sweden)

Jakov Karmelić

2012-05-01

Full Text Available Within the whole world container traffic, the largest share of containers is in the status of repositioning. Container repositioning results from the need for harmonization between the point of empty container accumulation and the point of demand, and waiting time for the availability of the first next transport of cargo. This status of containers on the container market is the consequence of imbalances in the worldwide trade distribution on most important shipping routes. The need for fast and effective reallocation of empty containers causes high costs and often represents an obstacle affecting the efficiency of port container terminals and inland carriers.In accordance with the above issue, this paper is mainly focused on the analysis of the data concerning global container capacities and the roots of container equipment imbalances, with the aim of determining the importance of empty container management and the need for empty container micro-logistic planning at the spread port area.

Type VI secretion systems of human gut Bacteroidales segregate into three genetic architectures, two of which are contained on mobile genetic elements.

Science.gov (United States)

Coyne, Michael J; Roelofs, Kevin G; Comstock, Laurie E

2016-01-15

Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems employed by Gram negative bacteria to intoxicate other bacteria or eukaryotic cells. T6SSs were recently discovered in a few Bacteroidetes strains, thereby extending the presence of these systems beyond Proteobacteria. The present study was designed to analyze in a global nature the diversity, abundance, and properties of T6SSs in the Bacteroidales, the most predominant Gram negative bacterial order of the human gut. By performing extensive bioinformatics analyses and creating hidden Markov models for Bacteroidales Tss proteins, we identified 130 T6SS loci in 205 human gut Bacteroidales genomes. Of the 13 core T6SS proteins of Proteobacteria, human gut Bacteroidales T6SS loci encode orthologs of nine, and an additional five other core proteins not present in Proteobacterial T6SSs. The Bacteroidales T6SS loci segregate into three distinct genetic architectures with extensive DNA identity between loci of a given genetic architecture. We found that divergent DNA regions of a genetic architecture encode numerous types of effector and immunity proteins and likely include new classes of these proteins. TheT6SS loci of genetic architecture 1 are contained on highly similar integrative conjugative elements (ICEs), as are the T6SS loci of genetic architecture 2, whereas the T6SS loci of genetic architecture 3 are not and are confined to Bacteroides fragilis. Using collections of co-resident Bacteroidales strains from human subjects, we provide evidence for the transfer of genetic architecture 1 T6SS loci among co-resident Bacteroidales species in the human gut. However, we also found that established ecosystems can harbor strains with distinct T6SS of all genetic architectures. This is the first study to comprehensively analyze of the presence and diversity of T6SS loci within an order of bacteria and to analyze T6SSs of bacteria from a natural community. These studies demonstrate that more than

Human Relations Class. A Syllabus.

Science.gov (United States)

Guillen, Mary A.

A junior high level human relations class develops human interaction and oral communication skills. A week-by-week syllabus contains the following components: introduction of the students to each other and to the principles of body language, transactional analysis, and group interaction; behavior contracts; group dynamics topics and exercises;…

Human-specific HERV-K insertion causes genomic variations in the human genome.

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Wonseok Shin

Full Text Available Human endogenous retroviruses (HERV sequences account for about 8% of the human genome. Through comparative genomics and literature mining, we identified a total of 29 human-specific HERV-K insertions. We characterized them focusing on their structure and flanking sequence. The results showed that four of the human-specific HERV-K insertions deleted human genomic sequences via non-classical insertion mechanisms. Interestingly, two of the human-specific HERV-K insertion loci contained two HERV-K internals and three LTR elements, a pattern which could be explained by LTR-LTR ectopic recombination or template switching. In addition, we conducted a polymorphic test and observed that twelve out of the 29 elements are polymorphic in the human population. In conclusion, human-specific HERV-K elements have inserted into human genome since the divergence of human and chimpanzee, causing human genomic changes. Thus, we believe that human-specific HERV-K activity has contributed to the genomic divergence between humans and chimpanzees, as well as within the human population.

Chronic periapical periodontitis containing mature human hair shaft: a case report.

Science.gov (United States)

Sharif, Mohammad Owaise; Yar, Riaz; Oliver, Richard

2011-04-01

A case is reported of a 44-year-old male who was referred with persistent pus discharge associated with his UL2 which had been root treated on two occasions. Radiographic examination revealed a radiolucency of approximately 8 mm diameter. An apicectomy was performed and histopathological examination revealed the presence of mature birefringent hair-shaft structures within a chronic periapical periodontitis. This article presents a rare occurrence, the presence of human hair in the periapical tissues.

RESEARCH ARTICLE

Indian Academy of Sciences (India)

Navya

Expression analysis of Argonaute, Dicer-like and RNA-dependent RNA polymerase ... environmental stresses such as salinity, drought, cold, heat and other biotic and abiotic .... mechanisms and other physiological processes in this important ...

Molecular characterization of a novel reassortant H1N2 influenza virus containing genes from the 2009 pandemic human H1N1 virus in swine from eastern China.

Science.gov (United States)

Peng, Xiuming; Wu, Haibo; Xu, Lihua; Peng, Xiaorong; Cheng, Linfang; Jin, Changzhong; Xie, Tiansheng; Lu, Xiangyun; Wu, Nanping

2016-06-01

Pandemic outbreaks of H1N1 swine influenza virus have been reported since 2009. Reassortant H1N2 viruses that contain genes from the pandemic H1N1 virus have been isolated in Italy and the United States. However, there is limited information regarding the molecular characteristics of reassortant H1N2 swine influenza viruses in eastern China. Active influenza surveillance programs in Zhejiang Province identified a novel H1N2 influenza virus isolated from pigs displaying clinical signs of influenza virus infection. Whole-genome sequencing was performed and this strain was compared with other influenza viruses available in GenBank. Phylogenetic analysis suggested that the novel strain contained genes from the 2009 pandemic human H1N1 and swine H3N2 viruses. BALB/c mice were infected with the isolated virus to assess its virulence in mice. While the novel H1N2 isolate replicated well in mice, it was found to be less virulent. These results provide additional evidence that swine serve as intermediate hosts or 'mixing vessels' for novel influenza viruses. They also emphasize the importance of surveillance in the swine population for use as an early warning system for influenza outbreaks in swine and human populations.

Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

Science.gov (United States)

Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

2000-01-01

To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

78 FR 9355 - Influenza Viruses Containing the Hemagglutinin From the Goose/Guangdong/1/96 Lineage

Science.gov (United States)

2013-02-08

... DEPARTMENT OF HEALTH AND HUMAN SERVICES [Docket: CDC-2012-0010] 42 CFR Part 73 Influenza Viruses... influenza (HPAI) H5N1 viruses that contain a hemagglutinin (HA) from the Goose/Guangdong/1/96 lineage, and... concerning highly pathogenic avian influenza (HPAI) H5N1 viruses that contain a hemagglutinin (HA) from the...

A container for containing and protecting a radioactive substance

International Nuclear Information System (INIS)

1974-01-01

The invention relates to a container adapted to contain and protect a radio-active substance. That container comprises a heat sensitive device for automatically (and, preferably, sealingly) enclosing and protecting the radio-active substance, should room temperature reach a predetermined level. Thus, the radio-active substance cannot escape in case of fire. Preferably, a bolt is also provided, capable of being actuated at a temperature slightly above the temperature actuating the protective device so as to maintain the radioactive substance protected. This can be applied to containers containing a radio-active substance such as polonium 210 [fr

Nuclear reactor container

International Nuclear Information System (INIS)

Ishiyama, Takenori.

1989-01-01

This invention concerns a nuclear reactor container in which heat is removed from a container by external water injection. Heat is removed from the container by immersing the lower portion of the container into water and scattering spary water from above. Thus, the container can be cooled by the spray water falling down along the outer wall of the container to condensate and cool vapors filled in the container upon occurrence of accidents. Further, since the inside of the container can be cooled also during usual operation, it can also serve as a dry well cooler. Accordingly, heat is removed from the reactor container upon occurrence of accidents by the automatic operation of a spray device corresponding to the change of the internal temperature and the pressure in the reactor container. Further, since all of these devices are disposed out of container, maintenance is also facilitated. (I.S.)

Effect of organic carbon on sorption of human adenovirus to soil particles and laboratory containers

Science.gov (United States)

A key factor controlling the relationship between virus release and human exposure is how virus particles interact with soils, sediments and other solid particles in the environment and in engineered treatment systems. Finding no previous investigations of human adenovirus (HAdV)...

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

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Alexandra Steffens

2015-07-01

Full Text Available Members of the highly conserved class of BEACH domain containing proteins (BDCPs have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1, associates to mRNA processing bodies (P-bodies, and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

Storage Space Allocation of Inbound Container in Railway Container Terminal

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Li Wang

2014-01-01

Full Text Available Efficient storage strategy of railway container terminals is important in balancing resource utilization, reducing waiting time, and improving handling efficiency. In this paper, we consider the formulation and solution algorithm for storage space allocation problem of inbound containers in railway container terminal. The problem is formulated as two-stage optimization models, whose objectives are balancing the workload of inbound containers and reducing the overlapping amounts. An algorithm implement process based on rolling horizon approach is designed to solve the proposed models. Computational experiments on an actual railway container terminal show that the proposed approach is effective to solve space allocation problem of inbound container and is significant for the operation and organization of railway container terminals.

Human Rights Texts: Converting Human Rights Primary Source Documents into Data.

Science.gov (United States)

Fariss, Christopher J; Linder, Fridolin J; Jones, Zachary M; Crabtree, Charles D; Biek, Megan A; Ross, Ana-Sophia M; Kaur, Taranamol; Tsai, Michael

2015-01-01

We introduce and make publicly available a large corpus of digitized primary source human rights documents which are published annually by monitoring agencies that include Amnesty International, Human Rights Watch, the Lawyers Committee for Human Rights, and the United States Department of State. In addition to the digitized text, we also make available and describe document-term matrices, which are datasets that systematically organize the word counts from each unique document by each unique term within the corpus of human rights documents. To contextualize the importance of this corpus, we describe the development of coding procedures in the human rights community and several existing categorical indicators that have been created by human coding of the human rights documents contained in the corpus. We then discuss how the new human rights corpus and the existing human rights datasets can be used with a variety of statistical analyses and machine learning algorithms to help scholars understand how human rights practices and reporting have evolved over time. We close with a discussion of our plans for dataset maintenance, updating, and availability.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

International Nuclear Information System (INIS)

Quaresma, Alexandre J.C.; Bressan, G.C.; Gava, L.M.; Lanza, D.C.F.; Ramos, C.H.I; Kobarg, Joerg

2009-01-01

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 - GWbs marker protein and TIA-1 stress granule component

Epidemiological studies on viral infections and co-infections : Human immunodeficiency virus, hepatitis C virus and human papillomavirus

NARCIS (Netherlands)

van Santen, D.K.

2018-01-01

The research described in this thesis aimed to increase our understanding of the incidence, disease progression and treatment of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and human papillomavirus (HPV) infections and co-infections in key populations. Chapter 1 contains an overview

Pyrrolizidine alkaloid-containing toxic plants (Senecio, Crotalaria, Cynoglossum, Amsinckia, Heliotropium, and Echium spp.).

Science.gov (United States)

Stegelmeier, Bryan L

2011-07-01

Pyrrolizidine alkaloid (PA)-containing plants are found throughout the world and are probably the most common plant cause of poisoning of livestock, wildlife, and humans. PAs are potent liver toxins that under some conditions can be carcinogenic. This article briefly introduces high-risk North American PA-containing plants, summarizing their toxicity and subsequent pathology. Current diagnostic techniques, treatments, and strategies to avoid losses to PA poisoning are also reviewed. Published by Elsevier Inc.

Human chromosome-specific changes in a human-hamster hybrid cell line (AL) assessed by fluorescent in situ hybridization (fish)

International Nuclear Information System (INIS)

Geard, Charles R.; Jenkins, Gloria

1995-01-01

Purpose: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. Methods and Materials: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137 Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. Results: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. Conclusions: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1

21 CFR 864.9900 - Cord blood processing system and storage container.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cord blood processing system and storage container. 864.9900 Section 864.9900 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That...

A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans.

Science.gov (United States)

Charron, Craig S; Dawson, Harry D; Albaugh, George P; Solverson, Patrick M; Vinyard, Bryan T; Solano-Aguilar, Gloria I; Molokin, Aleksey; Novotny, Janet A

2015-11-01

Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. We designed a study to probe the mechanisms of garlic action in humans. We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 μL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h. The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P garlic, respectively). OSM is a pleiotropic cytokine that inhibits several tumor cell lines in culture. These data indicate that the bioactivity of garlic is multifaceted and includes activation of genes related to immunity, apoptosis, and xenobiotic metabolism in humans and Mono Mac 6 cells. This trial is registered at clinicaltrials.gov as NCT01293591. © 2015 American Society for Nutrition.

An Alternative to the Human Hemoglobin Test in the Investigation of Bloodstains Treated with Active Oxygen: The Human Glycophorin A Test

Directory of Open Access Journals (Sweden)

Ana Castelló

2011-01-01

Full Text Available In criminal investigations, there are three stages involved when studying bloodstains: search and orientation, confirmation, and individualization. Confirmatory tests have two aims: to show that the stain contains a human biological fluid and to confirm the type of biological fluid. The need to determine the nature of the evidence is reflected in the latest bibliography, where the possibility of employing mRNA and miRNA markers for this purpose is proposed. While these new proposals are being investigated, the kits for determining human hemoglobin currently provide a simple solution for resolving this issue. With these kits, the possibility of obtaining false positives and false negatives is well known. However, recently, a new problem has been detected. This involves the interference caused by new cleaning products that contain sodium percarbonate (or active oxygen when determining human hemoglobin. With the aim to resolve this problem, this work studied the ability of the human glycophorin A test to determine human blood in samples that have been treated with active oxygen. Our results show that the human glycophorin A test has a greater resistance to the destructive effect of the new detergents containing active oxygen; consequently, it provides an alternative to be taken into consideration in the confirmatory diagnoses of bloodstains.

British Container Breeding Mosquitoes: The Impact of Urbanisation and Climate Change on Community Composition and Phenology

Science.gov (United States)

Townroe, Susannah; Callaghan, Amanda

2014-01-01

The proliferation of artificial container habitats in urban areas has benefitted urban adaptable mosquito species globally. In areas where mosquitoes transmit viruses and parasites, it can promote vector population productivity and fuel mosquito-borne disease outbreaks. In Britain, storage of water in garden water butts is increasing, potentially expanding mosquito larval habitats and influencing population dynamics and mosquito-human contact. Here we show that the community composition, abundance and phenology of mosquitoes breeding in experimental water butt containers were influenced by urbanisation. Mosquitoes in urban containers were less species-rich but present in significantly higher densities (100.4±21.3) per container than those in rural containers (77.7±15.1). Urban containers were dominated by Culex pipiens (a potential vector of West Nile Virus [WNV]) and appear to be increasingly exploited by Anopheles plumbeus (a human-biting potential WNV and malaria vector). Culex phenology was influenced by urban land use type, with peaks in larval abundances occurring earlier in urban than rural containers. Among other factors, this was associated with an urban heat island effect which raised urban air and water temperatures by 0.9°C and 1.2°C respectively. Further increases in domestic water storage, particularly in urban areas, in combination with climate changes will likely alter mosquito population dynamics in the UK. PMID:24759617

Fibre-concrete container

International Nuclear Information System (INIS)

2000-01-01

In this leaflet the fibre-concrete container for radioactive wastes is described. The fibre container is made of fibre-concrete that contains cement, aggregate, sand, filter, flame-silica, super-plastificator, water and scattered metal fibres. The fibre-concrete container has a dice shape with outer dimension 1.7 x 1.7 x 1.7 m. It is mounted of a container body, a container cover and two caps. Total weight of container is 4,240 kg, maximum weight of loaded container do not must exceed 15,000 kg. The physical and mechanical properties of the fibre-concrete container are described in detail. The fibre-concrete container manufactured for storing of low and intermediate radioactive wastes. A fibre-concrete container utilization to store of radioactive wastes solves these problems: increase of stability of stored packages of radioactive waste; watertightness within 300 years at least; static stability of bearing space; better utilization of bearing spaces; insulation of radioactive waste in a case of seismic and geological event; increase of fire resistance; and transport of radioactive waste

Containment Loads Analysis for CANDU6 Reactor using CONTAIN 2.0

International Nuclear Information System (INIS)

Kim, Tae H.; Yang, Chae Y.

2013-01-01

The containment plays an important role to limit the release of radioactive materials to the environment during design basis accidents (DBAs). Therefore, the containment has to maintain its integrity under DBA conditions. Generally, a containment functional DBA evaluation includes calculations of the key containment loads, i. e., pressure and temperature effects associated with a postulated large rupture of the primary or secondary coolant system piping. In this paper, the behavior of containment pressure and temperature was evaluated for loss of coolant accidents (LOCAs) of the Wolsong unit 1 in order to assess the applicability of CONTAIN 2.0 code for the containment loads analysis of the CANDU6 reactor. The containment pressure and temperature of the Wolsong unit 1 were evaluated using the CONTAIN 2.0 code and the results were compared with the CONTEMPT4 code. The peak pressure and temperature calculated by CONTAIN 2.0 agreed well with those of CONTEMPT4 calculation. The overall result of this analysis shows that the CONTAIN 2.0 code can apply to the containment loads analysis for the CANDU6 reactor

Fecal microbiota changes with the consumption of follow-up formulas containing Bifidobacterium spp. and/or galactooligosaccharides by rats and a follow-up infant formula containing Bifidobacterium spp. by human infants

NARCIS (Netherlands)

Perez-Conesa, D.; Lopez, G.; Ros, G.H.; Abellan, P.; Hartemink, R.

2006-01-01

Seven groups of rats were fed during 1 mo using 1 infant formula containing Bifidobacterium bifidum and Bifidobacterium longum, 3 infant formulas containing 4-galactosyllactose at 1.2%, 5.0%, and 10.0%, and 3 infant formulas containing both ingredients. During 3 periods, corresponding to day 8 to

Effect of prolonged exposure to sublethal concentrations of DDT and DDE on protein expression in human pancreatic beta cells.

Science.gov (United States)

Pavlikova, Nela; Smetana, Pavel; Halada, Petr; Kovar, Jan

2015-10-01

Pollution of the environment represents one of less explored potential reasons for the worldwide epidemic of type 2 diabetes. One of the most prevalent organochlorine pollutants remains the pesticide DDT and its degradation product DDE. Despite some epidemiologic correlations between levels of DDT and DDE in human organism and the prevalence of diabetes, there is almost no information about the exact targets of these compounds inside pancreatic beta cells. To detect functional areas of pancreatic beta cells that could be affected by exposure to DDT and DDE, we analyzed changes in protein expression in the NES2Y human pancreatic beta cell line exposed to three sublethal concentrations (0.1 μM, 1 μM, 10 μM) of DDT and DDE for 1 month. Protein separation and identification was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectrometry. With these techniques, four proteins were found downregulated after exposure to 10 μM DDT: three cytoskeletal proteins (cytokeratin 8, cytokeratin 18 and actin) and one protein involved in glycolysis (alpha-enolase). Two proteins were downregulated after exposure to 10 μM DDE: cytokeratin 18 and heterogenous nuclear ribonucleoprotein H1 (HNRH1). These changes correlate with previously described effects of other stress conditions (e.g. exposure to palmitate, hyperglycemia, imidazoline derivative, and cytokines) on protein expression in pancreatic beta cells. We conclude that cytoskeletal proteins and their processing, glucose metabolism, and mRNA processing may represent targets affected by exposure to conditions hostile to pancreatic beta cells, including exposure to DDT and DDE. Copyright © 2015 Elsevier Inc. All rights reserved.

GenMapDB: a database of mapped human BAC clones

OpenAIRE

Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

2001-01-01

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

International Nuclear Information System (INIS)

Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

2015-01-01

The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients.

Science.gov (United States)

Englund, Emelie; Reitsma, Bart; King, Ben C; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G; Jirström, Karin; Blom, Anna M

2015-10-19

The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

Subcellular SIMS imaging of gadolinium isotopes in human glioblastoma cells treated with a gadolinium containing MRI agent

Science.gov (United States)

Smith, Duane R.; Lorey, Daniel R.; Chandra, Subhash

2004-06-01

Neutron capture therapy is an experimental binary radiotherapeutic modality for the treatment of brain tumors such as glioblastoma multiforme. Recently, neutron capture therapy with gadolinium-157 has gained attention, and techniques for studying the subcellular distribution of gadolinium-157 are needed. In this preliminary study, we have been able to image the subcellular distribution of gadolinium-157, as well as the other six naturally abundant isotopes of gadolinium, with SIMS ion microscopy. T98G human glioblastoma cells were treated for 24 h with 25 mg/ml of the metal ion complex diethylenetriaminepentaacetic acid Gd(III) dihydrogen salt hydrate (Gd-DTPA). Gd-DTPA is a contrast enhancing agent used for MRI of brain tumors, blood-brain barrier impairment, diseases of the central nervous system, etc. A highly heterogeneous subcellular distribution was observed for gadolinium-157. The nuclei in each cell were distinctly lower in gadolinium-157 than in the cytoplasm. Even within the cytoplasm the gadolinium-157 was heterogeneously distributed. The other six naturally abundant isotopes of gadolinium were imaged from the same cells and exhibited a subcellular distribution consistent with that observed for gadolinium-157. These observations indicate that SIMS ion microscopy may be a viable approach for subcellular studies of gadolinium containing neutron capture therapy drugs and may even play a major role in the development and validation of new gadolinium contrast enhancing agents for diagnostic MRI applications.

Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

International Nuclear Information System (INIS)

Higuchi, K.; Hospattankar, A.V.; Law, S.W.; Meglin, N.; Cortright, J.; Brewer, H.B. Jr.

1988-01-01

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

Directory of Open Access Journals (Sweden)

Susan K. Eszterhas

2011-09-01

Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.