Sequential chromatin immunoprecipitation to detect SUMOylated MeCP2 in neurons

Directory of Open Access Journals (Sweden)

Tao Wu

2016-03-01

Full Text Available The small ubiquitin-like modifier (SUMO is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM. Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2 is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.

Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high-performance liquid chromatography.

Science.gov (United States)

Kim, Soung Min; Jeong, Dasul; Kim, Min Keun; Lee, Sang Shin; Lee, Suk Keun

2017-08-08

Oral squamous cell carcinoma (OSCC) is one of the most dangerous cancers in the body, producing serious complications with individual behaviors. Many different pathogenetic factors are involved in the carcinogenesis of OSCC. Cancer cells derived from oral keratinocytes can produce different carcinogenic signaling pathways through differences in protein expression, but their protein expression profiles cannot be easily explored with ordinary detection methods. The present study compared the protein expression profiles between two different types of OSCCs, which were analyzed through immunoprecipitation high-performance liquid chromatography (IP-HPLC). Two types of squamous cell carcinoma (SCC) occurred in a mandibular (SCC-1) and maxillary gingiva (SCC-2), but their clinical features and progression were quite different from each other. SCC-1 showed a large gingival ulceration with severe halitosis and extensive bony destruction, while SCC-2 showed a relatively small papillary gingival swelling but rapidly grew to form a large submucosal mass, followed by early cervical lymph node metastasis. In the histological observation, SCC-1 was relatively well differentiated with a severe inflammatory reaction, while SCC-2 showed severely infiltrative growth of each cancer islets accompanied with a mild inflammatory reaction. IP-HPLC analysis revealed contrary protein expression profiles analyzed by 72 different oncogenic proteins. SCC-1 showed more cellular apoptosis and invasive growth than SCC-2 through increased expression of caspases, MMPs, p53 signaling, FAS signaling, TGF-β1 signaling, and angiogenesis factors, while SCC-2 showed more cellular growth and survival than SCC-1 through the increased expression of proliferating factors, RAS signaling, eIF5A signaling, WNT signaling, and survivin. The increased trends of cellular apoptosis and invasiveness in the protein expression profiles of SCC-1 were implicative of its extensive gingival ulceration and bony destruction

Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation.

Science.gov (United States)

Fujisawa, Masaki; Nakano, Toshitsugu; Ito, Yasuhiro

2011-01-30

During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Using immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own

Identification of potential target genes for the tomato fruit-ripening regulator RIN by chromatin immunoprecipitation

Directory of Open Access Journals (Sweden)

Nakano Toshitsugu

2011-01-01

Full Text Available Abstract Background During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results Using immunoprecipitated (IPed DNA fragments recovered by chromatin immunoprecipitation (ChIP with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

Science.gov (United States)

Huang, Bill X.; Kim, Hee-Yong

2013-01-01

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence [version 2; referees: 2 approved

Directory of Open Access Journals (Sweden)

Heidi A. Neubauer

2017-03-01

Full Text Available Sphingosine kinase 2 (SK2 is a ubiquitously expressed lipid kinase that has important, albeit complex and poorly understood, roles in regulating cell survival and cell death. In addition to being able to promote cell cycle arrest and apoptosis under certain conditions, it has recently been shown that SK2 can promote neoplastic transformation and tumorigenesis in vivo. Therefore, well validated and reliable tools are required to study and better understand the true functions of SK2. Here, we compare two commercially available SK2 antibodies: a rabbit polyclonal antibody from Proteintech that recognizes amino acids 266-618 of human SK2a, and a rabbit polyclonal antibody from ECM Biosciences that recognizes amino acids 36-52 of human SK2a. We examine the performance of these antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence staining of endogenous SK2, using human HEK293 and HeLa cell lines, as well as mouse embryonic fibroblasts (MEFs. Furthermore, we assess the specificity of these antibodies to the target protein through the use of siRNA-mediated SK2 knockdown and SK2 knockout (Sphk2-/- MEFs. Our results demonstrate that the Proteintech anti-SK2 antibody reproducibly displayed superior sensitivity and selectivity towards SK2 in immunoblot analyses, while the ECM Biosciences anti-SK2 antibody was reproducibly superior for SK2 immunoprecipitation and detection by immunofluorescence staining. Notably, both antibodies produced non-specific bands and staining in the MEFs, which was not observed with the human cell lines. Therefore, we conclude that the Proteintech SK2 antibody is a valuable reagent for use in immunoblot analyses, and the ECM Biosciences SK2 antibody is a useful tool for SK2 immunoprecipitation and immunofluorescence staining, at least in the human cell lines employed in this study.

Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications.

Science.gov (United States)

McCullough, Shaun D; On, Doan M; Bowers, Emma C

2017-05-02

Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin-associated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs. Furthermore, the differences in ChIP protocols can confound efforts to increase reproducibility across studies. The streamlined ChIP procedure presented here can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells) and clinical samples (peripheral leukocytes) in toxicology. We also provide detailed guidance on the optimization of critical protocol parameters, such as chromatin fixation, fragmentation, and immunoprecipitation, to increase efficiency and improve reproducibility. Expanding toxicoepigenetic studies to more readily include histone modifications will facilitate a more comprehensive understanding of the role of the epigenome in environmental exposure effects and the integration of epigenetic data in mechanistic toxicology, adverse outcome pathways, and risk assessment. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Nanobody®-based chromatin immunoprecipitation/micro-array analysis for genome-wide identification of transcription factor DNA binding sites

Science.gov (United States)

Nguyen-Duc, Trong; Peeters, Eveline; Muyldermans, Serge; Charlier, Daniel; Hassanzadeh-Ghassabeh, Gholamreza

2013-01-01

Nanobodies® are single-domain antibody fragments derived from camelid heavy-chain antibodies. Because of their small size, straightforward production in Escherichia coli, easy tailoring, high affinity, specificity, stability and solubility, nanobodies® have been exploited in various biotechnological applications. A major challenge in the post-genomics and post-proteomics era is the identification of regulatory networks involving nucleic acid–protein and protein–protein interactions. Here, we apply a nanobody® in chromatin immunoprecipitation followed by DNA microarray hybridization (ChIP-chip) for genome-wide identification of DNA–protein interactions. The Lrp-like regulator Ss-LrpB, arguably one of the best-studied specific transcription factors of the hyperthermophilic archaeon Sulfolobus solfataricus, was chosen for this proof-of-principle nanobody®-assisted ChIP. Three distinct Ss-LrpB-specific nanobodies®, each interacting with a different epitope, were generated for ChIP. Genome-wide ChIP-chip with one of these nanobodies® identified the well-established Ss-LrpB binding sites and revealed several unknown target sequences. Furthermore, these ChIP-chip profiles revealed auxiliary operator sites in the open reading frame of Ss-lrpB. Our work introduces nanobodies® as a novel class of affinity reagents for ChIP. Taking into account the unique characteristics of nanobodies®, in particular, their short generation time, nanobody®-based ChIP is expected to further streamline ChIP-chip and ChIP-Seq experiments, especially in organisms with no (or limited) possibility of genetic manipulation. PMID:23275538

Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP)

NARCIS (Netherlands)

Kaufmann, K.; Muiño, J.M.; Østerås, M.; Farinelli, L.; Krajewski, P.; Angenent, G.C.

2010-01-01

Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences

Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry

DEFF Research Database (Denmark)

Kristensen, B.K.; Askerlund, P.; Bykova, N.V.

2004-01-01

.2 mM CuSO4 for 10 min at room temperature). The oxidised proteins in both samples were tagged with dinitrophenylhydrazine (DNP), which forms a covalent bond with carbonyl groups. The DNP-tagged proteins were immunoprecipitated using anti-DNP antibodies and digested with trypsin. The mixture...... of peptides was analysed by nano-HPLC coupled online to an ESI-Quad-TOF mass spectrometer. The peptides were separated by stepwise ion exchange chromatography followed by reverse phase chromatography (2D-LC), and analysed by MS/MS. Proteins were identified by un-interpreted fragment ion database searches...... blots showed that neither the isolation of mitochondria, nor their subfractionation introduced carbonyl groups. We therefore conclude that a number of proteins are oxidised in the matrix of rice leaf mitochondria in vivo and further identify a group of proteins that are particularly susceptible to mild...

Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA regions of the host genome.

Science.gov (United States)

Benferhat, Rima; Josse, Thibaut; Albaud, Benoit; Gentien, David; Mansuroglu, Zeyni; Marcato, Vasco; Souès, Sylvie; Le Bonniec, Bernard; Bouloy, Michèle; Bonnefoy, Eliette

2012-10-01

Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways, such as cell adhesion, axonal guidance, development, and coagulation were closely related to RVFV-induced disorders. In particular, we show in this work that NSs targeted and modified the expression of genes coding for coagulation factors, demonstrating for the first time that this hemorrhagic virus impairs the host coagulation cascade at the transcriptional level.

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

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Lee, Hong-Won; Kyung, Taeyoon; Yoo, Janghyun; Kim, Tackhoon; Chung, Chaeuk; Ryu, Ji Young; Lee, Hanki; Park, Kihyun; Lee, Sangkyu; Jones, Walton D.; Lim, Dae-Sik; Hyeon, Changbong; Do Heo, Won; Yoon, Tae-Young

2013-01-01

Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level. PMID:23422673

Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

Science.gov (United States)

Burlibașa, Liliana; Suciu, Ilinca

2015-12-01

Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter.

Science.gov (United States)

Rogge, George A; Shen, Li-Ling; Kuhar, Michael J

2010-07-16

Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

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Vidal Mônica Scarpelli Martinelli

2003-01-01

Full Text Available Chromoblastomycosis (CBM is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID, counterimmunoelectrophoresis (CIE and immunoenzymatic test (ELISA have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

A novel mitochondrial protein of Neurospora crassa immunoprecipitates with known enzyme subunits but is not antigenic

International Nuclear Information System (INIS)

Nixon, E.

1989-01-01

14 C labeled 4'-phosphopantetheine (PAN) is detectable as 2 bands after SDS-PAGE of mitochondrial proteins. The bands comigrate with subunit 6 of cytochrome oxidase (COX) and a small ATPase subunit in tube gel slices of immunoprecipitates. However, other work demonstrated these bands to be due to modification of a novel protein, related to acyl carrier protein (ACP) of spinach and E. coli, that exists in two forms. To resolve this discrepancy, 1-dimensional (1D) slab and 2-dimensional (2D) SDS-PAGE was used for increased resolution over tube gels. Total mitochondrial protein gels from PAN labeled cells were western blotted, probed for COX, and autoradiographed. In 1D there is exact migration of PAN with COX6. In 2D PAN overlaps a protein distinct from and not antigenically related to COX subunits. These data suggest it is the ACP-like protein that in PAN-modified. Its possible association with COX during assembly will be discussed

[Immunoprecipitation mapping of the TRX-associated chromosome elements in the fork head gene promoter in the Drosophila melanogaster salivary gland cells].

Science.gov (United States)

Riakhovskiĭ, A A; Tillib, S V

2007-09-01

Using the method of immunoprecipitation of the in vivo crosslinked and sheared by sonication chromatin, mapping of potential trithorax-associated regulatory elements within the extended (9 kb) promoter region of the fork head gene (fkh) in the Drosophila melanogaster salivary gland cells was performed. Relative homogeneity of the salivary gland cells, along with the parallel use of the antibodies to different domains of the same trithorax protein (TRX), and the introduction of cross-hybridization steps for additional specific enrichment of initial DNA libraries, provided improvement of the method effectiveness and identification of one major and two less expressed potential TRX-binding sites.

Dramatic repositioning of c-Myb to different promoters during the cell cycle observed by combining cell sorting with chromatin immunoprecipitation.

Directory of Open Access Journals (Sweden)

Anita M Quintana

2011-02-01

Full Text Available The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about changes in c-Myb activity during the cell cycle. To map the association of c-Myb with specific target genes during the cell cycle, we developed a novel Fix-Sort-ChIP approach, in which asynchronously growing cells were fixed with formaldehyde, stained with Hoechst 33342 and separated into different cell cycle fractions by flow sorting, then processed for chromatin immunoprecipitation (ChIP assays. We found that c-Myb actively repositions, binding to some genes only in specific cell cycle phases. In addition, the specificity of c-Myb is dramatically different in small subpopulations of cells, for example cells in the G2/M phase of the cell cycle, than in the bulk population. The repositioning of c-Myb during the cell cycle is not due to changes in its expression and also occurs with ectopically expressed, epitope-tagged versions of c-Myb. The repositioning occurs in established cell lines, in primary human CD34+ hematopoietic progenitors and in primary human acute myeloid leukemia cells. The combination of fixation, sorting and ChIP analysis sheds new light on the dynamic nature of gene regulation during the cell cycle and provides a new type of tool for the analysis of gene regulation in small subsets of cells, such as cells in a specific phase of the cell cycle.

From human monocytes to genome-wide binding sites--a protocol for small amounts of blood: monocyte isolation/ChIP-protocol/library amplification/genome wide computational data analysis.

Directory of Open Access Journals (Sweden)

Sebastian Weiterer

Full Text Available Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner.The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA.

Importance Performance Analysis as a Trade Show Performance Evaluation and Benchmarking Tool

OpenAIRE

Tafesse, Wondwesen; Skallerud, Kåre; Korneliussen, Tor

2010-01-01

Author's accepted version (post-print). The purpose of this study is to introduce importance performance analysis as a trade show performance evaluation and benchmarking tool. Importance performance analysis considers exhibitors’ performance expectation and perceived performance in unison to evaluate and benchmark trade show performance. The present study uses data obtained from exhibitors of an international trade show to demonstrate how importance performance analysis can be used to eval...

Encapsulation of Adenovirus Serotype 5 in Anionic Lecithin Liposomes using a Bead-Based Immunoprecipitation Technique Enhances Transfection Efficiency

Science.gov (United States)

Mendez, N.; Herrera, V.; Zhang, L.; Hedjran, F.; Feuer, R.; Blair, S.; Trogler, W.; Reid, T.

2014-01-01

Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140–180nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4× higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations. PMID:25154663

Encapsulation of adenovirus serotype 5 in anionic lecithin liposomes using a bead-based immunoprecipitation technique enhances transfection efficiency.

Science.gov (United States)

Mendez, Natalie; Herrera, Vanessa; Zhang, Lingzhi; Hedjran, Farah; Feuer, Ralph; Blair, Sarah L; Trogler, William C; Reid, Tony R; Kummel, Andrew C

2014-11-01

Oncolytic viruses (OVs) constitute a promising class of cancer therapeutics which exploit validated genetic pathways known to be deregulated in many cancers. To overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of adenovirus in non-toxic anionic lecithin-cholesterol-PEG liposomes ranging from 140 to 180 nm in diameter have been prepared by self-assembly around the viral capsid. The encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. 78% of adenovirus plaque forming units were encapsulated and retained infectivity after IP processing. Additionally, encapsulated viruses have shown enhanced transfection efficiency up to 4 × higher compared to non-encapsulated Ads. Extracting non-encapsulated viruses from solution may prevent an adverse in vivo immune response and may enhance treatment for multiple administrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Overexpressed HDAC8 in cervical cancer cells shows functional redundancy of tubulin deacetylation with HDAC6.

Science.gov (United States)

Vanaja, G R; Ramulu, Hemalatha Golaconda; Kalle, Arunasree M

2018-05-02

Histone deacetylases (HDACs) are involved in epigenetic gene regulation via deacetylation of acetylated lysine residues of both histone and non-histone proteins. Among the 18 HDACs identified in humans, HDAC8, a class I HDAC, is best understood structurally and enzymatically. However, its precise subcellular location, function in normal cellular physiology, its protein partners and substrates still remain elusive. The subcellular localization of HDAC8 was studied using immunofluorescence and confocal imaging. The binding parterns were identified employing immunoprecipitation (IP) followed by MALDI-TOF analysis and confirmed using in-silico protein-protein interaction studies, HPLC-based in vitro deacetylation assay, intrinsic fluorescence spectrophotometric analysis, Circular dichroism (CD) and Surface Plasmon Resonance (SPR). Functional characterization of the binding was carried out using immunoblot and knockdown by siRNA. Using one way ANOVA statistical significance (n = 3) was determined. Here, we show that HDAC8 and its phosphorylated form (pHDAC8) localized predominantly in the cytoplasm in cancerous, HeLa, and non-cancerous (normal), HEK293T, cells, although nucleolar localization was observed in HeLa cells. The study identified Alpha tubulin as a novel interacting partner of HDAC8. Further, the results indicated binding and deacetylation of tubulin at ac-lys40 by HDAC8. Knockdown of HDAC8 by siRNA, inhibition of HDAC8 and/or HDAC6 by PCI-34051 and tubastatin respectively, cell-migration, cell morphology and cell cycle analysis clearly explained HDAC8 as tubulin deacetylase in HeLa cells and HDAC6 in HEK 293 T cells. HDAC8 shows functional redundancy with HDAC6 when overexpressed in cervical cancer cells, HeLa, and deacetylaes ac-lys40 of alpha tubulin leading to cervical cancer proliferation and progression.

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation.

Science.gov (United States)

Nohara, Kazunari; Chen, Zheng; Yoo, Seung-Hee

2017-07-06

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

KAUST Repository

Abu Samra, Dina Bashir Kamil; Al Kilani, Alia; Hamdan, Samir; Sakashita, Kosuke; Gadhoum, Samah Z.; Merzaban, Jasmeen

2015-01-01

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

KAUST Repository

Abu Samra, Dina Bashir Kamil

2015-06-29

Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

Science.gov (United States)

Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

2012-06-01

There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

Pax6 interacts with Iba1 and shows age-associated alterations in brain of aging mice.

Science.gov (United States)

Maurya, Shashank Kumar; Mishra, Rajnikant

2017-07-01

The Pax6, a transcriptional regulator and multifunctional protein, has been found critical for neurogenesis, neuro-degeneration, mental retardation, neuroendocrine tumors, glioblastoma and astrocytomas. The age-associated alteration in the expression of Pax6 in neuron and glia has also been observed in the immunologically privileged brain. Therefore, it is presumed that Pax6 may modulate brain immunity by activation of microglia either directly interacting with genes or proteins of microglia or indirectly though inflammation associated with neurodegeneration. This report describes evaluation of expression, co-localization and interactions of Pax6 with Ionized binding protein1 (Iba1) in brain of aging mice by Immunohistochemistry, Chromatin Immuno-precipitation (ChIP) and Co-immunoprecipitation (Co-IP), respectively. The co-localization of Pax6 with Iba1 was observed in the cerebellum, cerebral cortex, hippocampus, midbrain and olfactory lobe. The Pax6 and Iba1 also interact physically. The age-dependent alteration in their expression and co-localization were also observed in mice. Results indicate Pax6-dependent activities of Iba1 in the remodelling of microglia during immunological surveillance of the brain. Copyright © 2017 Elsevier B.V. All rights reserved.

Fat stigmatization in television shows and movies: a content analysis.

Science.gov (United States)

Himes, Susan M; Thompson, J Kevin

2007-03-01

To examine the phenomenon of fat stigmatization messages presented in television shows and movies, a content analysis was used to quantify and categorize fat-specific commentary and humor. Fat stigmatization vignettes were identified using a targeted sampling procedure, and 135 scenes were excised from movies and television shows. The material was coded by trained raters. Reliability indices were uniformly high for the seven categories (percentage agreement ranged from 0.90 to 0.98; kappas ranged from 0.66 to 0.94). Results indicated that fat stigmatization commentary and fat humor were often verbal, directed toward another person, and often presented directly in the presence of the overweight target. Results also indicated that male characters were three times more likely to engage in fat stigmatization commentary or fat humor than female characters. To our knowledge, these findings provide the first information regarding the specific gender, age, and types of fat stigmatization that occur frequently in movies and television shows. The stimuli should prove useful in future research examining the role of individual difference factors (e.g., BMI) in the reaction to viewing such vignettes.

Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

Directory of Open Access Journals (Sweden)

Xin Li

2008-06-01

Full Text Available Abstract Background Target genes of a transcription factor (TF Pou5f1 (Oct3/4 or Oct4, which is essential for pluripotency maintenance and self-renewal of embryonic stem (ES cells, have previously been identified based on their response to Pou5f1 manipulation and occurrence of Chromatin-immunoprecipitation (ChIP-binding sites in promoters. However, many responding genes with binding sites may not be direct targets because response may be mediated by other genes and ChIP-binding site may not be functional in terms of transcription regulation. Results To reduce the number of false positives, we propose to separate responding genes into groups according to direction, magnitude, and time of response, and to apply the false discovery rate (FDR criterion to each group individually. Using this novel algorithm with stringent statistical criteria (FDR Pou5f1 suppression and published ChIP data, we identified 420 tentative target genes (TTGs for Pou5f1. The majority of TTGs (372 were down-regulated after Pou5f1 suppression, indicating that the Pou5f1 functions as an activator of gene expression when it binds to promoters. Interestingly, many activated genes are potent suppressors of transcription, which include polycomb genes, zinc finger TFs, chromatin remodeling factors, and suppressors of signaling. Similar analysis showed that Sox2 and Nanog also function mostly as transcription activators in cooperation with Pou5f1. Conclusion We have identified the most reliable sets of direct target genes for key pluripotency genes – Pou5f1, Sox2, and Nanog, and found that they predominantly function as activators of downstream gene expression. Thus, most genes related to cell differentiation are suppressed indirectly.

Detection of a rare BCR-ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS).

Science.gov (United States)

Breitkopf, Susanne B; Yuan, Min; Pihan, German A; Asara, John M

2012-10-02

Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.

Location analysis for the estrogen receptor-? reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

OpenAIRE

Mason, Christopher E.; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M.; Kallen, Roland G.; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B.

2010-01-01

Location analysis for estrogen receptor-? (ER?)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ER?-bound loci and quantify the incidence of ERE sequences under two stringencies of detection:

Nebula--a web-server for advanced ChIP-seq data analysis.

Science.gov (United States)

Boeva, Valentina; Lermine, Alban; Barette, Camille; Guillouf, Christel; Barillot, Emmanuel

2012-10-01

ChIP-seq consists of chromatin immunoprecipitation and deep sequencing of the extracted DNA fragments. It is the technique of choice for accurate characterization of the binding sites of transcription factors and other DNA-associated proteins. We present a web service, Nebula, which allows inexperienced users to perform a complete bioinformatics analysis of ChIP-seq data. Nebula was designed for both bioinformaticians and biologists. It is based on the Galaxy open source framework. Galaxy already includes a large number of functionalities for mapping reads and peak calling. We added the following to Galaxy: (i) peak calling with FindPeaks and a module for immunoprecipitation quality control, (ii) de novo motif discovery with ChIPMunk, (iii) calculation of the density and the cumulative distribution of peak locations relative to gene transcription start sites, (iv) annotation of peaks with genomic features and (v) annotation of genes with peak information. Nebula generates the graphs and the enrichment statistics at each step of the process. During Steps 3-5, Nebula optionally repeats the analysis on a control dataset and compares these results with those from the main dataset. Nebula can also incorporate gene expression (or gene modulation) data during these steps. In summary, Nebula is an innovative web service that provides an advanced ChIP-seq analysis pipeline providing ready-to-publish results. Nebula is available at http://nebula.curie.fr/ Supplementary data are available at Bioinformatics online.

Testing for myositis specific autoantibodies: Comparison between line blot and immunoprecipitation assays in 57 myositis sera.

Science.gov (United States)

Cavazzana, Ilaria; Fredi, Micaela; Ceribelli, Angela; Mordenti, Cristina; Ferrari, Fabio; Carabellese, Nice; Tincani, Angela; Satoh, Minoru; Franceschini, Franco

2016-06-01

To analyze the performance of a line blot assay for the identification of autoantibodies in sera of patients affected by myositis, compared with immunoprecipitation (IP) as gold standard. 66 sera of patients with myositis (23 polymyositis, 8 anti-synthetase syndromes, 29 dermatomyositis and 6 overlap syndromes) were tested by commercial LB (Euroimmun, Lubeck, Germany); 57 sera were analyzed also by IP of K562 cell extract radiolabeled with (35)S-methionine. Inter-rater agreement was calculated with Cohen's k coefficient. Myositis-specific antibodies (MSA) were detected in 36/57 sera (63%) by IP and in 39/66 sera (59%) by LB. The most frequent MSA found by LB were anti-Jo1 and anti-Mi2 found in 15% (10/66) of sera, followed by anti-NXP2 and anti-SRP detected in 106% (7/66) of sera. Anti-TIF1gamma and anti-MDA5 were found in 6 (9%) and 5 sera (7.6%), respectively. A good agreement between methods was found only for anti-TIF1γ, anti-MDA5 and anti-NXP-2 antibodies, while a moderate agreement was estimated for anti-Mi2 and anti-EJ. By contrast, a high discordance rate for the detection of anti-Jo1 antibodies was evident (k: 0.3). Multiple positivity for MSA were found in 11/66 (17%) by LB and 0/57 by IP (p: 0001). Comparing the clinical features of these 11 sera, we found total discrepancies between assays in 3 sera (27.3%), a relative discrepancy due to the occurrence of one discordant autoantibody (not confirmed by IP) in 5 cases (45.5%) and a total discrepancy between LB and IP results, but with a relative concordance with clinical features were found in other 3 sera (27.3%). The semiquantitative results do not support the interpretation of the data. The use of LB assay allowed the detection of new MSA, such as anti-MDA5, anti-MJ and anti-TIF1gamma antibodies, previously not found with routine methods. However, the high prevalence of multiple positivities and the high discondant rate of anti-Jo1 antibodies could create some misinterpretation of the results from the

Isolation and characterization of DUSP11, a novel p53 target gene

DEFF Research Database (Denmark)

Caprara, Greta; Zamponi, Raffaella; Melixetian, Marina

2009-01-01

target gene. Consistent with this, the expression of DUSP11 is induced in a p53-dependent manner after treatment with DNA damaging agents. Chromatin immunoprecipitation analysis showed that p53 binds to 2 putative p53 DNA binding sites in the promoter region of DUSP11. Colony formation and proliferation...

Proteome Analysis of the Plant Pathogenic Fungus Monilinia laxa Showing Host Specificity

Directory of Open Access Journals (Sweden)

Olja Bregar

2012-01-01

Full Text Available Brown rot fungus Monilinia laxa (Aderh. & Ruhl. Honey is an important plant pathogen in stone and pome fruits in Europe. We applied a proteomic approach in a study of M. laxa isolates obtained from apples and apricots in order to show the host specifity of the isolates and to analyse differentially expressed proteins in terms of host specifity, fungal pathogenicity and identification of candidate proteins for diagnostic marker development. Extracted mycelium proteins were separated by 2-D electrophoresis (2-DE and visualized by Coomassie staining in a non-linear pH range of 3–11 and Mr of 14–116 kDa. We set up a 2-DE reference map of M. laxa, resolving up to 800 protein spots, and used it for image analysis. The average technical coefficient of variance (13 % demonstrated a high reproducibility of protein extraction and 2-D polyacrylamide gel electrophoresis (2-DE PAGE, and the average biological coefficient of variance (23 % enabled differential proteomic analysis of the isolates. Multivariate statistical analysis (principal component analysis discriminated isolates from two different hosts, providing new data that support the existence of a M. laxa specialized form f. sp. mali, which infects only apples. A total of 50 differentially expressed proteins were further analyzed by LC-MS/MS, yielding 41 positive identifications. The identified mycelial proteins were functionally classified into 6 groups: amino acid and protein metabolism, energy production, carbohydrate metabolism, stress response, fatty acid metabolism and other proteins. Some proteins expressed only in apple isolates have been described as virulence factors in other fungi. The acetolactate synthase was almost 11-fold more abundant in apple-specific isolates than in apricot isolates and it might be implicated in M. laxa host specificity. Ten proteins identified only in apple isolates are potential candidates for the development of M. laxa host-specific diagnostic markers.

STAT5 induces miR-21 expression in cutaneous T cell lymphoma

DEFF Research Database (Denmark)

Lindahl, Lise M; Fredholm, Simon; Joseph, Claudine

2016-01-01

was inhibited by Tofacitinib (CP-690550), a clinical-grade JAK3 inhibitor. Chromatin immunoprecipitation (ChIP) analysis showed direct binding of STAT5 to the miR-21 promoter. Cytokine starvation ex vivo triggered a decrease in miR-21 expression, whereas IL-2 induced an increased miR-21 expression in primary SS...

fCCAC: functional canonical correlation analysis to evaluate covariance between nucleic acid sequencing datasets.

Science.gov (United States)

Madrigal, Pedro

2017-03-01

Computational evaluation of variability across DNA or RNA sequencing datasets is a crucial step in genomic science, as it allows both to evaluate reproducibility of biological or technical replicates, and to compare different datasets to identify their potential correlations. Here we present fCCAC, an application of functional canonical correlation analysis to assess covariance of nucleic acid sequencing datasets such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). We show how this method differs from other measures of correlation, and exemplify how it can reveal shared covariance between histone modifications and DNA binding proteins, such as the relationship between the H3K4me3 chromatin mark and its epigenetic writers and readers. An R/Bioconductor package is available at http://bioconductor.org/packages/fCCAC/ . pmb59@cam.ac.uk. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus.

OpenAIRE

Polatnick, J; Wool, S H

1985-01-01

Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h.

[FANCA gene mutation analysis in Fanconi anemia patients].

Science.gov (United States)

Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

2005-10-01

To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis.

Science.gov (United States)

Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

2016-07-14

Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis.

Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

International Nuclear Information System (INIS)

Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

2005-01-01

The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

Investigation of Fanconi anemia protein interactions by yeast two-hybrid analysis.

Science.gov (United States)

Huber, P A; Medhurst, A L; Youssoufian, H; Mathew, C G

2000-02-05

Fanconi anemia is a chromosomal breakage disorder with eight complementation groups (A-H), and three genes (FANCA, FANCC, and FANCG) have been identified. Initial investigations of the interaction between FANCA and FANCC, principally by co-immunoprecipitation, have proved controversial. We used the yeast two-hybrid assay to test for interactions of the FANCA, FANCC, and FANCG proteins. No activation of the reporter gene was observed in yeast co-expressing FANCA and FANCC as hybrid proteins, suggesting that FANCA does not directly interact with FANCC. However, a high level of activation was found when FANCA was co-expressed with FANCG, indicating strong, direct interaction between these proteins. Both FANCA and FANCG show weak but consistent interaction with themselves, suggesting that their function may involve dimerisation. The site of interaction of FANCG with FANCA was investigated by analysis of 12 mutant fragments of FANCG. Although both N- and C-terminal fragments did interact, binding to FANCA was drastically reduced, suggesting that more than one region of the FANCG protein is required for proper interaction with FANCA. Copyright 2000 Academic Press.

Analysis of HP1α regulation in human breast cancer cells

DEFF Research Database (Denmark)

Thomsen, Rune; Christensen, Dennis B; Rosborg, Sanne

2011-01-01

The three mammalian HP1 proteins, HP1α/CBX5, HP1β/CBX1, and HPγ/CBX3, are involved in chromatin packing and gene regulation. The HP1α protein is down-regulated in invasive compared to non-invasive breast cancer cells and HP1α is a suppressor of cell migration and invasion. In this report, we...... examined the background for HP1α protein down-regulation in invasive breast cancer cells. We identified a strict correlation between HP1α down-regulation at the protein level and the mRNA level. The HP1α mRNA down-regulation in invasive cancer cells was not caused by mRNA destabilization. Chromatin...... immunoprecipitation analysis of the HP1α gene showed a decrease in the histone mark for transcriptional activity H3-K36 tri-methylation and RNA polymerase II in invasive breast cancer cells which correlated with a decreased abundance of basal transcription factors at the HP1α promoter. E2F transcription factors...

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

Energy Technology Data Exchange (ETDEWEB)

Yoo, Byong Chul [Colorectal Cancer Branch, Research Institute, National Cancer Center, Goyang (Korea, Republic of); Yeo, Seung Gu [Dept. of Radiation Oncology, Soonchunhyang University College of Medicine, Soonchunhyang University Hospital, Cheonan (Korea, Republic of)

2017-09-15

The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

International Nuclear Information System (INIS)

Yoo, Byong Chul; Yeo, Seung Gu

2017-01-01

The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins

Luminescent Immunoprecipitation System (LIPS) for Detection of Autoantibodies Against ATP4A and ATP4B Subunits of Gastric Proton Pump H+,K+-ATPase in Atrophic Body Gastritis Patients

Science.gov (United States)

Lahner, Edith; Brigatti, Cristina; Marzinotto, Ilaria; Carabotti, Marilia; Scalese, Giulia; Davidson, Howard W; Wenzlau, Janet M; Bosi, Emanuele; Piemonti, Lorenzo; Annibale, Bruno; Lampasona, Vito

2017-01-01

Objectives: Circulating autoantibodies targeting the H+/K+-ATPase proton pump of gastric parietal cells are considered markers of autoimmune gastritis, whose diagnostic accuracy in atrophic body gastritis, the pathological lesion of autoimmune gastritis, remains unknown. This study aimed to assess autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in atrophic body gastritis patients and controls. Methods: One-hundred and four cases with atrophic body gastritis and 205 controls were assessed for serological autoantibodies specific for ATP4A or ATP4B subunits using luminescent immunoprecipitation system (LIPS). Recombinant luciferase-reporter-fused-antigens were expressed by in vitro transcription-translation (ATP4A) or after transfection in Expi293F cells (ATP4B), incubated with test sera, and immune complexes recovered using protein-A-sepharose. LIPS assays were compared with a commercial enzyme immunoassay (EIA) for parietal cell autoantibodies. Results: ATP4A and ATP4B autoantibody titers were higher in cases compared to controls (Pgastritis. Both assays had the highest sensitivity, at the cost of diagnostic accuracy (89 and 90% specificity), outperforming traditional EIA. Once validated, these LIPS assays should be valuable screening tools for detecting biomarkers of damaged atrophic oxyntic mucosa. PMID:28102858

Proteomic Analysis Shows Constitutive Secretion of MIF and p53-associated Activity of COX-2−/− Lung Fibroblasts

Directory of Open Access Journals (Sweden)

Mandar Dave

2017-12-01

Full Text Available The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF in lung fibroblasts derived from COX-2−/− but not wild-type (WT or COX-1−/− mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2−/− fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2−/− was “prostaglandin-independent.” GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2−/− cells. Furthermore, COX-2−/− fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

DEFF Research Database (Denmark)

Gobom, J; Kraeuter, K O; Persson, R

2000-01-01

A method was developed for mass spectrometric detection of neurotensin (NT)-like immunoreactivity and quantification of NT in human brain tissue. The method is based on immunoprecipitation followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF......-MS). The identity of the major component of the immunoprecipitates as neurotensin was confirmed by fragment ion analysis on an electrospray ionization quadrupole time-of-flight instrument. MALDI-TOF-MS quantification of NT was achieved using stable-isotope-labeled NT as the internal standard, yielding an error...

Discovering transcription factor binding sites in highly repetitive regions of genomes with multi-read analysis of ChIP-Seq data.

Science.gov (United States)

Chung, Dongjun; Kuan, Pei Fen; Li, Bo; Sanalkumar, Rajendran; Liang, Kun; Bresnick, Emery H; Dewey, Colin; Keleş, Sündüz

2011-07-01

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip) as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads). This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads). Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.

Discovering transcription factor binding sites in highly repetitive regions of genomes with multi-read analysis of ChIP-Seq data.

Directory of Open Access Journals (Sweden)

Dongjun Chung

2011-07-01

Full Text Available Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq is rapidly replacing chromatin immunoprecipitation combined with genome-wide tiling array analysis (ChIP-chip as the preferred approach for mapping transcription-factor binding sites and chromatin modifications. The state of the art for analyzing ChIP-seq data relies on using only reads that map uniquely to a relevant reference genome (uni-reads. This can lead to the omission of up to 30% of alignable reads. We describe a general approach for utilizing reads that map to multiple locations on the reference genome (multi-reads. Our approach is based on allocating multi-reads as fractional counts using a weighted alignment scheme. Using human STAT1 and mouse GATA1 ChIP-seq datasets, we illustrate that incorporation of multi-reads significantly increases sequencing depths, leads to detection of novel peaks that are not otherwise identifiable with uni-reads, and improves detection of peaks in mappable regions. We investigate various genome-wide characteristics of peaks detected only by utilization of multi-reads via computational experiments. Overall, peaks from multi-read analysis have similar characteristics to peaks that are identified by uni-reads except that the majority of them reside in segmental duplications. We further validate a number of GATA1 multi-read only peaks by independent quantitative real-time ChIP analysis and identify novel target genes of GATA1. These computational and experimental results establish that multi-reads can be of critical importance for studying transcription factor binding in highly repetitive regions of genomes with ChIP-seq experiments.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

OpenAIRE

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-01-01

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly ...

Advertising Discourse Analysis of FES stores: Killing Love, Cowards Show

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Cristian Venegas Ahumada

2013-08-01

Full Text Available The objective is to analyze the structural and photographic discourse of the Autumn-Winter campaign 2008 of FES stores for young people. This was done by a semiotic theory and a critical-structural methodology of discourse. An analysis of 4 advertising photographs was done, and at once an analysis of the discourse “FES says no to violence against Women”, which explains the campaign’s target. The result is: The discourse was subjected to production condition (society of control and makes advertising a way to homogenize subjectivity of masses to consume. Recognition conditions demonstrate that this advertising discourse of symbolic violence means a type of violation of Men and Women Rights. An action like this requires commitment of Psychology in order to promote the social humanizing change, by means of university teaching and professional tasks.

#13ReasonsWhy Health Professionals and Educators are Tweeting: A Systematic Analysis of Uses and Perceptions of Show Content and Learning Outcomes.

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Walker, Kimberly K; Burns, Kelli

2018-04-27

This study is a content analysis of health professionals' and educators' tweets about a popular Netflix show that depicts teen suicide: 13 Reasons Why. A content analysis of 740 tweets was conducted to determine the main themes associated with professionals' and educators' tweets about the show, as well as the valence of the tweets. Additionally, a thematic analysis of linked content in tweets (n = 178) was conducted to explore additional content shared about the show and modeling outcomes. Results indicated the largest percentage of tweets was related to social learning, particularly about outcomes that could occur from viewing the show. The valence of the tweets about outcomes was more positive than negative. However, linked materials commonly circulated in tweets signified greater concern with unintended learning outcomes. Some of the linked content included media guidelines for reporting on suicide with recommendations that entertainment producers follow the guidelines. This study emphasizes the importance of including social learning objectives in future typologies of Twitter uses and demonstrates the importance of examining linked content in Twitter studies.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

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Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

2015-08-26

The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

Anti-signal recognition particle autoantibody ELISA validation and clinical associations.

Science.gov (United States)

Aggarwal, Rohit; Oddis, Chester V; Goudeau, Danielle; Fertig, Noreen; Metes, Ilinca; Stephens, Chad; Qi, Zengbiao; Koontz, Diane; Levesque, Marc C

2015-07-01

The aim of this study was to develop and validate a quantitative anti-signal recognition particle (SRP) autoantibody serum ELISA in patients with myositis and longitudinal association with myositis disease activity. We developed a serum ELISA using recombinant purified full-length human SRP coated on ELISA plates and a secondary antibody that bound human IgG to detect anti-SRP binding. Protein immunoprecipitation was used as the gold standard for the presence of anti-SRP. Serum samples from three groups were analysed: SRP(+) myositis subjects by immunoprecipitation, SRP(-) myositis subjects by immunoprecipitation and non-myositis controls. The ELISA's sensitivity, specificity, positive predictive value and negative predictive value were evaluated. Percentage agreement and test-retest reliability were assessed. Serial samples from seven SRP immunoprecipitation-positive subjects were also tested, along with serum muscle enzymes and manual muscle testing. Using immunoprecipitation, we identified 26 SRP(+) myositis patients and 77 SRP(-) controls (including 38 patients with necrotizing myopathy). Non-myositis control patients included SLE (n = 4) and SSc (n = 7) patients. Anti-SRP positivity by ELISA showed strong agreement (97.1%) with immunoprecipitation (κ = 0.94). The sensitivity, specificity, positive predictive value, and negative predictive value of the anti-SRP ELISA were 88, 100, 100 and 96, respectively. The area under the curve was 0.94, and test-retest reliability was strong (r = 0.91, P < 0.001). Serial samples showed that anti-SRP levels paralleled changes in muscle enzymes and manual muscle testing. We developed a quantitative ELISA for detecting serum anti-SRP autoantibodies and validated the assay in myositis. Longitudinal assessment of SRP levels by ELISA may be a useful biomarker for disease activity. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions

Factor analysis shows association between family activity environment and children's health behaviour.

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Hendrie, Gilly A; Coveney, John; Cox, David N

2011-12-01

To characterise the family activity environment in a questionnaire format, assess the questionnaire's reliability and describe its predictive ability by examining the relationships between the family activity environment and children's health behaviours - physical activity, screen time and fruit and vegetable intake. This paper describes the creation of a tool, based on previously validated scales, adapted from the food domain. Data are from 106 children and their parents (Adelaide, South Australia). Factor analysis was used to characterise factors within the family activity environment. Pearson-Product Moment correlations between the family environment and child outcomes, controlling for demographic variation, were examined. Three factors described the family activity environment - parental activity involvement, opportunity for role modelling and parental support for physical activity - and explained 37.6% of the variance. Controlling for demographic factors, the scale was significantly correlated with children's health behaviour - physical activity (r=0.27), screen time (r=-0.24) and fruit and vegetable intake (r=0.34). The family activity environment questionnaire shows high internal consistency and moderate predictive ability. This study has built on previous research by taking a more comprehensive approach to measuring the family activity environment. This research suggests the family activity environment should be considered in family-based health promotion interventions. © 2011 The Authors. ANZJPH © 2011 Public Health Association of Australia.

Radiographic analysis of odontogenic cysts showing displacement of the mandibular canal

International Nuclear Information System (INIS)

Cho, Bong Hae

2003-01-01

To assess the radiographic findings of odontogenic cysts showing displacement of the mandibular canal using computed tomographic (CT) and panoramic images. CT and panoramic images of 63 odontogenic cysts (27 dentigerous, 16 odontogenic keratocysts, and 20 radicular cysts) were analyzed to evaluate the following parameters: the dimension and shape of the cysts, and the effect of the cysts on the mandibular canal and cortical plates. Of the 63 cysts examined in the study, 35 (55.6%) showed inferior displacement of the mandibular canal and 46 (73.0%) showed perforation of the canal. There were statistically significant differenced between CT and panoramic images in depicting displacement and perforation of the mandibular canal. Cortical expansion was seen in 46 cases (73.0%) and cortical perforation in 23 cases (36.5%). The radicular cysts showed cortical expansion and perforation less frequently than the other cyst groups. Large cysts of mandible should be evaluated by multiplanar CT images in order to detect the mandibular canal and cortical bone involvement.

From root to fruit: RNA-Seq analysis shows that arbuscular mycorrhizal symbiosis may affect tomato fruit metabolism.

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Zouari, Inès; Salvioli, Alessandra; Chialva, Matteo; Novero, Mara; Miozzi, Laura; Tenore, Gian Carlo; Bagnaresi, Paolo; Bonfante, Paola

2014-03-21

Tomato (Solanum lycopersicum) establishes a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. The formation of the mycorrhizal association in the roots leads to plant-wide modulation of gene expression. To understand the systemic effect of the fungal symbiosis on the tomato fruit, we used RNA-Seq to perform global transcriptome profiling on Moneymaker tomato fruits at the turning ripening stage. Fruits were collected at 55 days after flowering, from plants colonized with Funneliformis mosseae and from control plants, which were fertilized to avoid responses related to nutrient deficiency. Transcriptome analysis identified 712 genes that are differentially expressed in fruits from mycorrhizal and control plants. Gene Ontology (GO) enrichment analysis of these genes showed 81 overrepresented functional GO classes. Up-regulated GO classes include photosynthesis, stress response, transport, amino acid synthesis and carbohydrate metabolism functions, suggesting a general impact of fungal symbiosis on primary metabolisms and, particularly, on mineral nutrition. Down-regulated GO classes include cell wall, metabolism and ethylene response pathways. Quantitative RT-PCR validated the RNA-Seq results for 12 genes out of 14 when tested at three fruit ripening stages, mature green, breaker and turning. Quantification of fruit nutraceutical and mineral contents produced values consistent with the expression changes observed by RNA-Seq analysis. This RNA-Seq profiling produced a novel data set that explores the intersection of mycorrhization and fruit development. We found that the fruits of mycorrhizal plants show two transcriptomic "signatures": genes characteristic of a climacteric fleshy fruit, and genes characteristic of mycorrhizal status, like phosphate and sulphate transporters. Moreover, mycorrhizal plants under low nutrient conditions produce fruits with a nutrient content similar to those from non-mycorrhizal plants under high nutrient conditions

Analysis of ChIP-seq Data in R/Bioconductor.

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de Santiago, Ines; Carroll, Thomas

2018-01-01

The development of novel high-throughput sequencing methods for ChIP (chromatin immunoprecipitation) has provided a very powerful tool to study gene regulation in multiple conditions at unprecedented resolution and scale. Proactive quality-control and appropriate data analysis techniques are of critical importance to extract the most meaningful results from the data. Over the last years, an array of R/Bioconductor tools has been developed allowing researchers to process and analyze ChIP-seq data. This chapter provides an overview of the methods available to analyze ChIP-seq data based primarily on software packages from the open-source Bioconductor project. Protocols described in this chapter cover basic steps including data alignment, peak calling, quality control and data visualization, as well as more complex methods such as the identification of differentially bound regions and functional analyses to annotate regulatory regions. The steps in the data analysis process were demonstrated on publicly available data sets and will serve as a demonstration of the computational procedures routinely used for the analysis of ChIP-seq data in R/Bioconductor, from which readers can construct their own analysis pipelines.

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

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Vijee Mohan

Full Text Available Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS, carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1 involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

Recruitment Methods and Show Rates to a Prostate Cancer Early Detection Program for High-Risk Men: A Comprehensive Analysis

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Giri, Veda N.; Coups, Elliot J.; Ruth, Karen; Goplerud, Julia; Raysor, Susan; Kim, Taylor Y.; Bagden, Loretta; Mastalski, Kathleen; Zakrzewski, Debra; Leimkuhler, Suzanne; Watkins-Bruner, Deborah

2009-01-01

Purpose Men with a family history (FH) of prostate cancer (PCA) and African American (AA) men are at higher risk for PCA. Recruitment and retention of these high-risk men into early detection programs has been challenging. We report a comprehensive analysis on recruitment methods, show rates, and participant factors from the Prostate Cancer Risk Assessment Program (PRAP), which is a prospective, longitudinal PCA screening study. Materials and Methods Men 35–69 years are eligible if they have a FH of PCA, are AA, or have a BRCA1/2 mutation. Recruitment methods were analyzed with respect to participant demographics and show to the first PRAP appointment using standard statistical methods Results Out of 707 men recruited, 64.9% showed to the initial PRAP appointment. More individuals were recruited via radio than from referral or other methods (χ2 = 298.13, p < .0001). Men recruited via radio were more likely to be AA (p<0.001), less educated (p=0.003), not married or partnered (p=0.007), and have no FH of PCA (p<0.001). Men recruited via referrals had higher incomes (p=0.007). Men recruited via referral were more likely to attend their initial PRAP visit than those recruited by radio or other methods (χ2 = 27.08, p < .0001). Conclusions This comprehensive analysis finds that radio leads to higher recruitment of AA men with lower socioeconomic status. However, these are the high-risk men that have lower show rates for PCA screening. Targeted motivational measures need to be studied to improve show rates for PCA risk assessment for these high-risk men. PMID:19758657

Analysis of IRS-1-mediated phosphatidylinositol 3-kinase activation in the adipose tissue of polycystic ovary syndrome patients complicated with insulin resistance

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Yongli, Chu [Yantai Yuhuangding Hospital, Yantai (China). Dept. of Obstetrics and Gynecology; Hongyu, Qiu; Yongyu, Sun; Min, Li; Hongfa, Li

2004-04-01

Objective: To investigate the insulin receptor substance-1 (IRS-1)-mediated phosphatidylinositol-3 (PI-3) kinase activity in adipose tissue of polycystic ovary syndrome (PCOS) patients, and to explore molecular mechanisms of insulin resistance of PCOS. Methods: Blood and adipose tissue samples from patients with PCOS with insulin resistance (n=19), PCOS without insulin resistance (n=10) and controls (n=15) were collected. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) were measured by chemiluminescence assay. Fasting insulin (FIN) was measured by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Insulin resistance index (IR) was calculated using homeostasis model assessment (HOMA) to analyze the relationship between these markers and insulin resistance. The tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation and enhanced chemiluminescent immunoblotting technique. PI-3 kinase activity was detected by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed by statistical methods. Results: 1) The levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS without insulin resistance were significantly higher than those of control group (all P<0.05); the levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS with insulin resistance were significantly higher than those of PCOS without insulin resistance (all P<0.05). 2) The tyrosine phosphorylation analysis of IRS-1 showed that IRS-1 tyrosine phosphorylation was significantly decreased in PCOS with insulin resistance compared to that of PCOS without insulin resistance and control groups (P<0.01). 3) PI-3 kinase activity was significantly decreased (P<0.01) and negatively correlated with HOMA-IR. Conclusion: In consequence of the weaker signal caused by the change of upper stream signal molecule IRS-1 tyrosine phosphorylation, PI-3 kinase activity decreased, it affects the insulin signal

Analysis of IRS-1-mediated phosphatidylinositol 3-kinase activation in the adipose tissue of polycystic ovary syndrome patients complicated with insulin resistance

International Nuclear Information System (INIS)

Chu Yongli; Qiu Hongyu; Sun Yongyu; Li Min; Li Hongfa

2004-01-01

Objective: To investigate the insulin receptor substance-1 (IRS-1)-mediated phosphatidylinositol-3 (PI-3) kinase activity in adipose tissue of polycystic ovary syndrome (PCOS) patients, and to explore molecular mechanisms of insulin resistance of PCOS. Methods: Blood and adipose tissue samples from patients with PCOS with insulin resistance (n=19), PCOS without insulin resistance (n=10) and controls (n=15) were collected. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) were measured by chemiluminescence assay. Fasting insulin (FIN) was measured by radioimmunoassay. Fasting plasma glucose (FPG) was measured by oxidase assay. Insulin resistance index (IR) was calculated using homeostasis model assessment (HOMA) to analyze the relationship between these markers and insulin resistance. The tyrosine phosphorylation of IRS-1 was measured by immunoprecipitation and enhanced chemiluminescent immunoblotting technique. PI-3 kinase activity was detected by immunoprecipitation, thin-layer chromatography and gamma scintillation counting. The results were analyzed by statistical methods. Results: 1) The levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS without insulin resistance were significantly higher than those of control group (all P<0.05); the levels of serum LH, LH/FSH, T, FIN and HOMA-IR in PCOS with insulin resistance were significantly higher than those of PCOS without insulin resistance (all P<0.05). 2) The tyrosine phosphorylation analysis of IRS-1 showed that IRS-1 tyrosine phosphorylation was significantly decreased in PCOS with insulin resistance compared to that of PCOS without insulin resistance and control groups (P<0.01). 3) PI-3 kinase activity was significantly decreased (P<0.01) and negatively correlated with HOMA-IR. Conclusion: In consequence of the weaker signal caused by the change of upper stream signal molecule IRS-1 tyrosine phosphorylation, PI-3 kinase activity decreased, it affects the insulin signal

Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling networks

DEFF Research Database (Denmark)

Hammond, Dean E; Hyde, Russell; Kratchmarova, Irina

2010-01-01

549 lung adenocarcinoma cells with EGF or HGF. In total, we obtained quantitative information for 274 proteins, which respond to either or both stimuli by >1.5 fold changes in enrichment, following immuno-precipitation with antiphosphotyrosine antibodies. The data reveal a high degree of overlap...

HIV-1 phylogenetic analysis shows HIV-1 transits through the meninges to brain and peripheral tissues.

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Lamers, Susanna L; Gray, Rebecca R; Salemi, Marco; Huysentruyt, Leanne C; McGrath, Michael S

2011-01-01

Brain infection by the human immunodeficiency virus type 1 (HIV-1) has been investigated in many reports with a variety of conclusions concerning the time of entry and degree of viral compartmentalization. To address these diverse findings, we sequenced HIV-1 gp120 clones from a wide range of brain, peripheral and meningeal tissues from five patients who died from several HIV-1 associated disease pathologies. High-resolution phylogenetic analysis confirmed previous studies that showed a significant degree of compartmentalization in brain and peripheral tissue subpopulations. Some intermixing between the HIV-1 subpopulations was evident, especially in patients that died from pathologies other than HIV-associated dementia. Interestingly, the major tissue harboring virus from both the brain and peripheral tissues was the meninges. These results show that (1) HIV-1 is clearly capable of migrating out of the brain, (2) the meninges are the most likely primary transport tissues, and (3) infected brain macrophages comprise an important HIV reservoir during highly active antiretroviral therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

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Sharma, Shruti; Gupta, Ravi; Deswal, Renu

2017-05-01

In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

Science.gov (United States)

Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

2017-07-11

Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT

Practical guidelines for the comprehensive analysis of ChIP-seq data.

Directory of Open Access Journals (Sweden)

Timothy Bailey

Full Text Available Mapping the chromosomal locations of transcription factors, nucleosomes, histone modifications, chromatin remodeling enzymes, chaperones, and polymerases is one of the key tasks of modern biology, as evidenced by the Encyclopedia of DNA Elements (ENCODE Project. To this end, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq is the standard methodology. Mapping such protein-DNA interactions in vivo using ChIP-seq presents multiple challenges not only in sample preparation and sequencing but also for computational analysis. Here, we present step-by-step guidelines for the computational analysis of ChIP-seq data. We address all the major steps in the analysis of ChIP-seq data: sequencing depth selection, quality checking, mapping, data normalization, assessment of reproducibility, peak calling, differential binding analysis, controlling the false discovery rate, peak annotation, visualization, and motif analysis. At each step in our guidelines we discuss some of the software tools most frequently used. We also highlight the challenges and problems associated with each step in ChIP-seq data analysis. We present a concise workflow for the analysis of ChIP-seq data in Figure 1 that complements and expands on the recommendations of the ENCODE and modENCODE projects. Each step in the workflow is described in detail in the following sections.

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

OpenAIRE

Tran, Vuong; Gan, Qiang; Chen, Xin

2012-01-01

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. ...

Genome-wide analysis of adolescent psychotic-like experiences shows genetic overlap with psychiatric disorders.

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Pain, Oliver; Dudbridge, Frank; Cardno, Alastair G; Freeman, Daniel; Lu, Yi; Lundstrom, Sebastian; Lichtenstein, Paul; Ronald, Angelica

2018-03-31

This study aimed to test for overlap in genetic influences between psychotic-like experience traits shown by adolescents in the community, and clinically-recognized psychiatric disorders in adulthood, specifically schizophrenia, bipolar disorder, and major depression. The full spectra of psychotic-like experience domains, both in terms of their severity and type (positive, cognitive, and negative), were assessed using self- and parent-ratings in three European community samples aged 15-19 years (Final N incl. siblings = 6,297-10,098). A mega-genome-wide association study (mega-GWAS) for each psychotic-like experience domain was performed. Single nucleotide polymorphism (SNP)-heritability of each psychotic-like experience domain was estimated using genomic-relatedness-based restricted maximum-likelihood (GREML) and linkage disequilibrium- (LD-) score regression. Genetic overlap between specific psychotic-like experience domains and schizophrenia, bipolar disorder, and major depression was assessed using polygenic risk score (PRS) and LD-score regression. GREML returned SNP-heritability estimates of 3-9% for psychotic-like experience trait domains, with higher estimates for less skewed traits (Anhedonia, Cognitive Disorganization) than for more skewed traits (Paranoia and Hallucinations, Parent-rated Negative Symptoms). Mega-GWAS analysis identified one genome-wide significant association for Anhedonia within IDO2 but which did not replicate in an independent sample. PRS analysis revealed that the schizophrenia PRS significantly predicted all adolescent psychotic-like experience trait domains (Paranoia and Hallucinations only in non-zero scorers). The major depression PRS significantly predicted Anhedonia and Parent-rated Negative Symptoms in adolescence. Psychotic-like experiences during adolescence in the community show additive genetic effects and partly share genetic influences with clinically-recognized psychiatric disorders, specifically schizophrenia and

[Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig].

Science.gov (United States)

Luo, X; Wang, J Y; Zhang, F L; Xia, Y

2018-01-07

Objective: To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function. Methods: Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions. Results: The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein. Conclusion: EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

Systems level analysis of systemic sclerosis shows a network of immune and profibrotic pathways connected with genetic polymorphisms.

Directory of Open Access Journals (Sweden)

J Matthew Mahoney

2015-01-01

Full Text Available Systemic sclerosis (SSc is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6-12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected

Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC and its application for DNA methylation analysis

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Ottaviani Diego

2008-05-01

Full Text Available Abstract Background The major histocompatibility complex (MHC is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome. Methods To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP, methylated DNA immunoprecipitation (MeDIP, array comparative genomic hybridization (aCGH and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. Results Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs. Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. Conclusion A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.

Urine proteome analysis in Dent's disease shows high selective changes potentially involved in chronic renal damage.

Science.gov (United States)

Santucci, Laura; Candiano, Giovanni; Anglani, Franca; Bruschi, Maurizio; Tosetto, Enrica; Cremasco, Daniela; Murer, Luisa; D'Ambrosio, Chiara; Scaloni, Andrea; Petretto, Andrea; Caridi, Gianluca; Rossi, Roberta; Bonanni, Alice; Ghiggeri, Gian Marco

2016-01-01

Definition of the urinary protein composition would represent a potential tool for diagnosis in many clinical conditions. The use of new proteomic technologies allows detection of genetic and post-trasductional variants that increase sensitivity of the approach but complicates comparison within a heterogeneous patient population. Overall, this limits research of urinary biomarkers. Studying monogenic diseases are useful models to address this issue since genetic variability is reduced among first- and second-degree relatives of the same family. We applied this concept to Dent's disease, a monogenic condition characterised by low-molecular-weight proteinuria that is inherited following an X-linked trait. Results are presented here on a combined proteomic approach (LC-mass spectrometry, Western blot and zymograms for proteases and inhibitors) to characterise urine proteins in a large family (18 members, 6 hemizygous patients, 6 carrier females, and 6 normals) with Dent's diseases due to the 1070G>T mutation of the CLCN5. Gene ontology analysis on more than 1000 proteins showed that several clusters of proteins characterised urine of affected patients compared to carrier females and normal subjects: proteins involved in extracellular matrix remodelling were the major group. Specific analysis on metalloproteases and their inhibitors underscored unexpected mechanisms potentially involved in renal fibrosis. Studying with new-generation techniques for proteomic analysis of the members of a large family with Dent's disease sharing the same molecular defect allowed highly repetitive results that justify conclusions. Identification in urine of proteins actively involved in interstitial matrix remodelling poses the question of active anti-fibrotic drugs in Dent's patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Show-Bix &

DEFF Research Database (Denmark)

2014-01-01

The anti-reenactment 'Show-Bix &' consists of 5 dias projectors, a dial phone, quintophonic sound, and interactive elements. A responsive interface will enable the Dias projectors to show copies of original dias slides from the Show-Bix piece ”March på Stedet”, 265 images in total. The copies are...

'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.

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Rajesh Khanna

Full Text Available BACKGROUND: The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG, has also been implicated in synaptic vesicle (SV recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE from presynaptic nerve terminals and have used a novel fractional recovery (FR assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. METHODOLOGY/PRINCIPLE FINDINGS: The first step in FR analysis was to immunoprecipitate (IP the complex with an antibody against one protein component (the IP-protein. The immobilized complex was then exposed to a high salt (1150 mM stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins. A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized, and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. CONCLUSIONS/SIGNIFICANCE: We hypothesize that the SAE serves as a recognition site and also as a

Overexpression of hypoxia-inducible factor prolyl- hydoxylase ...

African Journals Online (AJOL)

Jane

2011-08-08

Aug 8, 2011 ... which is regulated by HIF prolyl-dydoxylase -mediated degradation. Taken together, our results ..... Chromatin immunoprecipitation analysis of gene ... phosphatidylinositol 3-kinase/Akt pathway. Endocrinology, 148(5):.

Regulated Ubiquitinylation-Dependent Corepressor and Coactivator Complex Exchange as a Potential Target in Estrogen-Dependent Breast Cancer

National Research Council Canada - National Science Library

Chang, Christine S

2006-01-01

.... Performed data analysis of genome-wide chromatin immunoprecipitation experiments with ERalpha, RNA polymerase III and histone modification markers and correlate the binding data with expression profiles upon estrogen stimulation.

Comportamento agressivo em shows musicais: analisando notícias de jornal impresso

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Carlos Eduardo Pimentel

2011-08-01

Full Text Available This paper reports a content analysis of the newspaper articles dealing with aggressive behavior in music shows. This analysis permits an understanding of the characteristics of these situations, the aggressive behaviors in shows, and the way in which the print media treat the subject. Despite of the newspapers’ influence on public opinion formation, that is, the transmission of attitudes and behavior repertoires, this kind of analysis is rarely reported in Brazilian social psychological literature. Some 31 newspaper articles were content analyzed as to aggressive behavior during music shows. Results indicate that aggressive behaviors occur in shows of most varied musical styles. Results are discussed on the basis of social cognitive theory.

Global Analysis of Photosynthesis Transcriptional Regulatory Networks

Science.gov (United States)

Imam, Saheed; Noguera, Daniel R.; Donohue, Timothy J.

2014-01-01

Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis. PMID:25503406

Global analysis of photosynthesis transcriptional regulatory networks.

Directory of Open Access Journals (Sweden)

Saheed Imam

2014-12-01

Full Text Available Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888, which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

Retrospective Analysis of Patient Presentations at the Sydney (Australia) Royal Easter Show from 2012 to 2014.

Science.gov (United States)

Crabtree, Nathan; Mo, Shirley; Ong, Leon; Jegathees, Thuvarahan; Wei, Daniel; Fahey, David; Liu, Jia Jenny

2017-04-01

Introduction Comprehensive studies on the relationship between patient demographics and subsequent treatment and disposition at a single mass-gathering event are lacking. The Sydney Royal Easter Show (SRES; Sydney Olympic Park, New South Wales, Australia) is an annual, 14-day, agricultural mass-gathering event occurring around the Easter weekend, attracting more than 800,000 patrons per year. In this study, patient records from the SRES were analyzed to examine relationships between weather, crowd size, day of week, and demographics on treatment and disposition. This information would help to predict factors affecting patient treatment and disposition to guide ongoing training of first responders and to evaluate the appropriateness of staffing skills mix at future events. Hypothesis Patient demographics, environmental factors, and attendance would influence the nature and severity of presentations at the SRES, which would influence staffing requirements. A retrospective analysis of 4,141 patient record forms was performed for patients who presented to St John Ambulance (Australian Capital Territory, Australia) at the SRES between 2012 and 2014 inclusive. Presentation type was classified using a previously published minimum data set. Data on weather and crowd size were obtained from the Australian Bureau of Meteorology (Melbourne, Victoria, Australia) and the SRES, respectively. Statistical analyses were performed using SPSS v22 (IBM; Armonk, New York USA). Between 2012 to 2014, over 2.5 million people attended the SRES with 4,141 patients treated onsite. As expected, the majority of presentations were injuries (49%) and illnesses (46%). Although patient demographics and presentation types did not change over time, the duration of treatment increased. A higher proportion of patients were discharged to hospital or home compared to the proportion of patients discharged back to the event. Patients from rural/regional locations (accounting for 15% of all patients) were

Detection of a high-molecular-weight LHRH precursor by cell-free translation of mRNA from human, rat, and mouse hypothalamus

International Nuclear Information System (INIS)

Curtis, A.; Szelke, M.; Fink, G.

1986-01-01

Large precursors have also been predicted using the immunoprecipitation technique which relies upon the identification of large immunoreactive molecules following in vitro translation of mRNA. The mRNA is presumed to represent the largest form of a nascent precursor polypeptide molecule irrespective of the number of biosynthetic cleavage steps which are necessary to liberate the active peptide. However, as has been shown for somatostatin, nonprotein modifications may be made which apparently increase molecular weight, such as glycosylation or phosphorylation of the molecule. The authors employed the immunoprecipitation technique to confirm earlier chromatographic studies that the hypothalamic decapeptide, luteinizing hormone releasing hormone (LHRH) is also synthesized by way of a large precursor form. The authors' finding show that the translation of hypothalamic mRNA produces a primary translation product with an apparent molecule weight of 28,000 which contains an amino acid sequence immunologically similar to that of biologically active LHRH. The procedure involved the incorporation of a radioactive amino acid into polypeptides synthesized by in vitro translation of hypothalamic messenger RNA. The resulting complex protein mixture was immunoprecipitated with a specific anti-LHRH serum, and the immunoprecipitate was identified by polyacrylamide gel electrophoresis and autoradiography

Genome wide analysis of acute myeloid leukemia reveal leukemia specific methylome and subtype specific hypomethylation of repeats.

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Marwa H Saied

Full Text Available Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6 with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001. We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2 = 0.7. We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

Science.gov (United States)

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-01

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Online Italian fandoms of American TV shows

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Eleonora Benecchi

2015-06-01

Full Text Available The Internet has changed media fandom in two main ways: it helps fans connect with each other despite physical distance, leading to the formation of international fan communities; and it helps fans connect with the creators of the TV show, deepening the relationship between TV producers and international fandoms. To assess whether Italian fan communities active online are indeed part of transnational online communities and whether the Internet has actually altered their relationship with the creators of the original text they are devoted to, qualitative analysis and narrative interviews of 26 Italian fans of American TV shows were conducted to explore the fan-producer relationship. Results indicated that the online Italian fans surveyed preferred to stay local, rather than using geography-leveling online tools. Further, the sampled Italian fans' relationships with the show runners were mediated or even absent.

Threonine phosphorylation of rat liver glycogen synthase

International Nuclear Information System (INIS)

Arino, J.; Arro, M.; Guinovart, J.J.

1985-01-01

32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

The Last Gender Picture Show to Closure Buffy the Vampire Slayer

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Sven Grampp

2016-03-01

Full Text Available This work presents in (and especially on one detail what it means to practice gender analysis as formal image analysis in the context of television shows. The chosen example for that is the closing scene of “Buffy the Vampire Slayer” (USA 1997-2003. This television show engages in a highly self-reflexive manner with common pop cultural representations of gender, which becomes particularly prevalent and radically condensed in the series finale.

ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana

DEFF Research Database (Denmark)

Jensen, Michael Krogh; Lindemose, Søren; De Masi, Federico

2013-01-01

ATAF1, an Arabidopsis thaliana NAC transcription factor, plays important roles in plant adaptation to environmental stress and development. To search for ATAF1 target genes, we used protein binding microarrays and chromatin-immunoprecipitation (ChIP). This identified T[A,C,G]CGT[A,G] and TT[A,C,G...... abscisic acid (ABA) phytohormone biosynthetic gene NCED3. ChIP-qPCR and expression analysis showed that ATAF1 binding to the NCED3 promoter correlated with increased NCED3 expression and ABA hormone levels. These results indicate that ATAF1 regulates ABA biosynthesis....

Grab That Mike: communicative issues in French Talk Show "Grab That Mike": questões comunicativas em um "Talk Show" Francês

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Ludwina Van Son

2004-12-01

Full Text Available In this analysis we have chosen a recent French talk show to illustrate how communication is turned into some new kind of "ideology"nowadays: in other words, you have to communicate if you consider yourself a citizen of today's world. The main characteristic of issue-centered talk shows being the destabilization of the implicit rules and participation framework, we observe how the so-called democratic right to express ourselves is (misused by the talk show host to secure the dynamics of the show. In order to reveal the host's manipulations, we have examined the verbal interactions between host and guests on the following issues: topic choice, turn-taking mechanisms and identity construction of the talk show's guests. In the perspective that this kind of talk show presents itself as a public space where direct democracy can be exercised, the analysis of the discursive strategies of the talk show host reveals the impact of a mediatic participation framework.Para a presente análise escolhemos um "talk show" francês recente para ilustrar como a comunicação se transforma em um novo tipo de ideologia: em outras palavras, você deve se comunicar se você se considera um cidadão do mundo de hoje. Sendo a principal característica do talk show a desestabilização das regras implícitas e o framework de participação, observamos como o tal direito democrático de expressão é interpretado pelo apresentador para manter a dinâmica do programa. Para revelar suas manipulações, examinamos as interações verbais entre apresentador e convidados nos seguintes temas: escolha do tópico, mecanismos de tomada de turno e construção de identidade dos convidados. É nessa perspectiva que este tipo de programa se apresenta como um espaço público onde a democracia direta pode ser exercitada; a análise das estratégias discursivas do apresentador do talk show revela o impacto de um framework de participação mediada.

Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses.

Science.gov (United States)

Brum, Jennifer R; Schenck, Ryan O; Sullivan, Matthew B

2013-09-01

Viruses influence oceanic ecosystems by causing mortality of microorganisms, altering nutrient and organic matter flux via lysis and auxiliary metabolic gene expression and changing the trajectory of microbial evolution through horizontal gene transfer. Limited host range and differing genetic potential of individual virus types mean that investigations into the types of viruses that exist in the ocean and their spatial distribution throughout the world's oceans are critical to understanding the global impacts of marine viruses. Here we evaluate viral morphological characteristics (morphotype, capsid diameter and tail length) using a quantitative transmission electron microscopy (qTEM) method across six of the world's oceans and seas sampled through the Tara Oceans Expedition. Extensive experimental validation of the qTEM method shows that neither sample preservation nor preparation significantly alters natural viral morphological characteristics. The global sampling analysis demonstrated that morphological characteristics did not vary consistently with depth (surface versus deep chlorophyll maximum waters) or oceanic region. Instead, temperature, salinity and oxygen concentration, but not chlorophyll a concentration, were more explanatory in evaluating differences in viral assemblage morphological characteristics. Surprisingly, given that the majority of cultivated bacterial viruses are tailed, non-tailed viruses appear to numerically dominate the upper oceans as they comprised 51-92% of the viral particles observed. Together, these results document global marine viral morphological characteristics, show that their minimal variability is more explained by environmental conditions than geography and suggest that non-tailed viruses might represent the most ecologically important targets for future research.

Expression and analysis of exogenous proteins in epidermal cells.

Science.gov (United States)

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

Bayesian analysis of repairable systems showing a bounded failure intensity

International Nuclear Information System (INIS)

Guida, Maurizio; Pulcini, Gianpaolo

2006-01-01

The failure pattern of repairable mechanical equipment subject to deterioration phenomena sometimes shows a finite bound for the increasing failure intensity. A non-homogeneous Poisson process with bounded increasing failure intensity is then illustrated and its characteristics are discussed. A Bayesian procedure, based on prior information on model-free quantities, is developed in order to allow technical information on the failure process to be incorporated into the inferential procedure and to improve the inference accuracy. Posterior estimation of the model-free quantities and of other quantities of interest (such as the optimal replacement interval) is provided, as well as prediction on the waiting time to the next failure and on the number of failures in a future time interval is given. Finally, numerical examples are given to illustrate the proposed inferential procedure

Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

Directory of Open Access Journals (Sweden)

Savinska L. O.

2015-08-01

Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

Marmosets, Raree shows and Pulcinelle: an Analysis and Edition of a Hitherto-Unpublished Carnival Play by Antonio de Zamora

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Fernando Plata

2013-05-01

Full Text Available This paper is an analyisis, annotation and edition of the Mojiganga del mundinovo (‘The raree show, a carnival play’ by Antonio de Zamora. The play was performed in 1698 Madrid by the troupe of Carlos Vallejo, along with the sacramental one-act play El templo vivo de Dios (‘The living temple of God’. The hitherto unpublished text is based on the only two extant manuscripts, located in archives in Madrid. Despite the play’s title, my analysis argues that the novelty in this play is not so much the raree show, a contraption popularized four decades earlier in Golden Age theater, as the marmosets. The death of two marmosets and the ensuing desolation of their owner, Ms. Estupenda, both trigger the play and provide it with a plot. The marmosets, too, point to a changing mentality in late 17th-century society, regarding the possession among ladies of marmosets and other monkeys as pets.

Genome-Wide Bimolecular Fluorescence Complementation-Based Proteomic Analysis of Toxoplasma gondii ROP18’s Human Interactome Shows Its Key Role in Regulation of Cell Immunity and Apoptosis

Directory of Open Access Journals (Sweden)

Jing Xia

2018-02-01

Full Text Available Toxoplasma gondii rhoptry protein ROP18 (TgROP18 is a key virulence factor secreted into the host cell during invasion, where it modulates the host cell response by interacting with its host targets. However, only a few TgROP18 targets have been identified. In this study, we applied a high-throughput protein–protein interaction (PPI screening in human cells using bimolecular fluorescence complementation (BiFC to identify the targets of Type I strain ROP18 (ROP18I and Type II strain ROP18 (ROP18II. From a pool of more than 18,000 human proteins, 492 and 141 proteins were identified as the targets of ROP18I and ROP18II, respectively. Gene ontology, search tool for the retrieval of interacting genes/proteins PPI network, and Ingenuity pathway analyses revealed that the majority of these proteins were associated with immune response and apoptosis. This indicates a key role of TgROP18 in manipulating host’s immunity and cell apoptosis, which might contribute to the immune escape and successful parasitism of the parasite. Among the proteins identified, the immunity-related proteins N-myc and STAT interactor, IL20RB, IL21, ubiquitin C, and vimentin and the apoptosis-related protein P2RX1 were further verified as ROP18I targets by sensitized emission-fluorescence resonance energy transfer (SE-FRET and co-immunoprecipitation. Our study substantially contributes to the current limited knowledge on human targets of TgROP18 and provides a novel tool to investigate the function of parasite effectors in human cells.

Meta-analysis of diffusion tensor imaging studies shows altered fractional anisotropy occurring in distinct brain areas in association with depression.

LENUS (Irish Health Repository)

Murphy, Melissa L

2011-09-01

Fractional anisotropy anomalies occurring in the white matter tracts in the brains of depressed patients may reflect microstructural changes underlying the pathophysiology of this disorder. We conducted a meta-analysis of fractional anisotropy abnormalities occurring in major depressive disorder using voxel-based diffusion tensor imaging studies. Using the Embase, PubMed and Google Scholar databases, 89 relevant data sets were identified, of which 7 (including 188 patients with major depressive disorder and 221 healthy controls) met our inclusion criteria. Authors were contacted to retrieve any additional data required. Coordinates were extracted from clusters of significant white matter fractional anisotropy differences between patients and controls. Relevant demographic, clinical and methodological variables were extracted from each study or obtained directly from authors. The meta-analysis was carried out using Signed Differential Mapping. Patients with depression showed decreased white matter fractional anisotropy values in the superior longitudinal fasciculus and increased fractional anisotropy values in the fronto-occipital fasciculus compared to controls. Using quartile and jackknife sensitivity analysis, we found that reduced fractional anisotropy in the left superior longitudinal fasciculus was very stable, with increases in the right fronto-occipital fasciculus driven by just one study. In conclusion, our meta-analysis revealed a significant reduction in fractional anisotropy values in the left superior longitudinal fasciculus, which may ultimately play an important role in the pathology of depression.

Software for rapid time dependent ChIP-sequencing analysis (TDCA).

Science.gov (United States)

Myschyshyn, Mike; Farren-Dai, Marco; Chuang, Tien-Jui; Vocadlo, David

2017-11-25

Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and associated methods are widely used to define the genome wide distribution of chromatin associated proteins, post-translational epigenetic marks, and modifications found on DNA bases. An area of emerging interest is to study time dependent changes in the distribution of such proteins and marks by using serial ChIP-seq experiments performed in a time resolved manner. Despite such time resolved studies becoming increasingly common, software to facilitate analysis of such data in a robust automated manner is limited. We have designed software called Time-Dependent ChIP-Sequencing Analyser (TDCA), which is the first program to automate analysis of time-dependent ChIP-seq data by fitting to sigmoidal curves. We provide users with guidance for experimental design of TDCA for modeling of time course (TC) ChIP-seq data using two simulated data sets. Furthermore, we demonstrate that this fitting strategy is widely applicable by showing that automated analysis of three previously published TC data sets accurately recapitulates key findings reported in these studies. Using each of these data sets, we highlight how biologically relevant findings can be readily obtained by exploiting TDCA to yield intuitive parameters that describe behavior at either a single locus or sets of loci. TDCA enables customizable analysis of user input aligned DNA sequencing data, coupled with graphical outputs in the form of publication-ready figures that describe behavior at either individual loci or sets of loci sharing common traits defined by the user. TDCA accepts sequencing data as standard binary alignment map (BAM) files and loci of interest in browser extensible data (BED) file format. TDCA accurately models the number of sequencing reads, or coverage, at loci from TC ChIP-seq studies or conceptually related TC sequencing experiments. TC experiments are reduced to intuitive parametric values that facilitate biologically

Can Automated Facial Expression Analysis Show Differences Between Autism and Typical Functioning?

Science.gov (United States)

Borsos, Zsófia; Gyori, Miklos

2017-01-01

Exploratory analyses of emotional expressions using a commercially available facial expression recognition software are reported, from the context of a serious game for screening purposes. Our results are based on a comparative analysis of two matched groups of kindergarten-age children (high-functioning children with autism spectrum condition: n=13; typically developing children: n=13). Results indicate that this technology has the potential to identify autism-specific emotion expression features, and may play a role in affective diagnostic and assistive technologies.

Systematic analysis of DEMETER-like DNA glycosylase genes shows lineage-specific Smi-miR7972 involved in SmDML1 regulation in Salvia miltiorrhiza.

Science.gov (United States)

Li, Jiang; Li, Caili; Lu, Shanfa

2018-05-08

DEMETER-like DNA glycosylases (DMLs) initiate the base excision repair-dependent DNA demethylation to regulate a wide range of biological processes in plants. Six putative SmDML genes, termed SmDML1-SmDML6, were identified from the genome of S. miltiorrhiza, an emerging model plant for Traditional Chinese Medicine (TCM) studies. Integrated analysis of gene structures, sequence features, conserved domains and motifs, phylogenetic analysis and differential expression showed the conservation and divergence of SmDMLs. SmDML1, SmDML2 and SmDML4 were significantly down-regulated by the treatment of 5Aza-dC, a general DNA methylation inhibitor, suggesting involvement of SmDMLs in genome DNA methylation change. SmDML1 was predicted and experimentally validated to be target of Smi-miR7972. Computational analysis of forty whole genome sequences and almost all of RNA-seq data from Lamiids revealed that MIR7972s were only distributed in some plants of the three orders, including Lamiales, Solanales and Boraginales, and the number of MIR7972 genes varied among species. It suggests that MIR7972 genes underwent expansion and loss during the evolution of some Lamiids species. Phylogenetic analysis of MIR7972s showed closer evolutionary relationships between MIR7972s in Boraginales and Solanales in comparison with Lamiales. These results provide a valuable resource for elucidating DNA demethylation mechanism in S. miltiorrhiza.

Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

DEFF Research Database (Denmark)

Nagirnaja, Liina; Venclovas, Česlovas; Rull, Kristiina

2012-01-01

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by appl...... of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGβ genes CGB5 and CGB8.......Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of h......CG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls...

Role of Nab2 in RNA metabolism in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Olszewski, Pawel

RNP assembly generates errors, which are tracked down by nuclear surveillance mechanisms. Thus, not surprisingly, protein components of mRNPs have multiple functions in the biogenesis of the particle but also interact with the surveillance machinery. An example of such a multifunctional factor is the S......RNP components showed changes in the Nab2 interactome in the absence of Rrp6, and also suggested that Nab2 binds to multiple sites on the mRNA. Wide-range proteomic analysis of Nab2 immunoprecipitates revealed association with C/D box snoRNP components. These novel interactions were supported by the presence...

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Science.gov (United States)

Gim, Jeong-An; Hong, Chang Pyo; Kim, Dae-Soo; Moon, Jae-Woo; Choi, Yuri; Eo, Jungwoo; Kwon, Yun-Jeong; Lee, Ja-Rang; Jung, Yi-Deun; Bae, Jin-Han; Choi, Bong-Hwan; Ko, Junsu; Song, Sanghoon; Ahn, Kung; Ha, Hong-Seok; Yang, Young Mok; Lee, Hak-Kyo; Park, Kyung-Do; Do, Kyoung-Tag; Han, Kyudong; Yi, Joo Mi; Cha, Hee-Jae; Ayarpadikannan, Selvam; Cho, Byung-Wook; Bhak, Jong; Kim, Heui-Soo

2015-01-01

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits. PMID:25666347

Two-dimensional electrophoretic analysis of transformation-sensitive polypeptides during chemically, spontaneously, and oncogene-induced transformation of rat liver epithelial cells

DEFF Research Database (Denmark)

Wirth, P J; Luo, L D; Fujimoto, Y

1992-01-01

; AFB), spontaneously, and oncogene (v-Ha-ras, v-raf, and v-myc/v-raf)-induced transformation of RLE cells. Two-dimensional mapping of [35S]methionine-labeled whole cell lysate, cell-free in vitro translation products and [32P]orthophosphate-labeled polypeptides revealed subsets of polypeptides specific...... for each transformation modality. A search of the RLE protein database indicated the specific subcellular location for the majority of these transformation-sensitive proteins. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin......- and intermediate filament-related polypeptides (vimentin, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Immunoprecipitation and Western immunoblot analysis of tropomyosin expression in four individual AFB-, as well as four spontaneously induced, and each...

Airline Overbooking Problem with Uncertain No-Shows

Directory of Open Access Journals (Sweden)

Chunxiao Zhang

2014-01-01

Full Text Available This paper considers an airline overbooking problem of a new single-leg flight with discount fare. Due to the absence of historical data of no-shows for a new flight, and various uncertain human behaviors or unexpected events which causes that a few passengers cannot board their aircraft on time, we fail to obtain the probability distribution of no-shows. In this case, the airlines have to invite some domain experts to provide belief degree of no-shows to estimate its distribution. However, human beings often overestimate unlikely events, which makes the variance of belief degree much greater than that of the frequency. If we still regard the belief degree as a subjective probability, the derived results will exceed our expectations. In order to deal with this uncertainty, the number of no-shows of new flight is assumed to be an uncertain variable in this paper. Given the chance constraint of social reputation, an overbooking model with discount fares is developed to maximize the profit rate based on uncertain programming theory. Finally, the analytic expression of the optimal booking limit is obtained through a numerical example, and the results of sensitivity analysis indicate that the optimal booking limit is affected by flight capacity, discount, confidence level, and parameters of the uncertainty distribution significantly.

Cystatin C–Adiponectin Complex in Plasma Associates with Coronary Plaque Instability

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Matsuoka, Tetsuro; Kayama, Kento; Onishi, Sumire; Matsuo, Natsumi

2017-01-01

Aim: Adiponectin (APN) is an adipocyte-derived bioactive molecule with antiatherogenic properties. We previously reported that cystatin C (CysC) abolished the anti-atherogenic effects of APN. We aimed to elucidate the clinical significance of CysC–APN complex in patients with coronary artery disease (CAD). Methods: We enrolled 43 stable CAD male patients to examine the relationship between CysC–APN complex and coronary plaque characteristics. Serum was immunoprecipitated by the anti-APN antibody and immunoblotted by the anti-CysC antibody to demonstrate the presence of CysC–APN complexes in vivo. To confirm their binding in vitro, HEK293T cell lysates overexpressing myc-APN and FLAG-CysC were immunoprecipitated with an anti-myc or anti-FLAG antibody, followed by immunoblotting with an anti-APN or anti-CysC antibody. Results: CysC was identified as a specific co-immunoprecipitant with APN by the anti-APN antibody in human serum. In vitro, FLAG-CysC was co-immunoprecipitated with myc-APN by the antimyc antibody and myc-APN was co-immunoprecipitated with FLAG-CysC by the anti-FLAG antibody. Among CAD patients, serum CysC–APN complex levels negatively correlated with fibrotic components of coronary plaques and positively correlated with either necrotic or lipidic plus necrotic components. Plaque burden negatively correlated with serum APN levels but not serum CysC–APN complex levels. Serum CysC levels had no association with plaque characteristics. In multivariate analysis, CysC–APN complex levels were identified as the strongest negative factor for fibrotic components and the strongest positive factor for both necrotic and lipidic plus necrotic components. Conclusion: Measuring serum CysC–APN complex levels is helpful for evaluating coronary plaque instability in CAD patients. PMID:28321013

Analysis of AtCry1 and Mutants

Science.gov (United States)

Burdick, Derek; Purvis, Adam; Ahmad, Margaret; Link, Justin J.; Engle, Dorothy

Cryptochrome is an incredibly versatile protein that influences numerous biological processes such as plant growth, bird migration, and sleep cycles. Due to the versatility of this protein, understanding the mechanism would allow for advances in numerous fields such as crop growth, animal behavior, and sleep disorders. It is known that cryptochrome requires blue light to function, but the exact processes in the regulation of biological activity are still not fully understood. It is believed that the c-terminal domain of the protein undergoes a conformational change when exposed to blue light which allows for biological function. Three different non-functioning mutants were tested during this study to gain insight on the mechanism of cryptochrome. Absorbance spectra showed a difference between two of the mutants and the wild type with one mutant showing little difference. Immunoprecipitation experiments were also conducted to identify the different c-terminal responses of the mutants. By studying non functioning mutants of this protein, the mechanism of the protein can be further characterized. This two-month research experience in Paris allowed us to experience international and interdisciplinary collaborations in science and immerse in a different culture. The Borcer Fund for Student Research, Xavier University, Cincinnati, OH, and John Hauck Foundation.

Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq

DEFF Research Database (Denmark)

Sittka, A; Lucchini, S; Papenfort, K

2008-01-01

would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high...

Meta-analysis of diffusion tensor imaging (DTI) studies shows altered fractional anisotropy occurring in distinct brain areas in association with depression

LENUS (Irish Health Repository)

Murphy, Melissa L

2011-09-27

Abstract Fractional anisotropy anomalies occurring in the white matter tracts in the brains of depressed patients may reflect microstructural changes underlying the pathophysiology of this disorder. We conducted a meta-analysis of fractional anisotropy abnormalities occurring in major depressive disorder using voxel-based diffusion tensor imaging studies. Using the Embase, PubMed and Google Scholar databases, 89 relevant data sets were identified, of which 7 (including 188 patients with major depressive disorder and 221 healthy controls) met our inclusion criteria. Authors were contacted to retrieve any additional data required. Coordinates were extracted from clusters of significant white matter fractional anisotropy differences between patients and controls. Relevant demographic, clinical and methodological variables were extracted from each study or obtained directly from authors. The meta-analysis was carried out using Signed Differential Mapping. Patients with depression showed decreased white matter fractional anisotropy values in the superior longitudinal fasciculus and increased fractional anisotropy values in the fronto-occipital fasciculus compared to controls. Using quartile and jackknife sensitivity analysis, we found that reduced fractional anisotropy in the left superior longitudinal fasciculus was very stable, with increases in the right fronto-occipital fasciculus driven by just one study. In conclusion, our meta-analysis revealed a significant reduction in fractional anisotropy values in the left superior longitudinal fasciculus, which may ultimately play an important role in the pathology of depression.

ChIP-seq Analysis in R (CSAR): An R package for the statistical detection of protein-bound genomic regions

NARCIS (Netherlands)

Muino, J.M.; Kaufmann, K.; Ham, van R.C.H.J.; Angenent, G.C.; Krajewski, P.

2011-01-01

Background In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally

ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

Directory of Open Access Journals (Sweden)

Jeron Andreas

2012-12-01

Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

Gut Transcriptome Analysis Shows Different Food Utilization Efficiency by the Grasshopper Oedaleous asiaticus (Orthoptera: Acrididae).

Science.gov (United States)

Huang, Xunbing; McNeill, Mark Richard; Ma, Jingchuan; Qin, Xinghu; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

2017-08-01

Oedaleus asiaticus B. Bienko is a persistent pest occurring in north Asian grasslands. We found that O. asiaticus feeding on Stipa krylovii Roshev. had higher approximate digestibility (AD), efficiency of conversion of ingested food (ECI), and efficiency of conversion of digested food (ECD), compared with cohorts feeding on Leymus chinensis (Trin.) Tzvel, Artemisia frigida Willd., or Cleistogenes squarrosa (Trin.) Keng. Although this indicated high food utilization efficiency for S. krylovii, the physiological processes and molecular mechanisms underlying these biological observations are not well understood. Transcriptome analysis was used to examine how gene expression levels in O. asiaticus gut are altered by feeding on the four plant species. Nymphs (fifth-instar female) that fed on S. krylovii had the largest variation in gene expression profiles, with a total of 88 genes significantly upregulated compared with those feeding on the other three plants, mainly including nutrition digestive genes of protein, carbohydrate, and lipid digestion. GO and KEGG enrichment also showed that feeding S. krylovii could upregulate the nutrition digestion-related molecular function, biological process, and pathways. These changes in transcripts levels indicate that the physiological processes of activating nutrition digestive enzymes and metabolism pathways can well explain the high food utilization of S. krylovii by O. asiaticus. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A comparison of genotyping-by-sequencing analysis methods on low-coverage crop datasets shows advantages of a new workflow, GB-eaSy.

Science.gov (United States)

Wickland, Daniel P; Battu, Gopal; Hudson, Karen A; Diers, Brian W; Hudson, Matthew E

2017-12-28

Genotyping-by-sequencing (GBS), a method to identify genetic variants and quickly genotype samples, reduces genome complexity by using restriction enzymes to divide the genome into fragments whose ends are sequenced on short-read sequencing platforms. While cost-effective, this method produces extensive missing data and requires complex bioinformatics analysis. GBS is most commonly used on crop plant genomes, and because crop plants have highly variable ploidy and repeat content, the performance of GBS analysis software can vary by target organism. Here we focus our analysis on soybean, a polyploid crop with a highly duplicated genome, relatively little public GBS data and few dedicated tools. We compared the performance of five GBS pipelines using low-coverage Illumina sequence data from three soybean populations. To address issues identified with existing methods, we developed GB-eaSy, a GBS bioinformatics workflow that incorporates widely used genomics tools, parallelization and automation to increase the accuracy and accessibility of GBS data analysis. Compared to other GBS pipelines, GB-eaSy rapidly and accurately identified the greatest number of SNPs, with SNP calls closely concordant with whole-genome sequencing of selected lines. Across all five GBS analysis platforms, SNP calls showed unexpectedly low convergence but generally high accuracy, indicating that the workflows arrived at largely complementary sets of valid SNP calls on the low-coverage data analyzed. We show that GB-eaSy is approximately as good as, or better than, other leading software solutions in the accuracy, yield and missing data fraction of variant calling, as tested on low-coverage genomic data from soybean. It also performs well relative to other solutions in terms of the run time and disk space required. In addition, GB-eaSy is built from existing open-source, modular software packages that are regularly updated and commonly used, making it straightforward to install and maintain

Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

International Nuclear Information System (INIS)

Safford, R.; de Silva, J.; Lucas, C.

1987-01-01

Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from ∼ 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH

Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803.

Science.gov (United States)

Mo, Ran; Yang, Mingkun; Chen, Zhuo; Cheng, Zhongyi; Yi, Xingling; Li, Chongyang; He, Chenliu; Xiong, Qian; Chen, Hui; Wang, Qiang; Ge, Feng

2015-02-06

Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC-MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

Directory of Open Access Journals (Sweden)

Le Zhan

Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

International Nuclear Information System (INIS)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E.; Flores, B.M.; Hagen, F.S.

1990-01-01

A λgt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35 S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface- 125 I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degree C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested

cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

Energy Technology Data Exchange (ETDEWEB)

Torian, B.E.; Stroeher, V.L.; Stamm, W.E. (Univ. of Washington, Seattle (USA)); Flores, B.M. (Louisiana State Univ. Medical Center, New Orleans (USA)); Hagen, F.S. (Zymogenetics Incorporated, Seattle, WA (USA))

1990-08-01

A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

Gene expression analysis of two extensively drug-resistant tuberculosis isolates show that two-component response systems enhance drug resistance.

Science.gov (United States)

Yu, Guohua; Cui, Zhenling; Sun, Xian; Peng, Jinfu; Jiang, Jun; Wu, Wei; Huang, Wenhua; Chu, Kaili; Zhang, Lu; Ge, Baoxue; Li, Yao

2015-05-01

Global analysis of expression profiles using DNA microarrays was performed between a reference strain H37Rv and two clinical extensively drug-resistant isolates in response to three anti-tuberculosis drug exposures (isoniazid, capreomycin, and rifampicin). A deep analysis was then conducted using a combination of genome sequences of the resistant isolates, resistance information, and related public microarray data. Certain known resistance-associated gene sets were significantly overrepresented in upregulated genes in the resistant isolates relative to that observed in H37Rv, which suggested a link between resistance and expression levels of particular genes. In addition, isoniazid and capreomycin response genes, but not rifampicin, either obtained from published works or our data, were highly consistent with the differentially expressed genes of resistant isolates compared to those of H37Rv, indicating a strong association between drug resistance of the isolates and genes differentially regulated by isoniazid and capreomycin exposures. Based on these results, 92 genes of the studied isolates were identified as candidate resistance genes, 10 of which are known resistance-related genes. Regulatory network analysis of candidate resistance genes using published networks and literature mining showed that three two-component regulatory systems and regulator CRP play significant roles in the resistance of the isolates by mediating the production of essential envelope components. Finally, drug sensitivity testing indicated strong correlations between expression levels of these regulatory genes and sensitivity to multiple anti-tuberculosis drugs in Mycobacterium tuberculosis. These findings may provide novel insights into the mechanism underlying the emergence and development of drug resistance in resistant tuberculosis isolates and useful clues for further studies on this issue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

Science.gov (United States)

Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

2014-08-01

The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage. © 2014 BSPP AND JOHN WILEY & SONS LTD.

BIOMETRICAL ANALYSIS OF ARABIAN FOALS AND THEIR LATER SUCCESSES IN SHOWS AND ON RACE TRACK

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M. PIESZKA

2007-10-01

Full Text Available Arabian horses are one of the most important products of Polish horse breeding.Many of them are International and World champions in shows; others are very wellknown as courageous race horses. To obtain such champions it is necessary to takeunder consideration many factors affecting the final results. The objective of thisstudy was to evaluate the effect of biometrical measurements of the foals at birthaccording to their future successes in shows and on racetrack. The study was carriedout on 143 horses winning in shows and in races. Body weight, height at withers,girth and canon circumferences taken at birth of these horses were analysed.Additionally coat colour was studied. All studied animals were divided into threegroups according to each measurement and the differences between such groupswere evaluated according points obtained for particular place at shows and place inraces. It was stated that horses heavier at birth and with higher girth circumferencegot more successes both at shows and on racetrack. Horses with higher height atwithers at birth were more successful in shows while animals with higher canoncircumference won oftener at race track. It was observed that the most courageousrace horses were bay while most champions were grey.

Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

International Nuclear Information System (INIS)

Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

1988-01-01

Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 [ 35 S]methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of [ 3 H]palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from 125 I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of [ 3 H]palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins

A Cbx8-containing polycomb complex facilitates the transition to gene activation during ES cell differentiation.

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Catherine Creppe

2014-12-01

Full Text Available Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq. Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.

Pedagogical Techniques Employed by the Television Show "MythBusters"

Science.gov (United States)

Zavrel, Erik

2016-11-01

"MythBusters," the long-running though recently discontinued Discovery Channel science entertainment television program, has proven itself to be far more than just a highly rated show. While its focus is on entertainment, the show employs an array of pedagogical techniques to communicate scientific concepts to its audience. These techniques include: achieving active learning, avoiding jargon, employing repetition to ensure comprehension, using captivating demonstrations, cultivating an enthusiastic disposition, and increasing intrinsic motivation to learn. In this content analysis, episodes from the show's 10-year history were examined for these techniques. "MythBusters" represents an untapped source of pedagogical techniques, which science educators may consider availing themselves of in their tireless effort to better reach their students. Physics educators in particular may look to "MythBusters" for inspiration and guidance in how to incorporate these techniques into their own teaching and help their students in the learning process.

The fading affect bias shows positive outcomes at the general but not the individual level of analysis in the context of social media.

Science.gov (United States)

Gibbons, Jeffrey A; Horowitz, Kyle A; Dunlap, Spencer M

2017-08-01

Unpleasant affect fades faster than pleasant affect (e.g., Walker, Vogl, & Thompson, 1997); this effect is referred to as the Fading Affect Bias (FAB; Walker, Skowronski, Gibbons, Vogl, & Thompson, 2003a). Research shows that the FAB is consistently related to positive/healthy outcomes at a general but not at a specific level of analysis based on event types and individual differences (e.g., Gibbons et al., 2013). Based on the positive outcomes for FAB and negative outcomes for social media (Bolton et al., 2013; Huang, 2010), the current study examined FAB in the context of social media events along with related individual differences. General positive outcomes were shown in the form of robust FAB effects across social media and non-social media events, a larger FAB for non-social media events than for social media events, negative correlations of FAB with depression, anxiety, and stress as well as a positive correlation of FAB with self-esteem. However, the lack of a negative correlation between FAB and anxiety for social media events in a 3-way interaction did not show positive outcomes at a specific level of analysis. Rehearsal ratings mediated the 3-way interaction. Implications are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of changes to mRNA levels and CTCF occupancy upon TFII-I knockdown

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Maud Marques

2015-06-01

Full Text Available CTCF is a key regulator of nuclear chromatin structure, chromatin organization and gene regulation. The impact of CTCF on transcriptional output is quite varied, ranging from repression, to transcriptional pausing and transactivation. The multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique properties. We identified the general transcription factor TFII-I as an interacting partner of CTCF. To gain an understanding of the function of TFII-I in regulating gene expression and CTCF binding genome wide, we conducted microarray experiments following TFII-I knockdown and chromatin immunoprecipitation of CTCF followed by next generation sequencing (ChIP-seq from the same TFII-I depleted cells. Here, we described the experimental design and the quality control and analysis that were performed on the dataset. The data is publicly available through the GEO database with accession number GSE60918. The interpretation and description of these data are included in a manuscript in revision (1.

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

Science.gov (United States)

Davis, W C; Naessens, J; Brown, W C; Ellis, J A; Hamilton, M J; Cantor, G H; Barbosa, J I; Ferens, W; Bohach, G A

1996-08-01

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

High-Resolution Observations of a Filament showing Activated Barb

Science.gov (United States)

Joshi, Anand; Martin, Sara F.; Mathew, Shibu; Srivastava, Nandita

2012-07-01

Analysis of a filament showing an activated barb using observations from the Dutch Open Telescope (DOT) on 2010 August 20 are presented. The DOT takes Doppler images in Hα, among other wavelengths, in a region about 110 × 110 arcsec^{2} in area, at a cadence of 30~seconds. The offline image restoration technique of speckle reconstruction is applied to obtain diffraction limited images. The filament developed a new barb in 10~minutes, which disappeared within the next 35~minutes. Such a rapid formation and disappearance of a filament barb is unusual, and has not been reported earlier. Line-of-sight velocity maps were constructed from the Doppler images of the target filament. We observe flows in the filament spine towards the barb location prior to its formation, and flows in the barb towards the spine during its disappearance. Photospheric magnetograms from Heliospheric Magnetic Imager on board the Solar Dynamics Observatory, at a cadence of 45~seconds, were used to determine the changes in magnetic flux in the region surrounding the barb location. The variation of magnetic flux in this duration supports the view that barbs are rooted in minor magnetic polarity. Our analysis shows that barbs can be short-lived and formation and disappearance of the barb was associated with cancellation of magnetic flux.

Genomic clones of bovine parvovirus: Construction and effect of deletions and terminal sequence inversions on infectivity

International Nuclear Information System (INIS)

Shull, B.C.; Chen, K.C.; Lederman, M.; Stout, E.R.; Bates, R.C.

1988-01-01

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytophathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Expression of capsid proteins from tranfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [ 35 S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus

Tokyo Motor Show 2003; Tokyo Motor Show 2003

Energy Technology Data Exchange (ETDEWEB)

Joly, E.

2004-01-01

The text which follows present the different techniques exposed during the 37. Tokyo Motor Show. The report points out the great tendencies of developments of the Japanese automobile industry. The hybrid electric-powered vehicles or those equipped with fuel cells have been highlighted by the Japanese manufacturers which allow considerable budgets in the research of less polluting vehicles. The exposed models, although being all different according to the manufacturer, use always a hybrid system: fuel cell/battery. The manufacturers have stressed too on the intelligent systems for navigation and safety as well as on the design and comfort. (O.M.)

Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication.

Science.gov (United States)

Pichereaux, Carole; Hernández-Domínguez, Eric E; Santos-Diaz, Maria Del Socorro; Reyes-Agüero, Antonio; Astello-García, Marizel; Guéraud, Françoise; Negre-Salvayre, Anne; Schiltz, Odile; Rossignol, Michel; Barba de la Rosa, Ana Paulina

2016-06-30

The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. Opuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under

Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids.

Science.gov (United States)

Rosebrock, J A; Parker, C L; Kute, T E

1981-01-01

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

Spatial Heterodyne Observation of Water (SHOW) from a high altitude aircraft

Science.gov (United States)

Bourassa, A. E.; Langille, J.; Solheim, B.; Degenstein, D. A.; Letros, D.; Lloyd, N. D.; Loewen, P.

2017-12-01

The Spatial Heterodyne Observations of Water instrument (SHOW) is limb-sounding satellite prototype that is being developed in collaboration between the University of Saskatchewan, York University, the Canadian Space Agency and ABB. The SHOW instrument combines a field-widened SHS with an imaging system to observe limb-scattered sunlight in a vibrational band of water (1363 nm - 1366 nm). Currently, the instrument has been optimized for deployment on NASA's ER-2 aircraft. Flying at an altitude of 70, 000 ft the ER-2 configuration and SHOW viewing geometry provides high spatial resolution (limb-measurements of water vapor in the Upper troposphere and lower stratosphere region. During an observation campaign from July 15 - July 22, the SHOW instrument performed 10 hours of observations from the ER-2. This paper describes the SHOW measurement technique and presents the preliminary analysis and results from these flights. These observations are used to validate the SHOW measurement technique and demonstrate the sampling capabilities of the instrument.

Genetic and molecular analyses of Escherichia coli N-acetylneuraminate lyase gene.

OpenAIRE

Kawakami, B; Kudo, T; Narahashi, Y; Horikoshi, K

1986-01-01

Two plasmids containing the N-acetylneuraminate lyase (NALase) gene (nanA) of Escherichia coli, pNL1 and pNL4, were constructed. Immunoprecipitation analysis indicated that the 35,000-dalton protein encoded in pNL4 was NALase. The synthesis of NALase in E. coli carrying these plasmids was constitutive.

Protective and therapeutic effects of fucoxanthin against sunburn caused by UV irradiation

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Mio Matsui

2016-09-01

Full Text Available Mild exposure to ultraviolet (UV radiation is also harmful and hazardous to the skin and often causes a photosensitivity disorder accompanied by sunburn. To understand the action of UV on the skin we performed a microarray analysis to isolate UV-sensitive genes. We show here that UV irradiation promoted sunburn and downregulated filaggrin (Flg; fucoxanthin (FX exerted a protective effect. In vitro analysis showed that UV irradiation of human dermal fibroblasts caused production of intracellular reactive oxygen species (ROS without cellular toxicity. ROS production was diminished by N-acetylcysteine (NAC or FX, but not by retinoic acid (RA. In vivo analysis showed that UV irradiation caused sunburn and Flg downregulation, and that FX, but not NAC, RA or clobetasol, exerted a protective effect. FX stimulated Flg promoter activity in a concentration-dependent manner. Flg promoter deletion and chromatin immunoprecipitation analysis showed that caudal type homeo box transcription factor 1 (Cdx1 was a key factor for Flg induction. Cdx1 was also downregulated in UV-exposed skin. Therefore, our data suggested that the protective effects of FX against UV-induced sunburn might be exerted by promotion of skin barrier formation through induction of Flg, unrelated to quenching of ROS or an RA-like action.

Breast Cancer-Derived Lung Metastases Show Increased Pyruvate Carboxylase-Dependent Anaplerosis

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Stefan Christen

2016-10-01

Full Text Available Cellular proliferation depends on refilling the tricarboxylic acid (TCA cycle to support biomass production (anaplerosis. The two major anaplerotic pathways in cells are pyruvate conversion to oxaloacetate via pyruvate carboxylase (PC and glutamine conversion to α-ketoglutarate. Cancers often show an organ-specific reliance on either pathway. However, it remains unknown whether they adapt their mode of anaplerosis when metastasizing to a distant organ. We measured PC-dependent anaplerosis in breast-cancer-derived lung metastases compared to their primary cancers using in vivo 13C tracer analysis. We discovered that lung metastases have higher PC-dependent anaplerosis compared to primary breast cancers. Based on in vitro analysis and a mathematical model for the determination of compartment-specific metabolite concentrations, we found that mitochondrial pyruvate concentrations can promote PC-dependent anaplerosis via enzyme kinetics. In conclusion, we show that breast cancer cells proliferating as lung metastases activate PC-dependent anaplerosis in response to the lung microenvironment.

Novel primary thymic defect with T lymphocytes expressing gamma delta T cell receptor

DEFF Research Database (Denmark)

Geisler, C; Pallesen, G; Platz, P

1989-01-01

Flow cytometric analysis of the peripheral blood mononuclear cells in a six year old girl with a primary cellular immune deficiency showed a normal fraction of CD3 positive T cells. Most (70%) of the CD3 positive cells, however, expressed the gamma delta and not the alpha beta T cell receptor....... Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that most of the gamma delta T cell receptors existed as disulphide-linked heterodimers. Proliferative responses to mitogens were severely reduced, but specific antibody responses after vaccination could be detected...... deficiency associated with a high proportion of T cells expressing the gamma delta T cell receptor has been described in nude mice, and it is suggested that the immune deficiency of this patient may represent a human analogue....

CPR in medical TV shows: non-health care student perspective.

Science.gov (United States)

Alismail, Abdullah; Meyer, Nicole C; Almutairi, Waleed; Daher, Noha S

2018-01-01

There are over a dozen medical shows airing on television, many of which are during prime time. Researchers have recently become more interested in the role of these shows, and the awareness on cardiopulmonary resuscitation. Several cases have been reported where a lay person resuscitated a family member using medical TV shows as a reference. The purpose of this study is to examine and evaluate college students' perception on cardiopulmonary resuscitation and when to shock using an automated external defibrillator based on their experience of watching medical TV shows. A total of 170 students (nonmedical major) were surveyed in four different colleges in the United States. The survey consisted of questions that reflect their perception and knowledge acquired from watching medical TV shows. A stepwise regression was used to determine the significant predictors of "How often do you watch medical drama TV shows" in addition to chi-square analysis for nominal variables. Regression model showed significant effect that TV shows did change students' perception positively ( p <0.001), and they would select shock on asystole as the frequency of watching increases ( p =0.023). The findings of this study show that high percentage of nonmedical college students are influenced significantly by medical shows. One particular influence is the false belief about when a shock using the automated external defibrillator (AED) is appropriate as it is portrayed falsely in most medical shows. This finding raises a concern about how these shows portray basic life support, especially when not following American Heart Association (AHA) guidelines. We recommend the medical advisors in these shows to use AHA guidelines and AHA to expand its expenditures to include medical shows to educate the public on the appropriate action to rescue an out-of-hospital cardiac arrest patient.

Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

Directory of Open Access Journals (Sweden)

Rick S Mitchell

2004-08-01

Full Text Available The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV, avian sarcoma-leukosis virus (ASLV, and murine leukemia virus (MLV. Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

Show Horse Welfare: Horse Show Competitors' Understanding, Awareness, and Perceptions of Equine Welfare.

Science.gov (United States)

Voigt, Melissa A; Hiney, Kristina; Richardson, Jennifer C; Waite, Karen; Borron, Abigail; Brady, Colleen M

2016-01-01

The purpose of this study was to gain a better understanding of stock-type horse show competitors' understanding of welfare and level of concern for stock-type show horses' welfare. Data were collected through an online questionnaire that included questions relating to (a) interest and general understanding of horse welfare, (b) welfare concerns of the horse show industry and specifically the stock-type horse show industry, (c) decision-making influences, and (d) level of empathic characteristics. The majority of respondents indicated they agree or strongly agree that physical metrics should be a factor when assessing horse welfare, while fewer agreed that behavioral and mental metrics should be a factor. Respondent empathy levels were moderate to high and were positively correlated with the belief that mental and behavioral metrics should be a factor in assessing horse welfare. Respondents indicated the inhumane practices that most often occur at stock-type shows include excessive jerking on reins, excessive spurring, and induced excessive unnatural movement. Additionally, respondents indicated association rules, hired trainers, and hired riding instructors are the most influential regarding the decisions they make related to their horses' care and treatment.

CASSys: an integrated software-system for the interactive analysis of ChIP-seq data

Directory of Open Access Journals (Sweden)

Alawi Malik

2011-06-01

Full Text Available The mapping of DNA-protein interactions is crucial for a full understanding of transcriptional regulation. Chromatin-immunoprecipitation followed bymassively parallel sequencing (ChIP-seq has become the standard technique for analyzing these interactions on a genome-wide scale. We have developed a software system called CASSys (ChIP-seq data Analysis Software System spanning all steps of ChIP-seq data analysis. It supersedes the laborious application of several single command line tools. CASSys provides functionality ranging from quality assessment and -control of short reads, over the mapping of reads against a reference genome (readmapping and the detection of enriched regions (peakdetection to various follow-up analyses. The latter are accessible via a state-of-the-art web interface and can be performed interactively by the user. The follow-up analyses allow for flexible user defined association of putative interaction sites with genes, visualization of their genomic context with an integrated genome browser, the detection of putative binding motifs, the identification of over-represented Gene Ontology-terms, pathway analysis and the visualization of interaction networks. The system is client-server based, accessible via a web browser and does not require any software installation on the client side. To demonstrate CASSys’s functionality we used the system for the complete data analysis of a publicly available Chip-seq study that investigated the role of the transcription factor estrogen receptor-α in breast cancer cells.

Gene network analysis shows immune-signaling and ERK1/2 as novel genetic markers for multiple addiction phenotypes: alcohol, smoking and opioid addiction.

Science.gov (United States)

Reyes-Gibby, Cielito C; Yuan, Christine; Wang, Jian; Yeung, Sai-Ching J; Shete, Sanjay

2015-06-05

Addictions to alcohol and tobacco, known risk factors for cancer, are complex heritable disorders. Addictive behaviors have a bidirectional relationship with pain. We hypothesize that the associations between alcohol, smoking, and opioid addiction observed in cancer patients have a genetic basis. Therefore, using bioinformatics tools, we explored the underlying genetic basis and identified new candidate genes and common biological pathways for smoking, alcohol, and opioid addiction. Literature search showed 56 genes associated with alcohol, smoking and opioid addiction. Using Core Analysis function in Ingenuity Pathway Analysis software, we found that ERK1/2 was strongly interconnected across all three addiction networks. Genes involved in immune signaling pathways were shown across all three networks. Connect function from IPA My Pathway toolbox showed that DRD2 is the gene common to both the list of genetic variations associated with all three addiction phenotypes and the components of the brain neuronal signaling network involved in substance addiction. The top canonical pathways associated with the 56 genes were: 1) calcium signaling, 2) GPCR signaling, 3) cAMP-mediated signaling, 4) GABA receptor signaling, and 5) G-alpha i signaling. Cancer patients are often prescribed opioids for cancer pain thus increasing their risk for opioid abuse and addiction. Our findings provide candidate genes and biological pathways underlying addiction phenotypes, which may be future targets for treatment of addiction. Further study of the variations of the candidate genes could allow physicians to make more informed decisions when treating cancer pain with opioid analgesics.

Location analysis for the estrogen receptor-α reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

Science.gov (United States)

Mason, Christopher E.; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M.; Kallen, Roland G.; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B.

2010-01-01

Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERα-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: ERE sequence. We demonstrate that ∼50% of all ERα-bound loci do not have a discernable ERE and show that most ERα-bound EREs are not perfect consensus EREs. Approximately one-third of all ERα-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERα-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERα binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers. PMID:20047966

Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements.

Science.gov (United States)

Mason, Christopher E; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M; Kallen, Roland G; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B

2010-04-01

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

Cytogenetically Unrelated Clones in Acute Myeloid Leukemia Showing Different Responses to Chemotherapy

Directory of Open Access Journals (Sweden)

Kohei Kasahara

2016-01-01

Full Text Available We report a case of acute myeloid leukemia (AML with two cytogenetically unrelated clones. The patient was a 45-year-old male who was diagnosed with acute monoblastic leukemia (AMoL. Initial G-band analysis showed 51,XY,+6,+8,inv(9(p12q13c,+11,+13,+19[12]/52,idem,+Y[8], but G-band analysis after induction therapy showed 45,XY,-7,inv(9(p12q13c[19]/46,XY,inv(9(p12q13c[1]. Retrospective FISH analysis revealed a cryptic monosomy 7 clone in the initial AML sample. The clone with multiple trisomies was eliminated after induction therapy and never recurred, but a clone with monosomy 7 was still detected in myelodysplastic marrow with a normal blast percentage. Both clones were successfully eliminated after related peripheral blood stem cell transplantation, but the patient died of relapsed AML with monosomy 7. We concluded that one clone was de novo AMoL with chromosome 6, 8, 11, 13, and 19 trisomy and that the other was acute myeloid leukemia with myelodysplasia-related changes　(AML-MRC with chromosome 7 monosomy showing different responses to chemotherapy. Simultaneous onset of cytogenetically unrelated hematological malignancies that each have a different disease status is a rare phenomenon but is important to diagnose for a correct understanding of the disease status and for establishing an appropriate treatment strategy.

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

Science.gov (United States)

Perez-Arnaiz, Patricia; Kaplan, Daniel L

2016-11-20

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

Long noncoding RNA TUG1 is a diagnostic factor in lung adenocarcinoma and suppresses apoptosis via epigenetic silencing of BAX.

Science.gov (United States)

Liu, Huan; Zhou, Guizhi; Fu, Xin; Cui, Haiyan; Pu, Guangrui; Xiao, Yao; Sun, Wei; Dong, Xinhua; Zhang, Libin; Cao, Sijia; Li, Guiqin; Wu, Xiaowei; Yang, Xu

2017-11-24

Lung cancer is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel diagnostic markers and therapeutic targets. Increasing evidences have indicated that long non-coding RNAs (lncRNAs) play an important role in initiation and progression of lung cancer. However, the role of lncRNA Taurine upregulated 1 (TUG1) in lung adenocarcinoma (LAD) progression is not well known. In this study, we determined the diagnostic value of TUG1 in LAD patients, and further uncovered the underlying functional mechanism. Our results showed that TUG1 was significantly upregulated in LAD cells and serum samples. Receiver operator characteristic (ROC) analysis suggested a relatively higher area under the curve (AUC) of TUG1 (0.756) contrast to cyfra21-1 (0.619). In addition, high TUG1 level was associated with enhanced tumor size, degree of differentiation, lymph node metastases, distant metastasis and TNM stage. Cell functional assays showed that knockdown of TUG1 suppressed LAD cell viability and promoted cell apoptosis. We then sought to reveal the underlying regulatory mechanism, and the pro-apoptotic protein BAX was then identified as the downstream target of TUG1. Gain and loss functional assays showed that inhibition of BAX reversed the induced apoptosis by TUG1 knockdown. Finally, RNA immunoprecipitation and Chromatin immunoprecipitation revealed that TUG1 suppressed BAX expression through physically interacting with EZH2. In conclusion, lncRNA TUG1 is a promising diagnostic marker for LAD patients and suppression of TUG1 levels could be a future direction to promote the prognosis of LAD patients.

Analysis of allergen immunotherapy studies shows increased clinical efficacy in highly symptomatic patients

DEFF Research Database (Denmark)

Howarth, P; Malling, Hans-Jørgen; Molimard, M

2011-01-01

them. Thus, clinical studies of AIT can neither establish baseline symptom levels nor limit the enrolment of patients to those with the most severe symptoms. Allergen immunotherapy treatment effects are therefore diluted by patients with low symptoms for a particular pollen season. The objective...... tertiles). The difference observed in the average score in each tertile in active vs placebo-treated patients was assessed. This allowed an estimation of the efficacy that could be achieved in patients from sites where symptoms were high during the pollen season. Results:  An increased treatment effect...... of this analysis was to assess the effect possible to achieve with AIT in the groups of patients presenting the most severe allergic symptoms. Methods:  Study centres were grouped into tertiles categorized according to symptom severity scores observed in the placebo patients in each centre (low, middle and high...

Identification and localization of a soluble antigen, Ag2, of 136 kDa from Plasmodium falciparum in vitro cultures

DEFF Research Database (Denmark)

Jakobsen, P H; Grellier, P; Theander, T G

1991-01-01

as a duplet with molecular masses of 136 and 120 kDa when tested by immunoblotting. Immunoprecipitation experiments on Triton X-100 extracted antigens from synchronized cultures showed that the antigen was synthesized in the schizont stage. Ag2 was located near the surface of schizonts in the parasitophorous...

Isolation and partial characterization of peripheral blood CD4+ T cell clones expressing γδT cell receptors

International Nuclear Information System (INIS)

Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki, Yoichiro.

1990-06-01

Rare T cell clones bearing both CD4 and T cell receptors (TCRγ and TCRδ) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCRδ1 antibodies. All the clones established were reactive with anti-TCRγδ1 antibody, whereas only about 20 % of the clones showed reactivity with anti-δTCS1 antibody. Unlike CD4 + T cells bearing TCRαβ, all the clones tested were lectin-dependent and showed CD3 antibody-redirected cytolytic activity. About 60 % exhibited natural killer cell-like activity. Immunoprecipitation analysis of TCRγδ showed that each clone expressed either a disulfide-linked or nondisulfide-linked heterodimer consisting of 37-44 kilodalton TCRγ and TCRδ chains. Southern blot analyses of TCRγ and TCRδ genes revealed some identical rearrangement patterns, suggesting the limited heterogeneity of CD4 + TCRγδ + T cells in peripheral blood. (author)

Social network analysis shows direct evidence for social transmission of tool use in wild chimpanzees.

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Catherine Hobaiter

2014-09-01

Full Text Available Social network analysis methods have made it possible to test whether novel behaviors in animals spread through individual or social learning. To date, however, social network analysis of wild populations has been limited to static models that cannot precisely reflect the dynamics of learning, for instance, the impact of multiple observations across time. Here, we present a novel dynamic version of network analysis that is capable of capturing temporal aspects of acquisition--that is, how successive observations by an individual influence its acquisition of the novel behavior. We apply this model to studying the spread of two novel tool-use variants, "moss-sponging" and "leaf-sponge re-use," in the Sonso chimpanzee community of Budongo Forest, Uganda. Chimpanzees are widely considered the most "cultural" of all animal species, with 39 behaviors suspected as socially acquired, most of them in the domain of tool-use. The cultural hypothesis is supported by experimental data from captive chimpanzees and a range of observational data. However, for wild groups, there is still no direct experimental evidence for social learning, nor has there been any direct observation of social diffusion of behavioral innovations. Here, we tested both a static and a dynamic network model and found strong evidence that diffusion patterns of moss-sponging, but not leaf-sponge re-use, were significantly better explained by social than individual learning. The most conservative estimate of social transmission accounted for 85% of observed events, with an estimated 15-fold increase in learning rate for each time a novice observed an informed individual moss-sponging. We conclude that group-specific behavioral variants in wild chimpanzees can be socially learned, adding to the evidence that this prerequisite for culture originated in a common ancestor of great apes and humans, long before the advent of modern humans.

Social network analysis shows direct evidence for social transmission of tool use in wild chimpanzees.

Science.gov (United States)

Hobaiter, Catherine; Poisot, Timothée; Zuberbühler, Klaus; Hoppitt, William; Gruber, Thibaud

2014-09-01

Social network analysis methods have made it possible to test whether novel behaviors in animals spread through individual or social learning. To date, however, social network analysis of wild populations has been limited to static models that cannot precisely reflect the dynamics of learning, for instance, the impact of multiple observations across time. Here, we present a novel dynamic version of network analysis that is capable of capturing temporal aspects of acquisition--that is, how successive observations by an individual influence its acquisition of the novel behavior. We apply this model to studying the spread of two novel tool-use variants, "moss-sponging" and "leaf-sponge re-use," in the Sonso chimpanzee community of Budongo Forest, Uganda. Chimpanzees are widely considered the most "cultural" of all animal species, with 39 behaviors suspected as socially acquired, most of them in the domain of tool-use. The cultural hypothesis is supported by experimental data from captive chimpanzees and a range of observational data. However, for wild groups, there is still no direct experimental evidence for social learning, nor has there been any direct observation of social diffusion of behavioral innovations. Here, we tested both a static and a dynamic network model and found strong evidence that diffusion patterns of moss-sponging, but not leaf-sponge re-use, were significantly better explained by social than individual learning. The most conservative estimate of social transmission accounted for 85% of observed events, with an estimated 15-fold increase in learning rate for each time a novice observed an informed individual moss-sponging. We conclude that group-specific behavioral variants in wild chimpanzees can be socially learned, adding to the evidence that this prerequisite for culture originated in a common ancestor of great apes and humans, long before the advent of modern humans.

The Not5 subunit of the ccr4-not complex connects transcription and translation.

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Zoltan Villanyi

2014-10-01

Full Text Available Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

A Content Analysis of How Sexual Behavior and Reproductive Health are Being Portrayed on Primetime Television Shows Being Watched by Teens and Young Adults.

Science.gov (United States)

Kinsler, Janni J; Glik, Deborah; de Castro Buffington, Sandra; Malan, Hannah; Nadjat-Haiem, Carsten; Wainwright, Nicole; Papp-Green, Melissa

2018-02-01

Television is a leading source of sexual education for teens and young adults, thus it is important to understand how sexual behavior and reproductive health are portrayed in popular primetime programming. This study is a media content analysis of the 19 top-rated scripted English-language primetime television shows aired between January 1, 2015 and May 31, 2015, and viewed by American youth audiences 12-24 years of age. The purpose of this study is to assess how sex/sexuality and reproductive health are being portrayed in a popular medium that reaches many adolescent and young adult audiences. Themes used for this analysis include youth pregnancy/parenting, mentoring/guidance of youth regarding sexual behavior, sex/sexuality, body image/identity, sexual violence/abuse/harassment, gender identity/sexual orientation, and reproductive health. Themes have been classified in one of the following six categories: visual cues, brief mentions, dialogue, minor storylines, major storylines, and multi-episode storylines. Our findings indicate that narratives providing educational information regarding the risks and consequences of sexual behavior were missing from the television shows we analyzed and that storylines promoting low risk sexual behavior were rare. Sexual violence and abuse, casual sex among adults, lack of contraception use, or no portrayal of consequences of risky behaviors were common. Compared to prior research, we found an emergent theme normalizing non-heterosexual gender identity and sexual orientation. Our findings have important implications as exposure to popular media shapes the perceptions and behaviors of teens and young adults. This study has the potential to shed light on the need to create stories and narratives in television shows watched by American teens and young adults with educational messages regarding the risks and consequences of sexual behavior.

Chromatin Immunoprecipitation (ChIP): Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR

Science.gov (United States)

Schoppee Bortz, Pamela D.; Wamhoff, Brian R.

2011-01-01

The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. PMID:22046253

Expression analysis of genes associated with human osteosarcoma tumors shows correlation of RUNX2 overexpression with poor response to chemotherapy

International Nuclear Information System (INIS)

Sadikovic, Bekim; Thorner, Paul; Chilton-MacNeill, Susan; Martin, Jeff W; Cervigne, Nilva K; Squire, Jeremy; Zielenska, Maria

2010-01-01

Human osteosarcoma is the most common pediatric bone tumor. There is limited understanding of the molecular mechanisms underlying osteosarcoma oncogenesis, and a lack of good diagnostic as well as prognostic clinical markers for this disease. Recent discoveries have highlighted a potential role of a number of genes including: RECQL4, DOCK5, SPP1, RUNX2, RB1, CDKN1A, P53, IBSP, LSAMP, MYC, TNFRSF1B, BMP2, HISTH2BE, FOS, CCNB1, and CDC5L. Our objective was to assess relative expression levels of these 16 genes as potential biomarkers of osteosarcoma oncogenesis and chemotherapy response in human tumors. We performed quantitative expression analysis in a panel of 22 human osteosarcoma tumors with differential response to chemotherapy, and 5 normal human osteoblasts. RECQL4, SPP1, RUNX2, and IBSP were significantly overexpressed, and DOCK5, CDKN1A, RB1, P53, and LSAMP showed significant loss of expression relative to normal osteoblasts. In addition to being overexpressed in osteosarcoma tumor samples relative to normal osteoblasts, RUNX2 was the only gene of the 16 to show significant overexpression in tumors that had a poor response to chemotherapy relative to good responders. These data underscore the loss of tumor suppressive pathways and activation of specific oncogenic mechanisms associated with osteosarcoma oncogenesis, while drawing attention to the role of RUNX2 expression as a potential biomarker of chemotherapy failure in osteosarcoma

Obstructing Androgen Receptor Activation in Prostate Cancer Cells Through Post-translational Modification by NEDD8

Science.gov (United States)

2012-11-01

FACS flow cytometer analysis . In addition, we will measure the steady state protein level of p53, p21, p27, and pRb. In the Jab1 silencing cell...affected by DHT treatment, and the endogenous AR level was not affected by Jab1 silencing. Interestingly, Western blot analysis of immunoprecipitated AR...Avantaggiati, and R. G. Pestell . 2003. Acetylation of androgen receptor enhances coactivator binding and promotes prostate cancer cell growth. Mol

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

Science.gov (United States)

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

Identification of Sumoylated Proteins in the Silkworm Bombyx mori

Science.gov (United States)

Tang, Xudong; Fu, Xuliang; Hao, Bifang; Zhu, Feng; Xiao, Shengyan; Xu, Li; Shen, Zhongyuan

2014-01-01

Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori. PMID:25470021

Analysis of genetic distance between Peruvian Alpaca (Vicugna Pacos showing two distinct fleece phenotypes, Suri and Huacaya, by means of microsatellite markers

Directory of Open Access Journals (Sweden)

Carlo Renieri

2011-10-01

Full Text Available Two coat phenotypes exist in Alpaca, Huacaya and Suri. The two coats show different fleece structure, textile characteristics and prices on the market. Although present scientific knowledge suggests a simple genetic model of inheritance, there is a tendency to manage and consider the two phenotypes as two different breeds. A 13 microsatellite panel was used in this study to assess genetic distance between Suri and Huacaya alpacas in a sample of non-related animals from two phenotypically pure flocks at the Illpa-Puno experimental station in Quimsachata, Peru. The animals are part of a germplasm established approximately 20 years ago and have been bred separately according to their coat type since then. Genetic variability parameters were also calculated. The data were statistically analyzed using the software Genalex 6.3, Phylip 3.69 and Fstat 2.9.3.2. The sample was tested for Hardy-Weinberg equilibrium (HWE and after strict Bonferroni correction only one locus (LCA37 showed deviation from equilibrium (Ploci associations showed significant disequilibrium. Observed heterozygosis (Ho= 0.766; SE=0.044, expected heterozygosis (He=0.769; SE=0.033, number of alleles (Na=9.667, SE=0.772 and Fixation index (F=0.004; SE=0.036 are comparable to data from previous studies. Measures of genetic distance were 0.06 for Nei’s and 0.03 for Cavalli-Sforza’s. The analysis of molecular variance reported no existing variance between populations. Considering the origin of the animals, their post domestication evolution and the reproductive practices in place, the results do not show genetic differentiation between the two populations for the studied loci.

Talk Show Science.

Science.gov (United States)

Moore, Mitzi Ruth

1992-01-01

Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

GSK3 beta forms a tetrameric complex with endogenous PS1-CTF/NTF and beta-catenin. Effects of the D257/D385A and FAD-linked mutations.

Science.gov (United States)

Tesco, G; Tanzi, R E

2000-01-01

We have previously shown that the endogenous C-terminal fragment of presenilin 1 co-immunoprecipitates with endogenous beta-catenin. Since PS1 has been suggested to be involved in beta-catenin stabilization, we further investigated whether GSK3 beta, responsible for beta-catenin phosphorylation and degradation, is part of the PS1/beta-catenin complex. In naïve H4 and CHO cells, PS1 co-immunoprecipitated with both endogenous beta-catenin and GSK3 beta. In addition, GSK3 beta endogenously binds to the PS1-CTF/NTF complex and beta-catenin in naïve CHO cells. GSK3 beta also co-immunoprecipitated with PS1 full length in CHO cell lines overexpressing PS1 wild type. Given that it has been recently shown that PS1 mutations of aspartate 257 or 385 result in prevention of PS1 endoproteolysis and inhibition of gamma-secretase activity, we also tested whether PS1 endoproteolysis is required for beta-catenin/GSK3 beta/PS1 binding and whether PS1 FAD-linked mutations affect GSK3 beta recruitment in the PS1/beta-catenin complex. GSK3 beta was detected in PS1 immunoprecipitates from H4 cell lines overexpressing PS1 wild type, delta E10, A286E, L246V and in CHO cell lines overexpressing aspartate or M146L mutations. The latter data show that the absence of PS1 endoproteolysis (D257A/D385A and delta E10) or the presence of PS1-FAD mutations does not interfere with beta-catenin/GSK3 beta/PS1 complex formation.

Chromatin Immunoprecipitation of Replication Factors Moving with the Replication Fork

OpenAIRE

Rapp, Jordan B.; Ansbach, Alison B.; Noguchi, Chiaki; Noguchi, Eishi

2009-01-01

Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization ...

Alcohol content in the 'Hyper-Reality' MTV show 'Geordie Shore'

OpenAIRE

Lowe, Eden; Britton, John; Cranwell, Jo

2018-01-01

Aim: To quantify the occurrence of alcohol content, including alcohol branding, in the popular primetime television UK Reality TV show 'Geordie Shore' Series 11. \\ud \\ud Methods: A 1-min interval coding content analysis of alcohol content in the entire DVD Series 11 of 'Geordie Shore' (10 episodes). Occurrence of alcohol use, implied use, other alcohol reference/paraphernalia or branding was recorded. \\ud \\ud Results: All categories of alcohol were present in all episodes. 'Any alcohol' conte...

Large-Scale No-Show Patterns and Distributions for Clinic Operational Research

Directory of Open Access Journals (Sweden)

Michael L. Davies

2016-02-01

Full Text Available Patient no-shows for scheduled primary care appointments are common. Unused appointment slots reduce patient quality of care, access to services and provider productivity while increasing loss to follow-up and medical costs. This paper describes patterns of no-show variation by patient age, gender, appointment age, and type of appointment request for six individual service lines in the United States Veterans Health Administration (VHA. This retrospective observational descriptive project examined 25,050,479 VHA appointments contained in individual-level records for eight years (FY07-FY14 for 555,183 patients. Multifactor analysis of variance (ANOVA was performed, with no-show rate as the dependent variable, and gender, age group, appointment age, new patient status, and service line as factors. The analyses revealed that males had higher no-show rates than females to age 65, at which point males and females exhibited similar rates. The average no-show rates decreased with age until 75–79, whereupon rates increased. As appointment age increased, males and new patients had increasing no-show rates. Younger patients are especially prone to no-show as appointment age increases. These findings provide novel information to healthcare practitioners and management scientists to more accurately characterize no-show and attendance rates and the impact of certain patient factors. Future general population data could determine whether findings from VHA data generalize to others.

Large-Scale No-Show Patterns and Distributions for Clinic Operational Research.

Science.gov (United States)

Davies, Michael L; Goffman, Rachel M; May, Jerrold H; Monte, Robert J; Rodriguez, Keri L; Tjader, Youxu C; Vargas, Dominic L

2016-02-16

Patient no-shows for scheduled primary care appointments are common. Unused appointment slots reduce patient quality of care, access to services and provider productivity while increasing loss to follow-up and medical costs. This paper describes patterns of no-show variation by patient age, gender, appointment age, and type of appointment request for six individual service lines in the United States Veterans Health Administration (VHA). This retrospective observational descriptive project examined 25,050,479 VHA appointments contained in individual-level records for eight years (FY07-FY14) for 555,183 patients. Multifactor analysis of variance (ANOVA) was performed, with no-show rate as the dependent variable, and gender, age group, appointment age, new patient status, and service line as factors. The analyses revealed that males had higher no-show rates than females to age 65, at which point males and females exhibited similar rates. The average no-show rates decreased with age until 75-79, whereupon rates increased. As appointment age increased, males and new patients had increasing no-show rates. Younger patients are especially prone to no-show as appointment age increases. These findings provide novel information to healthcare practitioners and management scientists to more accurately characterize no-show and attendance rates and the impact of certain patient factors. Future general population data could determine whether findings from VHA data generalize to others.

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

Science.gov (United States)

Barbieri, M Alejandro; Kong, Chen; Chen, Pin-I; Horazdovsky, Bruce F; Stahl, Philip D

2003-08-22

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

Production and characterization of a monoclonal antibody to H+ATPase

International Nuclear Information System (INIS)

Yurko, M.; Fitch, F; Gluck, S.

1986-01-01

Acidification of endocytic vesicles is carried out by an ATP-dependent proton pump, H+ATPase, an FOF1 type enzyme comprised of at least 5 major subunits of 70, 56, 45, 35, and 17 kDa. A monoclonal antibody, H6.1, to H+ATPase from bovine kidney medulla, was raised to enable the structural characterization and localization of the pump. Several criteria were used to show that H6.1 recognized H+ATPase. 1.) H6.1 immunoprecipitated N-ethylmaleimide-sensitive and vanadate- and azide-insensitive solubilized ATPase activity (and GTPase activity) from both crude and purified enzyme preparations. 2.) H6.1 immunoprecipitated oligomycin-insensitive ATP-dependent proton transporting vesicles made from bovine kidney medulla, rat kidney, and CHO cells. 3.) H6.1 specifically immuno-precipitated the 5 subunits of H+ATPase from a partially purified preparation of the enzyme that had been labelled with I-125. H6.1 was then used as an immunocytochemical probe for the localization of H+ATPase. In bovine kidney medullary collecting duct, there was an intense apical staining of selected cells. In proximal tubule and in cultured CHO cells there was a granular pattern of staining characteristic of endocytic vesicles and lysosomes, suggesting that the kidney and CHO cell proton pumps are structurally related

Obesity in show cats.

Science.gov (United States)

Corbee, R J

2014-12-01

Obesity is an important disease with a high prevalence in cats. Because obesity is related to several other diseases, it is important to identify the population at risk. Several risk factors for obesity have been described in the literature. A higher incidence of obesity in certain cat breeds has been suggested. The aim of this study was to determine whether obesity occurs more often in certain breeds. The second aim was to relate the increased prevalence of obesity in certain breeds to the official standards of that breed. To this end, 268 cats of 22 different breeds investigated by determining their body condition score (BCS) on a nine-point scale by inspection and palpation, at two different cat shows. Overall, 45.5% of the show cats had a BCS > 5, and 4.5% of the show cats had a BCS > 7. There were significant differences between breeds, which could be related to the breed standards. Most overweight and obese cats were in the neutered group. It warrants firm discussions with breeders and cat show judges to come to different interpretations of the standards in order to prevent overweight conditions in certain breeds from being the standard of beauty. Neutering predisposes for obesity and requires early nutritional intervention to prevent obese conditions. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

DEFF Research Database (Denmark)

Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

2010-01-01

Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

A new scintillation proximity assay-based approach for the detection of KRAS mutations

Energy Technology Data Exchange (ETDEWEB)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

2016-04-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

A new scintillation proximity assay-based approach for the detection of KRAS mutations

International Nuclear Information System (INIS)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee

2016-01-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125 I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

Molecular tools for carotenogenesis analysis in the zygomycete Mucor circinelloides.

Science.gov (United States)

Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M; Garre, Victoriano; López-García, Sergio; Navarro, Eusebio; Vila, Ana

2012-01-01

The carotene producer fungus Mucor circinelloides is the zygomycete more amenable to genetic manipulations by using molecular tools. Since the initial development of an effective procedure of genetic transformation, more than two decades ago, the availability of new molecular approaches such as gene replacement techniques and gene expression inactivation by RNA silencing, in addition to the sequencing of its genome, has made Mucor a valuable organism for the study of a number of processes. Here we describe in detail the main techniques and methods currently used to manipulate M. circinelloides, including transformation, gene replacement, gene silencing, RNAi, and immunoprecipitation.

Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscape alteration in bovine cells

Science.gov (United States)

Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology, we analyzed histone modification (acetylation) induced by butyrate and the large-scale mapping of the epigenomic landscape of normal histone H3 and acetylated histone H3K9 and H3K27. To d...

Investigating CSI: portrayals of DNA testing on a forensic crime show and their potential effects.

Science.gov (United States)

Ley, Barbara L; Jankowski, Natalie; Brewer, Paul R

2012-01-01

The popularity of forensic crime shows such as CSI has fueled debate about their potential social impact. This study considers CSI's potential effects on public understandings regarding DNA testing in the context of judicial processes, the policy debates surrounding crime laboratory procedures, and the forensic science profession, as well as an effect not discussed in previous accounts: namely, the show's potential impact on public understandings of DNA and genetics more generally. To develop a theoretical foundation for research on the "CSI effect," it draws on cultivation theory, social cognitive theory, and audience reception studies. It then uses content analysis and textual analysis to illuminate how the show depicts DNA testing. The results demonstrate that CSI tends to depict DNA testing as routine, swift, useful, and reliable and that it echoes broader discourses about genetics. At times, however, the show suggests more complex ways of thinking about DNA testing and genetics.

Quantitative phenotyping via deep barcode sequencing.

Science.gov (United States)

Smith, Andrew M; Heisler, Lawrence E; Mellor, Joseph; Kaper, Fiona; Thompson, Michael J; Chee, Mark; Roth, Frederick P; Giaever, Guri; Nislow, Corey

2009-10-01

Next-generation DNA sequencing technologies have revolutionized diverse genomics applications, including de novo genome sequencing, SNP detection, chromatin immunoprecipitation, and transcriptome analysis. Here we apply deep sequencing to genome-scale fitness profiling to evaluate yeast strain collections in parallel. This method, Barcode analysis by Sequencing, or "Bar-seq," outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex chemogenomic assay, Bar-seq quantitatively identifies drug targets, with performance superior to the benchmark microarray assay. We also show that Bar-seq is well-suited for a multiplex format. We completely re-sequenced and re-annotated the yeast deletion collection using deep sequencing, found that approximately 20% of the barcodes and common priming sequences varied from expectation, and used this revised list of barcode sequences to improve data quality. Together, this new assay and analysis routine provide a deep-sequencing-based toolkit for identifying gene-environment interactions on a genome-wide scale.

Talking with TV shows

DEFF Research Database (Denmark)

Sandvik, Kjetil; Laursen, Ditte

2014-01-01

User interaction with radio and television programmes is not a new thing. However, with new cross-media production concepts such as X Factor and Voice, this is changing dramatically. The second-screen logic of these productions encourages viewers, along with TV’s traditional one-way communication...... mode, to communicate on interactive (dialogue-enabling) devices such as laptops, smartphones and tablets. Using the TV show Voice as our example, this article shows how the technological and situational set-up of the production invites viewers to engage in new ways of interaction and communication...

Protective and therapeutic effects of fucoxanthin against sunburn caused by UV irradiation.

Science.gov (United States)

Matsui, Mio; Tanaka, Kosuke; Higashiguchi, Naoki; Okawa, Hisato; Yamada, Yoichi; Tanaka, Ken; Taira, Soichiro; Aoyama, Tomoko; Takanishi, Misaki; Natsume, Chika; Takakura, Yuuki; Fujita, Norihisa; Hashimoto, Takeshi; Fujita, Takashi

2016-09-01

Mild exposure to ultraviolet (UV) radiation is also harmful and hazardous to the skin and often causes a photosensitivity disorder accompanied by sunburn. To understand the action of UV on the skin we performed a microarray analysis to isolate UV-sensitive genes. We show here that UV irradiation promoted sunburn and downregulated filaggrin (Flg); fucoxanthin (FX) exerted a protective effect. In vitro analysis showed that UV irradiation of human dermal fibroblasts caused production of intracellular reactive oxygen species (ROS) without cellular toxicity. ROS production was diminished by N-acetylcysteine (NAC) or FX, but not by retinoic acid (RA). In vivo analysis showed that UV irradiation caused sunburn and Flg downregulation, and that FX, but not NAC, RA or clobetasol, exerted a protective effect. FX stimulated Flg promoter activity in a concentration-dependent manner. Flg promoter deletion and chromatin immunoprecipitation analysis showed that caudal type homeo box transcription factor 1 (Cdx1) was a key factor for Flg induction. Cdx1 was also downregulated in UV-exposed skin. Therefore, our data suggested that the protective effects of FX against UV-induced sunburn might be exerted by promotion of skin barrier formation through induction of Flg, unrelated to quenching of ROS or an RA-like action. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Bovine papillomavirus type 2 (BPV-2) E5 oncoprotein binds to the subunit D of the V₁-ATPase proton pump in naturally occurring urothelial tumors of the urinary bladder of cattle.

Science.gov (United States)

Roperto, Sante; Russo, Valeria; Borzacchiello, Giuseppe; Urraro, Chiara; Lucà, Roberta; Esposito, Iolanda; Riccardi, Marita Georgia; Raso, Cinzia; Gaspari, Marco; Ceccarelli, Dora Maria; Galasso, Rocco; Roperto, Franco

2014-01-01

Active infection by bovine papillomavirus type 2 (BPV-2) was documented for fifteen urinary bladder tumors in cattle. Two were diagnosed as papillary urothelial neoplasm of low malignant potential (PUNLMP), nine as papillary and four as invasive urothelial cancers. In all cancer samples, PCR analysis revealed a BPV-2-specific 503 bp DNA fragment. E5 protein, the major oncoprotein of the virus, was shown both by immunoprecipitation and immunohistochemical analysis. E5 was found to bind to the activated (phosphorylated) form of the platelet derived growth factor β receptor. PDGFβR immunoprecipitation from bladder tumor samples and from normal bladder tissue used as control revealed a protein band which was present in the pull-down from bladder cancer samples only. The protein was identified with mass spectrometry as "V₁-ATPase subunit D", a component of the central stalk of the V₁-ATPase vacuolar pump. The subunit D was confirmed in this complex by coimmunoprecipitation investigations and it was found to colocalize with the receptor. The subunit D was also shown to be overexpressed by Western blot, RT-PCR and immunofluorescence analyses. Immunoprecipitation and immunofluorescence also revealed that E5 oncoprotein was bound to the subunit D. For the first time, a tri-component complex composed of E5/PDGFβR/subunit D has been documented in vivo. Previous in vitro studies have shown that the BPV-2 E5 oncoprotein binds to the proteolipid c ring of the V₀-ATPase sector. We suggest that the E5/PDGFβR/subunit D complex may perturb proteostasis, organelle and cytosol homeostasis, which can result in altered protein degradation and in autophagic responses.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

Directory of Open Access Journals (Sweden)

Cordula Klockenbusch

2010-01-01

Full Text Available Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

Science.gov (United States)

Klockenbusch, Cordula; Kast, Juergen

2010-01-01

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879

Pharmacological and immunochemical characterization of α2* nicotinic acetylcholine receptors (nAChRs) in mouse brain

Science.gov (United States)

Whiteaker, Paul; Wilking, Jennifer A; Brown, Robert WB; Brennan, Robert J; Collins, Allan C; Lindstrom, Jon M; Boulter, Jim

2009-01-01

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of α2* nAChRs in these mouse brain regions. Methods: α2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions. Results: [125I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8±1.7 and 92±26 fmol̇mg-1 protein, respectively). Pharmacological characterization using the β2-selective drug A85380 to inhibit [125I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs. Protocols were established to immunoprecipitate β2 and β4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits. PMID:19498420

Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB.

OpenAIRE

Forsberg, K; Valyi-Nagy, I; Heldin, C H; Herlyn, M; Westermark, B

1993-01-01

Human WM9 melanoma cells, previously shown to be devoid of PDGF expression, were stably transfected with a PDGF-B cDNA under the transcriptional control of a cytomegalovirus promoter. Northern blot analysis revealed high expression of an mRNA of the expected size in the PDGF-B-transfected cells. Synthesis and secretion of PDGF-BB was confirmed by immunoprecipitation. Furthermore, conditioned medium from PDGF-B-transfected cells contained a mitogenic activity for fibroblasts. For analysis of t...

Genome-wide Analysis of RARβ Transcriptional Targets in Mouse Striatum Links Retinoic Acid Signaling with Huntington's Disease and Other Neurodegenerative Disorders.

Science.gov (United States)

Niewiadomska-Cimicka, Anna; Krzyżosiak, Agnieszka; Ye, Tao; Podleśny-Drabiniok, Anna; Dembélé, Doulaye; Dollé, Pascal; Krężel, Wojciech

2017-07-01

Retinoic acid (RA) signaling through retinoic acid receptors (RARs), known for its multiple developmental functions, emerged more recently as an important regulator of adult brain physiology. How RAR-mediated regulation is achieved is poorly known, partly due to the paucity of information on critical target genes in the brain. Also, it is not clear how reduced RA signaling may contribute to pathophysiology of diverse neuropsychiatric disorders. We report the first genome-wide analysis of RAR transcriptional targets in the brain. Using chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis of RARβ-null mutant mice, we identified genomic targets of RARβ in the striatum. Characterization of RARβ transcriptional targets in the mouse striatum points to mechanisms through which RAR may control brain functions and display neuroprotective activity. Namely, our data indicate with statistical significance (FDR 0.1) a strong contribution of RARβ in controlling neurotransmission, energy metabolism, and transcription, with a particular involvement of G-protein coupled receptor (p = 5.0e -5 ), cAMP (p = 4.5e -4 ), and calcium signaling (p = 3.4e -3 ). Many identified RARβ target genes related to these pathways have been implicated in Alzheimer's, Parkinson's, and Huntington's disease (HD), raising the possibility that compromised RA signaling in the striatum may be a mechanistic link explaining the similar affective and cognitive symptoms in these diseases. The RARβ transcriptional targets were particularly enriched for transcripts affected in HD. Using the R6/2 transgenic mouse model of HD, we show that partial sequestration of RARβ in huntingtin protein aggregates may account for reduced RA signaling reported in HD.

Fungal communities in wheat grain show significant co-existence patterns among species

DEFF Research Database (Denmark)

Nicolaisen, M.; Justesen, A. F.; Knorr, K.

2014-01-01

identified as ‘core’ OTUs as they were found in all or almost all samples and accounted for almost 99 % of all sequences. The remaining OTUs were only sporadically found and only in small amounts. Cluster and factor analyses showed patterns of co-existence among the core species. Cluster analysis grouped...... the 21 core OTUs into three clusters: cluster 1 consisting of saprotrophs, cluster 2 consisting mainly of yeasts and saprotrophs and cluster 3 consisting of wheat pathogens. Principal component extraction showed that the Fusarium graminearum group was inversely related to OTUs of clusters 1 and 2....

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

DEFF Research Database (Denmark)

Svensson, Charlotte; Ceder, Jens; Iglesias Gato, Diego

2014-01-01

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds...... in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently...... of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis....

Measuring performance at trade shows

DEFF Research Database (Denmark)

Hansen, Kåre

2004-01-01

Trade shows is an increasingly important marketing activity to many companies, but current measures of trade show performance do not adequately capture dimensions important to exhibitors. Based on the marketing literature's outcome and behavior-based control system taxonomy, a model is built...... that captures a outcome-based sales dimension and four behavior-based dimensions (i.e. information-gathering, relationship building, image building, and motivation activities). A 16-item instrument is developed for assessing exhibitors perceptions of their trade show performance. The paper presents evidence...

P-Glycoprotein/MDR1 regulates pokemon gene transcription through p53 expression in human breast cancer cells.

Science.gov (United States)

He, Shengnan; Liu, Feng; Xie, Zhenhua; Zu, Xuyu; Xu, Wei; Jiang, Yuyang

2010-08-27

P-glycoprotein (Pgp), encoded by the multidrug resistance 1 (MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

Physically Active Men Show Better Semen Parameters than Their Sedentary Counterparts.

Science.gov (United States)

Lalinde-Acevedo, Paula C; Mayorga-Torres, B Jose Manuel; Agarwal, Ashok; du Plessis, Stefan S; Ahmad, Gulfam; Cadavid, Ángela P; Cardona Maya, Walter D

2017-10-01

The quality of semen depends upon several factors such as environment, life style, physical activity, age, and occupation. The aim of this study was to analyze and compare the conventional and functional semen parameters in men practicing vigorous physical activity to those of sedentary men. In this descriptive cross-sectional study, semen samples of 17 physically active men and 15 sedentary men were collected for analysis. Semen analysis was performed according to the World Health Organization (WHO) guidelines, while functional parameters were evaluated by flow cytometry. Results showed that several semen parameters (semen volume, viability, progressive motility, total motility, normal morphology, and moribund cells) were superior in the physically active group in comparison with the sedentary group. Semen parameters such as viability, progressive motility and total motility, as well as the percentage of moribund spermatozoa were significantly different between both groups. However, sperm DNA damage, lipid peroxidation and mitochondrial potential were not significantly different among the groups. Nevertheless, the physical activity shows better semen parameters than sedentary group. Taken together, our results demonstrate that regular physical activity has beneficial impact in sperm fertility parameters and such a life style can enhance the fertility status of men. Copyright© by Royan Institute. All rights reserved.

The energy show

International Nuclear Information System (INIS)

1988-01-01

The Energy Show is a new look at the problems of world energy, where our supplies come from, now and in the future. The programme looks at how we need energy to maintain our standards of living. Energy supply is shown as the complicated set of problems it is - that Fossil Fuels are both raw materials and energy sources, that some 'alternatives' so readily suggested as practical options are in reality a long way from being effective. (author)

Comprehensive analysis of LANA interacting proteins essential for viral genome tethering and persistence.

Directory of Open Access Journals (Sweden)

Subhash C Verma

Full Text Available Kaposi's sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi's sarcoma (KS, Primary Effusion Lymphoma (PEL and Multicentric Castleman's Disease (MCD. KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA. LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs. We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET to demonstrate their association.

Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

LENUS (Irish Health Repository)

Murphy, Derek M

2009-01-01

BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified\\/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the

Suffering by comparison: Twitter users' reactions to the Victoria's Secret Fashion Show.

Science.gov (United States)

Chrisler, Joan C; Fung, Kaitlin T; Lopez, Alexandra M; Gorman, Jennifer A

2013-09-01

Social comparison theory suggests that evaluating the self in comparison with others (e.g., peers, celebrities, models) can influence body image. Experimental studies that have tested effects of viewing idealized images in the media often show that women feel worse about themselves after seeing images that illustrate the beauty ideal. Twitter presents a naturally occurring opportunity to study viewers' reactions. An analysis was conducted of 977 tweets sent immediately before and during the 2011 Victoria's Secret Fashion Show that reference the show. Although the majority were idiosyncratic remarks, many tweets contain evidence of upward social comparisons to the fashion models. There were tweets about body image, eating disorders, weight, desires for food or alcohol, and thoughts about self-harm. The results support social comparison theory, and suggest that vulnerable viewers could experience negative affect, or even engage in harmful behaviors, during or after viewing the show or others like it. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metachronous metastasis- and survival-analysis show prognostic importance of lymphadenectomy for colon carcinomas

Directory of Open Access Journals (Sweden)

Laubert Tilman

2012-03-01

Full Text Available Abstract Background Lymphadenectomy is performed to assess patient prognosis and to prevent metastasizing. Recently, it was questioned whether lymph node metastases were capable of metastasizing and therefore, if lymphadenectomy was still adequate. We evaluated whether the nodal status impacts on the occurrence of distant metastases by analyzing a highly selected cohort of colon cancer patients. Methods 1,395 patients underwent surgery exclusively for colon cancer at the University of Lübeck between 01/1993 and 12/2008. The following exclusion criteria were applied: synchronous metastasis, R1-resection, prior/synchronous second carcinoma, age Results Five-year survival rates for TM + and TM- were 21% and 73%, respectively (p Conclusions Besides a higher T-category, a positive N-stage independently implies a higher probability to develop distant metastases and correlates with poor survival. Our data thus show a prognostic relevance of lymphadenectomy which should therefore be retained until conclusive studies suggest the unimportance of lmyphadenectomy.

Risk Aversion in Game Shows

DEFF Research Database (Denmark)

Andersen, Steffen; Harrison, Glenn W.; Lau, Morten I.

2008-01-01

We review the use of behavior from television game shows to infer risk attitudes. These shows provide evidence when contestants are making decisions over very large stakes, and in a replicated, structured way. Inferences are generally confounded by the subjective assessment of skill in some games......, and the dynamic nature of the task in most games. We consider the game shows Card Sharks, Jeopardy!, Lingo, and finally Deal Or No Deal. We provide a detailed case study of the analyses of Deal Or No Deal, since it is suitable for inference about risk attitudes and has attracted considerable attention....

Glyceraldehyde-3-phosphate dehydrogenase from Chironomidae showed differential activity towards metals.

Science.gov (United States)

Chong, Isaac K W; Ho, Wing S

2013-09-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37°C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88% identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

Transcriptional activation of rat creatine kinase B by 17beta-estradiol in MCF-7 cells involves an estrogen responsive element and GC-rich sites.

Science.gov (United States)

Wang, F; Samudio, I; Safe, S

2001-01-01

The rat creatine kinase B (CKB) gene is induced by estrogen in the uterus, and constructs containing rat CKB gene promoter inserts are highly estrogen-responsive in cell culture. Analysis of the upstream -568 to -523 region of the promoter in HeLa cells has identified an imperfect palindromic estrogen response element (ERE) that is required for hormone inducibility. Analysis of the CKB gene promoter in MCF-7 breast cancer cells confirmed that pCKB7 (containing the -568 to -523 promoter insert) was estrogen-responsive in transient transfection studies. However, mutation and deletion analysis of this region of the promoter showed that two GC-rich sites and the concensus ERE were functional cis-elements that bound estrogen receptor alpha (ERalpha)/Sp1 and ERalpha proteins, respectively. The role of these elements was confirmed in gel mobility shift and chromatin immunoprecipitation assays and transfection studies in MDA-MB-231 and Schneider Drosophila SL-2 cells. These results show that transcriptional activation of CKB by estrogen is dependent, in part, on ERalpha/Sp1 action which is cell context-dependent. Copyright 2001 Wiley-Liss, Inc.

International trade shows: Structure, strategy and performance of exhibitors at individual booths vs. joint booths

DEFF Research Database (Denmark)

Hansen, Kåre

2000-01-01

This paper examines differences in exhibitors who participate at international trade shows at joint booths and those who participate at individual booths. The structure, strategy, and trade show performance of exhibitors at joint booths and those at individual booths are analysed. The analysis...... implications for exhibitors at interna-tional trade shows and export marketing programmes and other marketing programmes offering services to international trade show exhibitors....... of exhibitors at the international food shows SIAL (Paris) and ANUGA (Cologne) showed several significant differences with regard to structure and strategy. However, no significant differences in the performance assessments between the two partici-pation modes were found. The findings have important...

Economic impact and market analysis of a special event: The Great New England Air Show

Science.gov (United States)

Rodney B. Warnick; David C. Bojanic; Atul Sheel; Apurv Mather; Deepak Ninan

2010-01-01

We conducted a post-event evaluation for the Great New England Air Show to assess its general economic impact and to refine economic estimates where possible. In addition to the standard economic impact variables, we examined travel distance, purchase decision involvement, event satisfaction, and frequency of attendance. Graphic mapping of event visitors' home ZIP...

Proteomic analysis of protein interactions between Eimeria maxima sporozoites and chicken jejunal epithelial cells by shotgun LC-MS/MS.

Science.gov (United States)

Huang, Jingwei; Liu, Tingqi; Li, Ke; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-04-04

Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion.

Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

Science.gov (United States)

Nayak, Ajay P; Green, Brett J; Janotka, Erika; Hettick, Justin M; Friend, Sherri; Vesper, Steve J; Schmechel, Detlef; Beezhold, Donald H

2011-09-01

Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

TOX3 (TNRC9) overexpression in bladder cancer cells decreases cellular proliferation and triggers an interferon-like response

DEFF Research Database (Denmark)

Birkenkamp-Demtröder, Karin; Mansilla, Francisco; Andersen, Lars Dyrskjøt

2013-01-01

Background Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+-dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes...... urothelium. Microarray expression profiling of human bladder cancer cells overexpressing TOX3 followed by Pathway analysis showed that TOX3 overexpression mainly affected the Interferon Signaling Pathway. TOX3 upregulation induced the expression of several genes with a gamma interferon activation site (GAS......), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 overexpressing...

TOX3 (TNRC9) Over Expression in Bladder Cancer Cells Decreases Cellular Proliferation and Triggers an Interferon-Like Response

DEFF Research Database (Denmark)

Birkenkamp-Demtroder, Karin; Mansilla Castaño, Francisco; Dyrskjøt, Lars

2013-01-01

Background: Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+ dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes...... urothelium. Microarray expression profiling of human bladder cancer cells over expressing TOX3 followed by Pathway analysis showed that TOX3 Overexpression mainly affected the Interferon Signaling Pathway. TOX3 up regulation induced the expression of several genes with a gamma interferon activation site (GAS......), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 over...

A micro-epidemiological analysis of febrile malaria in Coastal Kenya showing hotspots within hotspots.

Science.gov (United States)

Bejon, Philip; Williams, Thomas N; Nyundo, Christopher; Hay, Simon I; Benz, David; Gething, Peter W; Otiende, Mark; Peshu, Judy; Bashraheil, Mahfudh; Greenhouse, Bryan; Bousema, Teun; Bauni, Evasius; Marsh, Kevin; Smith, David L; Borrmann, Steffen

2014-04-24

Malaria transmission is spatially heterogeneous. This reduces the efficacy of control strategies, but focusing control strategies on clusters or 'hotspots' of transmission may be highly effective. Among 1500 homesteads in coastal Kenya we calculated (a) the fraction of febrile children with positive malaria smears per homestead, and (b) the mean age of children with malaria per homestead. These two measures were inversely correlated, indicating that children in homesteads at higher transmission acquire immunity more rapidly. This inverse correlation increased gradually with increasing spatial scale of analysis, and hotspots of febrile malaria were identified at every scale. We found hotspots within hotspots, down to the level of an individual homestead. Febrile malaria hotspots were temporally unstable, but 4 km radius hotspots could be targeted for 1 month following 1 month periods of surveillance.DOI: http://dx.doi.org/10.7554/eLife.02130.001. Copyright © 2014, Bejon et al.

Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

Science.gov (United States)

Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

2018-06-01

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

A novel comparative pattern count analysis reveals a chronic ethanol-induced dynamic shift in immediate early NF-κB genome-wide promoter binding during liver regeneration.

Science.gov (United States)

Kuttippurathu, Lakshmi; Patra, Biswanath; Hoek, Jan B; Vadigepalli, Rajanikanth

2016-03-01

Liver regeneration after partial hepatectomy is a clinically important process that is impaired by adaptation to chronic alcohol intake. We focused on the initial time points following partial hepatectomy (PHx) to analyze the genome-wide binding activity of NF-κB, a key immediate early regulator. We investigated the effect of chronic alcohol intake on immediate early NF-κB genome-wide localization, in the adapted state as well as in response to partial hepatectomy, using chromatin immunoprecipitation followed by promoter microarray analysis. We found many ethanol-specific NF-κB binding target promoters in the ethanol-adapted state, corresponding to the regulation of biosynthetic processes, oxidation-reduction and apoptosis. Partial hepatectomy induced a diet-independent shift in NF-κB binding loci relative to the transcription start sites. We employed a novel pattern count analysis to exhaustively enumerate and compare the number of promoters corresponding to the temporal binding patterns in ethanol and pair-fed control groups. The highest pattern count corresponded to promoters with NF-κB binding exclusively in the ethanol group at 1 h post PHx. This set was associated with the regulation of cell death, response to oxidative stress, histone modification, mitochondrial function, and metabolic processes. Integration with the global gene expression profiles to identify putative transcriptional consequences of NF-κB binding patterns revealed that several of ethanol-specific 1 h binding targets showed ethanol-specific differential expression through 6 h post PHx. Motif analysis yielded co-incident binding loci for STAT3, AP-1, CREB, C/EBP-β, PPAR-γ and C/EBP-α, likely participating in co-regulatory modules with NF-κB in shaping the immediate early response to PHx. We conclude that adaptation to chronic ethanol intake disrupts the NF-κB promoter binding landscape with consequences for the immediate early gene regulatory response to the acute challenge of PHx.

Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

Directory of Open Access Journals (Sweden)

Wentao Wu

2017-10-01

Full Text Available The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens to 63 (Glycine max. The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals.

Science.gov (United States)

Wu, Wentao; Liu, Yaxue; Wang, Yuqian; Li, Huimin; Liu, Jiaxi; Tan, Jiaxin; He, Jiadai; Bai, Jingwen; Ma, Haoli

2017-10-08

The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three ( Physcomitrella patens ) to 63 ( Glycine max ). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

KIC 8164262: a heartbeat star showing tidally induced pulsations with resonant locking

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Hambleton, K.; Fuller, J.; Thompson, S.; Prša, A.; Kurtz, D. W.; Shporer, A.; Isaacson, H.; Howard, A. W.; Endl, M.; Cochran, W.; Murphy, S. J.

2018-02-01

We present the analysis of KIC 8164262, a heartbeat star with a high-amplitude (∼1 mmag), tidally resonant pulsation (a mode in resonance with the orbit) at 229 times the orbital frequency and a plethora of tidally induced g-mode pulsations (modes excited by the orbit). The analysis combines Kepler light curves with follow-up spectroscopic data from the Keck telescope, KPNO (Kitt Peak National Observatory) 4-m Mayall telescope and the 2.7-m telescope at the McDonald observatory. We apply the binary modelling software, PHOEBE, to the Kepler light curve and radial velocity data to determine a detailed binary star model that includes the prominent pulsation and Doppler boosting, alongside the usual attributes of a binary star model (including tidal distortion and reflection). The results show that the system contains a slightly evolved F star with an M secondary companion in a highly eccentric orbit (e = 0.886). We use the results of the binary star model in a companion paper (Fuller) where we show that the prominent pulsation can be explained by a tidally excited oscillation mode held near resonance by a resonance locking mechanism.

Grey('s) Identity: Complications of Learning and Becoming in a Popular Television Show

Science.gov (United States)

Jubas, Kaela

2013-01-01

In this article, the author outlines an analysis of the American show "Grey's Anatomy" as an example of how popular culture represents identity and the process of professional identity construction in a medical workplace, particularly the surgical service of a large urban hospital. In discussing identity, she connects professional identity to…

Characterization of phosphorylated isocitrate dehydrogenase and purification of the isocitrate dehydrogenase kinase/phosphatase of Escherichia coli

International Nuclear Information System (INIS)

Malloy, P.J.

1985-01-01

NADP + -specific isocitrate dehydrogenase (IDH; EC 1.1.1.42) was shown to be phosphorylated with ( 32 P)-orthophosphate in vivo in several strains of Escherichia coli. In strain KC 13, an adenylate cyclase deficient mutant, the specific activity of IDH decreased 70% when acetate was added to stationary phase cultures grown on glucose. The enzyme was immunoprecipitated from sonic extracts and shown to contain 32 P by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The results demonstrate that unlike many eukaryotic protein kinases, the protein kinase involved in the phosphorylation of IDH in E. coli does not require cyclic adenosine monophosphate for catalysis. Similarly, the phosphorylation of IDH was demonstrated in E. coli mutants deficient in either isocitrate lyase or malate synthase. The incorporation of 32 P in IDH was demonstrated following SDS-PAGE and autoradiography of the immunoprecipitated enzyme. These results suggest that the conditions required for the phosphorylation of IDH do not depend on the functioning of the glyoxylate shunt. Following in vivo 32 P-labeling of E. coli strain F143/KL259 in the presence of acetate, 32 P-labeled IDH was isolated from sonicated extracts of the cells. The 32 P-enzyme was carboxylmethylated and digested with trypsin. A single 32 P-labeled peptide was isolated from the tryptic digest. Amino acid analysis of the purified 32 P-labeled peptide showed that the peptide contains seven amino acids, including a single phosphorylated serine residue

Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

Science.gov (United States)

Choi, Won-Il; Kim, Youngsoo; Kim, Yuri; Yu, Mi-young; Park, Jungeun; Lee, Choong-Eun; Jeon, Bu-Nam; Koh, Dong-In; Hur, Man-Wook

2009-01-01

FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors. Copyright 2009 S. Karger AG, Basel.

Two memory associated genes regulated by amyloid precursor protein intracellular domain ovel insights into the pathogenesis of learning and memory impairment in Alzheimer's disease

Institute of Scientific and Technical Information of China (English)

Chuandong Zheng; Xi Gu; Zhimei Zhong; Rui Zhu; Tianming Gao; Fang Wang

2012-01-01

In this study, we employed chromatin immunoprecipitation, a useful method for studying the locations of transcription factors bound to specific DNA regions in specific cells, to investigate amyloid precursor protein intracellular domain binding sites in chromatin DNA from hippocampal neurons of rats, and to screen out five putative genes associated with the learning and memory functions. The promoter regions of the calcium/calmodulin-dependent protein kinase II alpha and glutamate receptor-2 genes were amplified by PCR from DNA products immunoprecipitated by amyloid precursor protein intracellular domain. An electrophoretic mobility shift assay and western blot analysis suggested that the promoter regions of these two genes associated with learning and memory were bound by amyloid precursor protein intracellular domain (in complex form). Our experimental findings indicate that the amyloid precursor protein intracellular domain is involved in the transcriptional regulation of learning- and memory-associated genes in hippocampal neurons. These data may provide new insights into the molecular mechanism underlying the symptoms of progressive memory loss in Alzheimer's disease.

Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurospora crassa

International Nuclear Information System (INIS)

Jackl, G.; Sebald, W.

1975-01-01

Soluble mitochondrial ATPase (F 1 ) isolated from Neurospora crassa is resolved by dodecyl-sulfate-gel electrophoresis into five polypeptide bands with apparent molecular weights of 59,000, 55,000, 36,000, 15,000 and 12,000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F 1 ATPase. Two of the associated polypeptides with apparent molecular weights of 19,000 and 11,000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex. (orig.) [de

Immunoassays in clinical chemistry (principles of immunoradiometric assays)

International Nuclear Information System (INIS)

Chapman, R.S.

1998-01-01

The use of antibodies as reagents in clinical chemistry for the quantitation of a wide range of analytes has now become widely established. Initially antibodies were employed in precipitation techniques, usually for the analysis of serum proteins, in solution or in the form of antibody containing gels, e.g. immunoprecipitation, immunodiffusion, and immunoelectrophoresis. Further developments have led to the highly sensitive techniques of radioimmunoassay and recently immunometric assay for the measurement of drugs, tumour markers and hormones. In general, those techniques without the addition of a label e.g. immunoprecipitation, immunodiffusion and immunoturbidimetry are the older techniques used for the measurement of serum proteins. These techniques are relatively insensitive, measuring at the g/L. level, and in the case of immunodiffusion are generally slow. Automation coupled with the development of chemistries to enhance precipitation has, however, reduced measurement times to minutes in modern laboratories. Nevertheless these methods have detection limits of the order of 1 g/L

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

Science.gov (United States)

Watanabe, Kazuhide; Biesinger, Jacob; Salmans, Michael L; Roberts, Brian S; Arthur, William T; Cleary, Michele; Andersen, Bogi; Xie, Xiaohui; Dai, Xing

2014-01-01

Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

Meta-analysis of grain yield QTL identified during agricultural drought in grasses showed consensus.

Science.gov (United States)

Swamy, B P Mallikarjuna; Vikram, Prashant; Dixit, Shalabh; Ahmed, H U; Kumar, Arvind

2011-06-16

In the last few years, efforts have been made to identify large effect QTL for grain yield under drought in rice. However, identification of most precise and consistent QTL across the environments and genetics backgrounds is essential for their successful use in Marker-assisted Selection. In this study, an attempt was made to locate consistent QTL regions associated with yield increase under drought by applying a genome-wide QTL meta-analysis approach. The integration of 15 maps resulted in a consensus map with 531 markers and a total map length of 1821 cM. Fifty-three yield QTL reported in 15 studies were projected on a consensus map and meta-analysis was performed. Fourteen meta-QTL were obtained on seven chromosomes. MQTL1.2, MQTL1.3, MQTL1.4, and MQTL12.1 were around 700 kb and corresponded to a reasonably small genetic distance of 1.8 to 5 cM and they are suitable for use in marker-assisted selection (MAS). The meta-QTL for grain yield under drought coincided with at least one of the meta-QTL identified for root and leaf morphology traits under drought in earlier reports. Validation of major-effect QTL on a panel of random drought-tolerant lines revealed the presence of at least one major QTL in each line. DTY12.1 was present in 85% of the lines, followed by DTY4.1 in 79% and DTY1.1 in 64% of the lines. Comparative genomics of meta-QTL with other cereals revealed that the homologous regions of MQTL1.4 and MQTL3.2 had QTL for grain yield under drought in maize, wheat, and barley respectively. The genes in the meta-QTL regions were analyzed by a comparative genomics approach and candidate genes were deduced for grain yield under drought. Three groups of genes such as stress-inducible genes, growth and development-related genes, and sugar transport-related genes were found in clusters in most of the meta-QTL. Meta-QTL with small genetic and physical intervals could be useful in Marker-assisted selection individually and in combinations. Validation and comparative

Analysis of nonlocal phonon thermal conductivity simulations showing the ballistic to diffusive crossover

Science.gov (United States)

Allen, Philip B.

2018-04-01

Simulations [e.g., X. W. Zhou et al., Phys. Rev. B 79, 115201 (2009), 10.1103/PhysRevB.79.115201] show nonlocal effects of the ballistic/diffusive crossover. The local temperature has nonlinear spatial variation not contained in the local Fourier law j ⃗(r ⃗) =-κ ∇ ⃗T (r ⃗) . The heat current j ⃗(r ⃗) depends not just on the local temperature gradient ∇ ⃗T (r ⃗) but also on temperatures at points r⃗' within phonon mean free paths, which can be micrometers long. This paper uses the Peierls-Boltzmann transport theory in nonlocal form to analyze the spatial variation Δ T (r ⃗) . The relaxation-time approximation (RTA) is used because the full solution is very challenging. Improved methods of extrapolation to obtain the bulk thermal conductivity κ are proposed. Callaway invented an approximate method of correcting RTA for the q ⃗ (phonon wave vector or crystal momentum) conservation of N (Normal as opposed to Umklapp) anharmonic collisions. This method is generalized to the nonlocal case where κ (k ⃗) depends on the wave vector of the current j ⃗(k ⃗) and temperature gradient i k ⃗Δ T (k ⃗) .

P-Glycoprotein/MDR1 Regulates Pokemon Gene Transcription Through p53 Expression in Human Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Wei Xu

2010-08-01

Full Text Available P-glycoprotein (Pgp, encoded by the multidrug resistance 1 (MDR1 gene, is an efflux transporter and plays an important role in pharmacokinetics. In this study, we demonstrated that the pokemon promoter activity, the pokemon mRNA and protein expression can be significantly inhibited by Pgp. Chromatin immunoprecipitation assay showed that Pgp can bind the pokemon prompter to repress pokemon transcription activity. Furthermore, Pgp regulated pokemon transcription activity through expression of p53 as seen by use of p53 siRNA transfected MCF-7 cells or p53 mutated MDA-MB-231 cells. Moreover, p53 was detected to bind with Pgp in vivo using immunoprecipitation assay. Taken together, we conclude that Pgp can regulate the expression of pokemon through the presence of p53, suggesting that Pgp is a potent regulator and may offer an effective novel target for cancer therapy.

Computational Methods for ChIP-seq Data Analysis and Applications

KAUST Repository

Ashoor, Haitham

2017-04-25

The development of Chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology has enabled the construction of genome-wide maps of protein-DNA interaction. Such maps provide information about transcriptional regulation at the epigenetic level (histone modifications and histone variants) and at the level of transcription factor (TF) activity. This dissertation presents novel computational methods for ChIP-seq data analysis and applications. The work of this dissertation addresses four main challenges. First, I address the problem of detecting histone modifications from ChIP-seq cancer samples. The presence of copy number variations (CNVs) in cancer samples results in statistical biases that lead to inaccurate predictions when standard methods are used. To overcome this issue I developed HMCan, a specially designed algorithm to handle ChIP-seq cancer data by accounting for the presence of CNVs. When using ChIP-seq data from cancer cells, HMCan demonstrates unbiased and accurate predictions compared to the standard state of the art methods. Second, I address the problem of identifying changes in histone modifications between two ChIP-seq samples with different genetic backgrounds (for example cancer vs. normal). In addition to CNVs, different antibody efficiency between samples and presence of samples replicates are challenges for this problem. To overcome these issues, I developed the HMCan-diff algorithm as an extension to HMCan. HMCan-diff implements robust normalization methods to address the challenges listed above. HMCan-diff significantly outperforms another state of the art methods on data containing cancer samples. Third, I investigate and analyze predictions of different methods for enhancer prediction based on ChIP-seq data. The analysis shows that predictions generated by different methods are poorly overlapping. To overcome this issue, I developed DENdb, a database that integrates enhancer predictions from different methods. DENdb also

Alcohol Content in the 'Hyper-Reality' MTV Show 'Geordie Shore'.

Science.gov (United States)

Lowe, Eden; Britton, John; Cranwell, Jo

2018-05-01

To quantify the occurrence of alcohol content, including alcohol branding, in the popular primetime television UK Reality TV show 'Geordie Shore' Series 11. A 1-min interval coding content analysis of alcohol content in the entire DVD Series 11 of 'Geordie Shore' (10 episodes). Occurrence of alcohol use, implied use, other alcohol reference/paraphernalia or branding was recorded. All categories of alcohol were present in all episodes. 'Any alcohol' content occurred in 78%, 'actual alcohol use' in 30%, 'inferred alcohol use' in 72%, and all 'other' alcohol references occurred in 59% of all coding intervals (ACIs), respectively. Brand appearances occurred in 23% of ACIs. The most frequently observed alcohol brand was Smirnoff which appeared in 43% of all brand appearances. Episodes categorized as suitable for viewing by adolescents below the legal drinking age of 18 years comprised of 61% of all brand appearances. Alcohol content, including branding, is highly prevalent in the UK Reality TV show 'Geordie Shore' Series 11. Two-thirds of all alcohol branding occurred in episodes age-rated by the British Board of Film Classification (BBFC) as suitable for viewers aged 15 years. The organizations OfCom, Advertising Standards Authority (ASA) and the Portman Group should implement more effective policies to reduce adolescent exposure to on-screen drinking. The drinks industry should consider demanding the withdrawal of their brands from the show. Alcohol content, including branding, is highly prevalent in the MTV reality TV show 'Geordie Shore' Series 11. Current alcohol regulation is failing to protect young viewers from exposure to such content.

Streptococcus pneumonia YlxR at 1.35 A shows a putative new fold.

Science.gov (United States)

Osipiuk, J; Górnicki, P; Maj, L; Dementieva, I; Laskowski, R; Joachimiak, A

2001-11-01

The structure of the YlxR protein of unknown function from Streptococcus pneumonia was determined to 1.35 A. YlxR is expressed from the nusA/infB operon in bacteria and belongs to a small protein family (COG2740) that shares a conserved sequence motif GRGA(Y/W). The family shows no significant amino-acid sequence similarity with other proteins. Three-wavelength diffraction MAD data were collected to 1.7 A from orthorhombic crystals using synchrotron radiation and the structure was determined using a semi-automated approach. The YlxR structure resembles a two-layer alpha/beta sandwich with the overall shape of a cylinder and shows no structural homology to proteins of known structure. Structural analysis revealed that the YlxR structure represents a new protein fold that belongs to the alpha-beta plait superfamily. The distribution of the electrostatic surface potential shows a large positively charged patch on one side of the protein, a feature often found in nucleic acid-binding proteins. Three sulfate ions bind to this positively charged surface. Analysis of potential binding sites uncovered several substantial clefts, with the largest spanning 3/4 of the protein. A similar distribution of binding sites and a large sharply bent cleft are observed in RNA-binding proteins that are unrelated in sequence and structure. It is proposed that YlxR is an RNA-binding protein.

Honored Teacher Shows Commitment.

Science.gov (United States)

Ratte, Kathy

1987-01-01

Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

Best in show but not best shape: a photographic assessment of show dog body condition.

Science.gov (United States)

Such, Z R; German, A J

2015-08-01

Previous studies suggest that owners often wrongly perceive overweight dogs to be in normal condition. The body shape of dogs attending shows might influence owners' perceptions, with online images of overweight show winners having a negative effect. This was an observational in silico study of canine body condition. 14 obese-prone breeds and 14 matched non-obese-probe breeds were first selected, and one operator then used an online search engine to identify 40 images, per breed, of dogs that had appeared at a major national UK show (Crufts). After images were anonymised and coded, a second observer subjectively assessed body condition, in a single sitting, using a previously validated method. Of 1120 photographs initially identified, 960 were suitable for assessing body condition, with all unsuitable images being from longhaired breeds. None of the dogs (0 per cent) were underweight, 708 (74 per cent) were in ideal condition and 252 (26 per cent) were overweight. Pugs, basset hounds and Labrador retrievers were most likely to be overweight, while standard poodles, Rhodesian ridgebacks, Hungarian vizslas and Dobermanns were least likely to be overweight. Given the proportion of show dogs from some breeds that are overweight, breed standards should be redefined to be consistent with a dog in optimal body condition. British Veterinary Association.

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

Science.gov (United States)

Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2017-03-01

The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

OpenAIRE

Demangeat, Gerard; Greif, Charles; Hemmer, O; Fritsch, C

1990-01-01

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is furthe...

Political Tv Talk Shows on Greek Television: Live Broadcasting as an ideological and Cognitive frame

Directory of Open Access Journals (Sweden)

Ioanna Vovou

2007-12-01

Full Text Available This paper presents a semiopragmatic analysis of the political talk shows on Greek television that reveals a symbolic conception of the TV studio and it’s value-attributing mechanisms through which principles of democratic dialogue are enacted inside this kind of social and media frame. Political talk shows create the notion of a complex temporality, larger than the concept of events presented live, afecting the political and social vision of the audience.

Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability

OpenAIRE

Fukawatase, Yoshihiro; Toyoda, Masashi; Okamura, Kohji; Nakamura, Ken-ichi; Nakabayashi, Kazuhiko; Takada, Shuji; Yamazaki-Inoue, Mayu; Masuda, Akira; Nasu, Michiyo; Hata, Kenichiro; Hanaoka, Kazunori; Higuchi, Akon; Takubo, Kaiyo; Umezawa, Akihiro

2014-01-01

Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a compa...

Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

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Hong Hao

Full Text Available A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons

DEFF Research Database (Denmark)

Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada

2016-01-01

Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol...... of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation...... of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K...

Effects of UVA irradiation, aryl azides, and reactive oxygen species on the orthogonal inactivation of the human immunodeficiency virus (HIV-1)

International Nuclear Information System (INIS)

Belanger, Julie M.; Raviv, Yossef; Viard, Mathias; Cruz, M. Jason de la; Nagashima, Kunio; Blumenthal, Robert

2011-01-01

Previously we reported that hydrophobic aryl azides partition into hydrophobic regions of the viral membrane of enveloped viruses and inactivate the virus upon UVA irradiation for 2 min. Prolonged irradiation (15 min) resulted in viral protein aggregation as visualized via Western blot analysis, due to reactive oxygen species (ROS) formation, with preservation of the surface antigenic epitopes. Herein, we demonstrate that these aggregates show detergent resistance and that this property may be useful towards the creation of a novel orthogonal virus inactivation strategy for use in preparing experimental vaccines. When ROS-modified HIV virus preparations were treated with 1% Triton X-100, there was an increase in the percent of viral proteins (gp41, p24) in the viral pellet after ultracentrifugation through sucrose. Transmission electron microscopy (TEM) of these detergent-resistant pellets shows some recognizable virus fragments, and immunoprecipitation studies of the gp41 aggregates suggest the aggregation is covalent in nature, involving short-range interactions.

The Lateral Occipital Complex shows no net response to object familiarity.

Science.gov (United States)

Margalit, Eshed; Shah, Manan P; Tjan, Bosco S; Biederman, Irving; Keller, Brenton; Brenner, Rorry

2016-09-01

In 1995, Malach et al. discovered an area whose fMRI BOLD response was greater when viewing intact, familiar objects than when viewing their scrambled versions (resembling texture). Since then hundreds of studies have explored this late visual region termed the Lateral Occipital Complex (LOC), which is now known to be critical for shape perception (James, Culham, Humphrey, Milner, & Goodale, 2003). Malach et al. (1995) discounted a role of familiarity by showing that "abstract" Henry Moore sculptures, unfamiliar to the subjects, also activated this region. This characterization of LOC as a region that responds to shape independently of familiarity has been accepted but never tested with control of the same low-level features. We assessed LOC's response to objects that had identical parts in two different arrangements, one familiar and the other novel. Malach was correct: There is no net effect of familiarity in LOC. However, a multivoxel correlation analysis showed that LOC does distinguish familiar from novel objects.

Genomewide Analysis of Aryl Hydrocarbon Receptor Binding Targets Reveals an Extensive Array of Gene Clusters that Control Morphogenetic and Developmental Programs

Science.gov (United States)

Sartor, Maureen A.; Schnekenburger, Michael; Marlowe, Jennifer L.; Reichard, John F.; Wang, Ying; Fan, Yunxia; Ma, Ci; Karyala, Saikumar; Halbleib, Danielle; Liu, Xiangdong; Medvedovic, Mario; Puga, Alvaro

2009-01-01

Background The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand. Objectives We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand. Methods The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts. Results We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes. Conclusions The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury. PMID:19654925

Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I

International Nuclear Information System (INIS)

Yamanashi, Yuji; Mori, Shigeo; Inoue, Kazushi; Yamamoto, Tadashi; Toyoshima, Kumao; Yoshida, Mitsuaki; Kishimoto, Tadamitsu

1989-01-01

This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells. A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with β-galactosidase. Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes. Incubation of the immunoprecipitate with [γ- 32 P]ATP resulted in the phosphorylation of this protein at tyrosine residues. Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages. Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

International Nuclear Information System (INIS)

Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

1988-01-01

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

Directory of Open Access Journals (Sweden)

Jingwei Cheng

2017-10-01

Full Text Available Merkel cell carcinoma (MCC frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT and an intact Small T antigen (ST. While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis.

Science.gov (United States)

Cheng, Jingwei; Park, Donglim Esther; Berrios, Christian; White, Elizabeth A; Arora, Reety; Yoon, Rosa; Branigan, Timothy; Xiao, Tengfei; Westerling, Thomas; Federation, Alexander; Zeid, Rhamy; Strober, Benjamin; Swanson, Selene K; Florens, Laurence; Bradner, James E; Brown, Myles; Howley, Peter M; Padi, Megha; Washburn, Michael P; DeCaprio, James A

2017-10-01

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

ChIP-PIT: Enhancing the Analysis of ChIP-Seq Data Using Convex-Relaxed Pair-Wise Interaction Tensor Decomposition.

Science.gov (United States)

Zhu, Lin; Guo, Wei-Li; Deng, Su-Ping; Huang, De-Shuang

2016-01-01

In recent years, thanks to the efforts of individual scientists and research consortiums, a huge amount of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experimental data have been accumulated. Instead of investigating them independently, several recent studies have convincingly demonstrated that a wealth of scientific insights can be gained by integrative analysis of these ChIP-seq data. However, when used for the purpose of integrative analysis, a serious drawback of current ChIP-seq technique is that it is still expensive and time-consuming to generate ChIP-seq datasets of high standard. Most researchers are therefore unable to obtain complete ChIP-seq data for several TFs in a wide variety of cell lines, which considerably limits the understanding of transcriptional regulation pattern. In this paper, we propose a novel method called ChIP-PIT to overcome the aforementioned limitation. In ChIP-PIT, ChIP-seq data corresponding to a diverse collection of cell types, TFs and genes are fused together using the three-mode pair-wise interaction tensor (PIT) model, and the prediction of unperformed ChIP-seq experimental results is formulated as a tensor completion problem. Computationally, we propose efficient first-order method based on extensions of coordinate descent method to learn the optimal solution of ChIP-PIT, which makes it particularly suitable for the analysis of massive scale ChIP-seq data. Experimental evaluation the ENCODE data illustrate the usefulness of the proposed model.

Reality show: um paradoxo nietzschiano

Directory of Open Access Journals (Sweden)

Ilana Feldman

2011-01-01

Full Text Available

O fenômeno dos reality shows - e a subseqüente relação entre imagem e verdade - assenta-se sobre uma série de paradoxos. Tais paradoxos podem ser compreendidos à luz do pensamento do filósofo alemão Friedrich Nietzsche, que, através dos usos de formulações paradoxais, concebia a realidade como um mundo de pura aparência e a verdade como um acréscimo ficcional, como um efeito. A ficção é então tomada, na filosofia de Nietzsche, não em seu aspecto falsificante e desrealizador - como sempre pleiteou nossa tradição metafísica -, mas como condição necessária para que certa espécie de invenção possa operar como verdade. Sendo assim, a própria expressão reality show, através de sua formulação paradoxal, engendra explicitamente um mundo de pura aparência, em que a verdade, a parte reality da proposição, é da ordem do suplemento, daquilo que se acrescenta ficcionalmente - como um adjetivo - a show. O ornamento, nesse caso, passa a ocupar o lugar central, apontando para o efeito produzido: o efeito-de-verdade. Seguindo, então, o pensamento nietzschiano e sua atualização na contemporaneidade, investigaremos de que forma os televisivos “shows de realidade” operam paradoxalmente, em consonância com nossas paradoxais práticas culturais.

Showing Value (Editorial

Directory of Open Access Journals (Sweden)

Denise Koufogiannakis

2009-06-01

Full Text Available When Su Cleyle and I first decided to start Evidence Based Library and Information Practice, one of the things we agreed upon immediately was that the journal be open access. We knew that a major obstacle to librarians using the research literature was that they did not have access to the research literature. Although Su and I are both academic librarians who can access a wide variety of library and information literature from our institutions, we belong to a profession where not everyone has equal access to the research in our field. Without such access to our own body of literature, how can we ever hope for practitioners to use research evidence in their decision making? It would have been contradictory to the principles of evidence based library and information practice to do otherwise.One of the specific groups we thought could use such an open access venue for discovering research literature was school librarians. School librarians are often isolated and lacking access to the research literature that may help them prove to stakeholders the importance of their libraries and their role within schools. Certainly, school libraries have been in decline and the use of evidence to show value is needed. As Ken Haycock noted in his 2003 report, The Crisis in Canada’s School Libraries: The Case for Reform and Reinvestment, “Across the country, teacher-librarians are losing their jobs or being reassigned. Collections are becoming depleted owing to budget cuts. Some principals believe that in the age of the Internet and the classroom workstation, the school library is an artifact” (9. Within this context, school librarians are looking to our research literature for evidence of the impact that school library programs have on learning outcomes and student success. They are integrating that evidence into their practice, and reflecting upon what can be improved locally. They are focusing on students and showing the impact of school libraries and

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

Science.gov (United States)

Waszak, Sebastian M; Kilpinen, Helena; Gschwind, Andreas R; Orioli, Andrea; Raghav, Sunil K; Witwicki, Robert M; Migliavacca, Eugenia; Yurovsky, Alisa; Lappalainen, Tuuli; Hernandez, Nouria; Reymond, Alexandre; Dermitzakis, Emmanouil T; Deplancke, Bart

2014-01-15

High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

Science.gov (United States)

Lin, H; Rao, V B; Black, L W

1999-06-04

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly

Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze

Directory of Open Access Journals (Sweden)

Soheil eKeihan Falsafi

2015-10-01

Full Text Available GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM task, a robust paradigm for evaluation of spatial learning.Trained or untrained (yoked control C57BL/6J male mice (n=10/group were subjected to the MTM task and sacrificed 6 hours following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA and a proximity ligation assay (PLA were also used to see if the two GABAB subunits are present in the receptor complex.Single bands were observed on Western blots, each representing GABAB1, GABAB1a or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

Beta 3 and PDI proteins isolated from human platelets bind with ECwt rotavirus in vitro

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Mayorga, Diana; Rubio, Linda; Guerrero-Fonseca, Carlos A; Acosta-Losada, Orlando

2010-01-01

Commercial integrin Beta 3 is currently not available and commercial PDI is too expensive, which is making access difficult to these proteins needed for conducting experiments aimed at the establishment of possible interactions between integrin Beta 3 and PDI and wild type rotavirus strains. Objective. To explore a methodology allowing isolation of proteins Beta 3 and PDI from human platelets to be used as antigens in the generation of rabbit polyclonal antibodies useful in the assessment of interactions between these proteins and rotavirus ECwt. Materials and methods. Proteins Beta 3 and PDI from human platelet lysates were separated using preparative electrophoresis under reducing conditions and then eluted. Interactions of these proteins with rotavirus ECwt were analyzed using co-immunoprecipitation, Western blotting and capture ELISA. Results. Proteins from human platelet lysates were separated by preparative electrophoresis under reducing conditions. The identification of proteins Beta 3 and PDI present in a gel slice was performed through their reaction with commercial antibodies in a Western blotting analysis. Protein purity was established after electro elution from a gel slice. Polyclonal antibodies against protein Beta 3 were generated in rabbit. Incubation of eluted proteins Beta 3 and PDI with rotavirus ECwt showed in co-immunoprecipitation and ELISA assays that these proteins bound virus in vitro. The same binding was showed to occur when rotavirus was incubated with isolated small intestinal villi from suckling mice. Conclusions. Relatively high amounts of proteins Beta 3 and PDI were partially purified from human platelets by preparative electrophoresis. The isolation of these proteins allowed the generation of polyclonal antibodies against Beta 3 in addition to the establishment of the in vitro interaction of proteins Beta 3 and PDI with rotavirus ECwt. This interaction was also demonstrated in vivo after incubating the virus with isolated small

What Are We Drinking? Beverages Shown in Adolescents' Favorite Television Shows.

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Eisenberg, Marla E; Larson, Nicole I; Gollust, Sarah E; Neumark-Sztainer, Dianne

2017-05-01

Media use has been shown to contribute to poor dietary intake; however, little attention has been paid to programming content. The portrayal of health behaviors in television (TV) programming contributes to social norms among viewers, which have been shown to influence adolescent behavior. This study reports on a content analysis of beverages shown in a sample of TV shows popular with a large, diverse group of adolescents, with attention to the types of beverages and differences across shows and characters. Favorite TV shows were assessed in an in-school survey in 2010. Three episodes of each of the top 25 shows were analyzed, using a detailed coding instrument. Beverage incidents (ie, beverage shown or described) were recorded. Beverage types included milk, sugar-sweetened beverages (SSBs), diet beverages, juice, water, alcoholic drinks, and coffee. Characters were coded with regard to gender, age group, race, and weight status. Shows were rated for a youth, general, or adult audience. χ 2 tests were used to compare the prevalence of each type of beverage across show ratings (youth, general, adult), and to compare characteristics of those involved in each type of beverage incident. Beverage incidents were common (mean=7.4 incidents/episode, range=0 to 25). Alcohol was the most commonly shown (38.8%); milk (5.8%) and juice (5.8%) were least common; 11.0% of incidents included SSBs. Significant differences in all types of beverage were found across characters' age groups. Almost half of young adults' (49.2%) or adults' (42.0%) beverage incidents included alcohol. Beverages are often portrayed on TV shows viewed by adolescents, and common beverages (alcohol, SSBs) may have adverse consequences for health. The portrayal of these beverages likely contributes to social norms regarding their desirability; nutrition and health professionals should talk with youth about TV portrayals to prevent the adoption of unhealthy beverage behaviors. Copyright © 2017 Academy of

What are we drinking? Beverages shown in adolescents’ favorite TV shows

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Eisenberg, Marla E.; Larson, Nicole I.; Gollust, Sarah E.; Neumark-Sztainer, Dianne

2016-01-01

Background Media use has been shown to contribute to poor dietary intake; however, little attention has been paid to programming content. The portrayal of health behaviors in television (TV) programming contributes to social norms among viewers, which have been shown to influence adolescent behavior. Objective This study reports on a content analysis of beverages shown in a sample of TV shows popular with a large, diverse group of adolescents, with attention to the types of beverages and differences across shows and characters. Design Favorite TV shows were assessed in an in-school survey in 2010. Three episodes of each of the top 25 shows were analyzed using a detailed coding instrument. Key measures Beverage incidents (i.e. beverage shown or described) were recorded. Beverage types included milk, sugar-sweetened beverages (SSB), diet beverages, juice, water, alcoholic drinks and coffee. Characters were coded with regards to gender, age group, race, and weight status. Shows were rated for a youth, general or adult audience. Statistical analyses Chi-square tests were used to compare the prevalence of each type of beverage across show ratings (youth, general, adult), and to compare characteristics of those involved in each type of beverage incident. Results Beverage incidents were common (mean=7.4 incidents/episode, range=0–25). Alcohol was the most commonly shown (38.8%); milk (5.8%) and juice (5.8%) were least common; 11.0% of incidents included SSB. Significant differences in all types of beverage were found across age groups. Almost half of young adults’ (49.2%) or adults’ (42.0%) beverage incidents included alcohol. Conclusions Beverages are often portrayed on TV shows viewed by adolescents, and common beverages (alcohol, SSB) may have adverse consequences for health. The portrayal of these beverages likely contributes to social norms regarding their desirability; nutrition and health professionals should talk with youth about TV portrayals to prevent the

Studying Protein-Protein Interactions by Biotin AP-Tagged Pulldown and LTQ-Orbitrap Mass Spectrometry.

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Xie, Zhongqiu; Jia, Yuemeng; Li, Hui

2017-01-01

The study of protein-protein interactions represents a key aspect of biological research. Identifying unknown protein binding partners using mass spectrometry (MS)-based proteomics has evolved into an indispensable strategy in drug discovery. The classic approach of immunoprecipitation with specific antibodies against the proteins of interest has limitations, such as the need for immunoprecipitation-qualified antibody. The biotin AP-tag pull-down system has the advantage of high specificity, ease of use, and no requirement for antibody. It is based on the high specificity, high affinity interaction between biotin and streptavidin. After pulldown, in-gel tryptic digestion and tandem mass spectrometry (MS/MS) analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein bands can be performed. In this work, we provide protocols that can be used for the identification of proteins that interact with FOXM1, a protein that has recently emerged as a potential biomarker and drug target in oncotherapy, as an example. We focus on the pull-down procedure and assess the efficacy of the pulldown with known FOXM1 interactors such as β-catenin. We use a high performance LTQ Orbitrap MSn system that combines rapid LTQ ion trap data acquisition with high mass accuracy Orbitrap analysis to identify the interacting proteins.

Promoter of CaZF, a chickpea gene that positively regulates growth and stress tolerance, is activated by an AP2-family transcription factor CAP2.

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Deepti Jain

Full Text Available Plants respond to different forms of stresses by inducing transcription of a common and distinct set of genes by concerted actions of a cascade of transcription regulators. We previously reported that a gene, CaZF encoding a C2H2-zinc finger family protein from chickpea (Cicer arietinum imparted high salinity tolerance when expressed in tobacco plants. We report here that in addition to promoting tolerance against dehydration, salinity and high temperature, the CaZF overexpressing plants exhibited similar phenotype of growth and development like the plants overexpressing CAP2, encoding an AP2-family transcription factor from chickpea. To investigate any relationship between these two genes, we performed gene expression analysis in the overexpressing plants, promoter-reporter analysis and chromatin immunoprecipitation. A number of transcripts that exhibited enhanced accumulation upon expression of CAP2 or CaZF in tobacco plants were found common. Transient expression of CAP2 in chickpea leaves resulted in increased accumulation of CaZF transcript. Gel mobility shift and transient promoter-reporter assays suggested that CAP2 activates CaZF promoter by interacting with C-repeat elements (CRTs in CaZF promoter. Chromatin immunoprecipitation (ChIP assay demonstrated an in vivo interaction of CAP2 protein with CaZF promoter.

Ankyrin repeat and SOCS box containing protein 4 (Asb-4 colocalizes with insulin receptor substrate 4 (IRS4 in the hypothalamic neurons and mediates IRS4 degradation

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Xia Zefeng

2011-09-01

Full Text Available Abstract Background The arcuate nucleus of the hypothalamus regulates food intake. Ankyrin repeat and SOCS box containing protein 4 (Asb-4 is expressed in neuropeptide Y and proopiomelanocortin (POMC neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. However, the target protein(s of Asb-4 in these neurons remains unknown. Insulin receptor substrate 4 (IRS4 is an adaptor molecule involved in the signal transduction by both insulin and leptin. In the present study we examined the colocalization and interaction of Asb-4 with IRS4 and the involvement of Asb-4 in insulin signaling. Results In situ hybridization showed that the expression pattern of Asb-4 was consistent with that of IRS4 in the rat brain. Double in situ hybridization showed that IRS4 colocalized with Asb-4, and both Asb-4 and IRS4 mRNA were expressed in proopiomelanocortin (POMC and neuropeptide Y (NPY neurons within the arcuate nucleus of the hypothalamus. In HEK293 cells co-transfected with Myc-tagged Asb-4 and Flag-tagged IRS4, Asb-4 co-immunoprecipitated with IRS4; In these cells endogenous IRS4 also co-immunoprecipitated with transfected Myc-Asb-4; Furthermore, Asb-4 co-immunoprecipitated with IRS4 in rat hypothalamic extracts. In HEK293 cells over expression of Asb-4 decreased IRS4 protein levels and deletion of the SOCS box abolished this effect. Asb-4 increased the ubiquitination of IRS4; Deletion of SOCS box abolished this effect. Expression of Asb-4 decreased both basal and insulin-stimulated phosphorylation of AKT at Thr308. Conclusions These data demonstrated that Asb-4 co-localizes and interacts with IRS4 in hypothalamic neurons. The interaction of Asb-4 with IRS4 in cell lines mediates the degradation of IRS4 and decreases insulin signaling.

Multiplexed ChIP-Seq Using Direct Nucleosome Barcoding: A Tool for High-Throughput Chromatin Analysis.

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Chabbert, Christophe D; Adjalley, Sophie H; Steinmetz, Lars M; Pelechano, Vicent

2018-01-01

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) or microarray hybridization (ChIP-on-chip) are standard methods for the study of transcription factor binding sites and histone chemical modifications. However, these approaches only allow profiling of a single factor or protein modification at a time.In this chapter, we present Bar-ChIP, a higher throughput version of ChIP-Seq that relies on the direct ligation of molecular barcodes to chromatin fragments. Bar-ChIP enables the concurrent profiling of multiple DNA-protein interactions and is therefore amenable to experimental scale-up, without the need for any robotic instrumentation.

Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene.

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Wang, Liangjun; Jahren, Neal; Miller, Ellen L; Ketel, Carrie S; Mallin, Daniel R; Simon, Jeffrey A

2010-06-01

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets.

Comparative Analysis of Chromatin Binding by Sex Comb on Midleg (SCM) and Other Polycomb Group Repressors at a Drosophila Hox Gene▿

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Wang, Liangjun; Jahren, Neal; Miller, Ellen L.; Ketel, Carrie S.; Mallin, Daniel R.; Simon, Jeffrey A.

2010-01-01

Sex Comb on Midleg (SCM) is a transcriptional repressor in the Polycomb group (PcG), but its molecular role in PcG silencing is not known. Although SCM can interact with Polycomb repressive complex 1 (PRC1) in vitro, biochemical studies have indicated that SCM is not a core constituent of PRC1 or PRC2. Nevertheless, SCM is just as critical for Drosophila Hox gene silencing as canonical subunits of these well-characterized PcG complexes. To address functional relationships between SCM and other PcG components, we have performed chromatin immunoprecipitation studies using cultured Drosophila Schneider line 2 (S2) cells and larval imaginal discs. We find that SCM associates with a Polycomb response element (PRE) upstream of the Ubx gene which also binds PRC1, PRC2, and the DNA-binding PcG protein Pleiohomeotic (PHO). However, SCM is retained at this Ubx PRE despite genetic disruption or knockdown of PHO, PRC1, or PRC2, suggesting that SCM chromatin targeting does not require prior association of these other PcG components. Chromatin immunoprecipitations (IPs) to test the consequences of SCM genetic disruption or knockdown revealed that PHO association is unaffected, but reduced levels of PRE-bound PRC2 and PRC1 were observed. We discuss these results in light of current models for recruitment of PcG complexes to chromatin targets. PMID:20351181

Modern techniques for the analysis of chromatin and nuclear organization in C. elegans.

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Askjaer, Peter; Ercan, Sevinç; Meister, Peter

2014-04-02

In recent years, Caenorhabditis elegans has emerged as a new model to investigate the relationships between nuclear architecture, cellular differentiation, and organismal development. On one hand, C. elegans with its fixed lineage and transparent body is a great model organism to observe gene functions in vivo in specific cell types using microscopy. On the other hand, two different techniques have been applied in nematodes to identify binding sites for chromatin-associated proteins genome-wide: chromatin immunoprecipitation (ChIP), and Dam-mediated identification (DamID). We summarize here all three techniques together as they are complementary. We also highlight strengths and differences of the individual approaches.

Global Trend Analysis of Multi-decade Soil Temperature Records Show Soils Resistant to Warming

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Frey, S. D.; Jennings, K.

2017-12-01

Soil temperature is an important determinant of many subterranean ecological processes including plant growth, nutrient cycling, and carbon sequestration. Soils are expected to warm in response to increasing global surface temperatures; however, despite the importance of soil temperature to ecosystem processes, less attention has been given to examining changes in soil temperature over time. We collected long-term (> 20 years) soil temperature records from approximately 50 sites globally, many with multiple depths (5 - 100 cm), and examined temperature trends over the last few decades. For each site and depth we calculated annual summer means and conducted non-parametric Mann Kendall trend and Sen slope analysis to assess changes in summer soil temperature over the length of each time series. The mean summer soil temperature trend across all sites and depths was not significantly different than zero (mean = 0.004 °C year-1 ± 0.033 SD), suggesting that soils have not warmed over the observation period. Of the subset of sites that exhibit significant increases in temperature over time, site location, depth of measurement, time series length, and neither start nor end date seem to be related to trend strength. These results provide evidence that the thermal regime of soils may have a stronger buffering capacity than expected, having important implications for the global carbon cycle and feedbacks to climate change.

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

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Kazuhide Watanabe

Full Text Available Deregulation of canonical Wnt/CTNNB1 (beta-catenin pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells.We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis.Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

Coilin phosphorylation mediates interaction with SMN and SmB′

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Toyota, Cory G.; Davis, Misty D.; Cosman, Angela M.; Hebert, Michael D.

2010-01-01

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB. PMID:19997741

Coilin phosphorylation mediates interaction with SMN and SmB'.

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Toyota, Cory G; Davis, Misty D; Cosman, Angela M; Hebert, Michael D

2010-04-01

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells

International Nuclear Information System (INIS)

Sklyanskaya, E.I.; Varich, N.L.; Amvrosieva, T.V.; Kaverin, N.V.

1985-01-01

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14 C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, the authors absorbed the lysates of doubly infected [ 3 H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected

Intracerebral metastasis showing restricted diffusion: Correlation with histopathologic findings

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Duygulu, G. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Ovali, G. Yilmaz [Radiology Department, Celal Bayar University Medicine School, Manisa (Turkey)], E-mail: gulgun.yilmaz@bayar.edu.tr; Calli, C.; Kitis, O.; Yuenten, N. [Radiology Department, Ege University Medicine School, Izmir (Turkey); Akalin, T. [Pathology Department, Ege University Medicine School, Izmir (Turkey); Islekel, S. [Neurosurgery Department, Ege University Medicine School, Izmir (Turkey)

2010-04-15

Objective: We aimed to detect the frequency of restricted diffusion in intracerebral metastases and to find whether there is correlation between the primary tumor pathology and diffusion-weighted MR imaging (DWI) findings of these metastases. Material and methods: 87 patients with intracerebral metastases were examined with routine MR imaging and DWI. 11 hemorrhagic metastatic lesions were excluded. The routine MR imaging included three plans before and after contrast enhancement. The DWI was performed with spin-echo EPI sequence with three b values (0, 500 and 1000), and ADC maps were calculated. 76 patients with metastases were grouped according to primary tumor histology and the ratios of restricted diffusion were calculated according to these groups. ADCmin values were measured within the solid components of the tumors and the ratio of metastases with restricted diffusion to that which do not show restricted diffusion were calculated. Fisher's exact and Mann-Whitney U tests were used for the statistical analysis. Results: Restricted diffusion was observed in a total of 15 metastatic lesions (19, 7%). Primary malignancy was lung carcinoma in 10 of these cases (66, 6%) (5 small cell carcinoma, 5 non-small cell carcinoma), and breast carcinoma in three cases (20%). Colon carcinoma and testicular teratocarcinoma were the other two primary tumors in which restricted diffusion in metastasis was detected. There was no statistical significant difference between the primary pathology groups which showed restricted diffusion (p > 0.05). ADCmin values of solid components of the metastasis with restricted diffusion and other metastasis without restricted diffusion also showed no significant statistical difference (0.72 {+-} 0.16 x 10{sup -3} mm{sup 2}/s and 0.78 {+-} 21 x 10{sup -3} mm{sup 2}/s respectively) (p = 0.325). Conclusion: Detection of restricted diffusion on DWI in intracerebral metastasis is not rare, particularly if the primary tumor is lung or breast

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

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Cho, Seong-Jun; Kang, Hana [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Kim, Min Young [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan (Korea, Republic of); Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of); Pyo, Suhkneung [College of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do (Korea, Republic of); Yang, Kwang Hee, E-mail: kwangheey@khnp.co.kr [KHNP Radiation Health Institute, Korea Hydro & Nuclear Power Co, Seoul (Korea, Republic of)

2016-04-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a {sup 137}Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation.

Science.gov (United States)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-04-01

To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Splenocytes and IM-9 cells were uniformly irradiated with various doses of a (137)Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast. Copyright © 2016 Elsevier Inc. All rights reserved.

Site-Specific Phosphorylation of Ikaros Induced by Low-Dose Ionizing Radiation Regulates Cell Cycle Progression of B Lymphoblast Through CK2 and AKT Activation

International Nuclear Information System (INIS)

Cho, Seong-Jun; Kang, Hana; Kim, Min Young; Lee, Jung Eun; Kim, Sung Jin; Nam, Seon Young; Kim, Ji Young; Kim, Hee Sun; Pyo, Suhkneung; Yang, Kwang Hee

2016-01-01

Purpose: To determine how low-dose ionizing radiation (LDIR) regulates B lympho-proliferation and its molecular mechanism related with Ikaros, transcription factor. Methods and Materials: Splenocytes and IM-9 cells were uniformly irradiated with various doses of a "1"3"7Cs γ-source, and cell proliferation was analyzed. To determine the LDIR-specific phosphorylation of Ikaros, immunoprecipitation and Western blot analysis were performed. To investigate the physiologic function of LDIR-mediatied Ikaros phosphorylation, Ikaros mutants at phosphorylation sites were generated, and cell cycle analysis was performed. Results: First, we found that LDIR enhances B lymphoblast proliferation in an Ikaros-dependent manner. Moreover, we found that LDIR elevates the phosphorylation level of Ikaros protein. Interestingly, we showed that CK2 and AKT are involved in LDIR-induced Ikaros phosphorylation and capable of regulating DNA binding activity of Ikaros via specific phosphorylation. Finally, we identified LDIR-specific Ikaros phosphorylation sites at S391/S393 and showed that the Ikaros phosphorylations at these sites control Ikaros's ability to regulate G1/S cell cycle progression. Conclusion: Low-dose ionizing radiation specifically phosphorylates Ikaros protein at Ser 391/393 residues to regulate cell cycle progression in B lymphoblast.

Meta-analysis of field experiments shows no change in racial discrimination in hiring over time.

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Quillian, Lincoln; Pager, Devah; Hexel, Ole; Midtbøen, Arnfinn H

2017-10-10

This study investigates change over time in the level of hiring discrimination in US labor markets. We perform a meta-analysis of every available field experiment of hiring discrimination against African Americans or Latinos ( n = 28). Together, these studies represent 55,842 applications submitted for 26,326 positions. We focus on trends since 1989 ( n = 24 studies), when field experiments became more common and improved methodologically. Since 1989, whites receive on average 36% more callbacks than African Americans, and 24% more callbacks than Latinos. We observe no change in the level of hiring discrimination against African Americans over the past 25 years, although we find modest evidence of a decline in discrimination against Latinos. Accounting for applicant education, applicant gender, study method, occupational groups, and local labor market conditions does little to alter this result. Contrary to claims of declining discrimination in American society, our estimates suggest that levels of discrimination remain largely unchanged, at least at the point of hire.

Calcium-binding proteins from human platelets

International Nuclear Information System (INIS)

Gogstad, G.O.; Krutnes, M.B.; Solum, N.O.

1983-01-01

Calcium-binding platelet proteins were examined by crossed immunoelectrophoresis of solubilized platelets against antibodies to whole platelets followed by incubation of the immunoplates with 45 Ca 2 + and autoradiography. When the immunoplates had been pretreated with EDTA at pH 9.0 in order to remove divalent cations, three immunoprecipitates were markedly labelled with 45 Ca 2 + . These corresponded to the glycoprotein IIb-IIIa complex, glycoprotein Ia and a presently unidentified antigen termed G18. These antigens were membrane-bound and surface-oriented. When an excess of EDTA was introduced in the incubation media the results revealed that the glycoprotein IIb-IIIa complex and antigen G18, but not glycoprotein Ia, contained sites with a stronger affinity for calcium than has EDTA at pH 7.4 Immunoprecipitates of the separate glycoproteins IIb and IIIa both bound calcium in the same manner as the glycoprotein IIb-IIIa complex. As another approach, platelet-rich plasma was incubated with 45 Ca 2 + prior to crossed immunoelectrophoresis of the solubilized platelets. A single immunoprecipitate was wekly labelled. This did not correspond to any of the immunoprecipitates which were visible after staining with Coomassie blue. The labelling of this antigen was markedly increased when the platelt-rich plasma had been preincubated with EDTA and in this case a weak labelling of the glycoprotein IIB-IIIa precipitate also became apparent. No increased incorporation of calcium occured in any of these immunoprecipitates when the platelets were aggregated with ADP in the presence of 45 Ca 2 + . (orig.)

Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

Science.gov (United States)

Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

2015-06-01

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Transcriptional Analysis of the MrpJ Network: Modulation of Diverse Virulence-Associated Genes and Direct Regulation of mrp Fimbrial and flhDC Flagellar Operons in Proteus mirabilis

Science.gov (United States)

Bode, Nadine J.; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen

2015-01-01

The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. PMID:25847961

Analysis of audiometric database shows evidence of employee fraud

Science.gov (United States)

Erdreich, John

2003-10-01

Following a lengthy strike, several hundred delivery drivers filed workers compensation claims for occupational hearing loss. We were asked to evaluate the noise exposure of the drivers during their in-plant tasks. In-plant exposures were not predictive of any hearing loss. A comparison of audiometric data for the claimants revealed consistent hearing loss independent of duration of employment or age. These discrepancies between observations and common understanding of dose-response relationships between noise exposure and hearing loss led to further investigation, ultimately resulting in the dismissal of all claims against the employer who then filed an action against the claimant's attorneys and physician under the Racketeering in Corrupt Organizations Act (RICO). The details of the legal complaint, which reads like a detective novel, can be found at the United States District Court for the Southern District of New York [93 Civ. 7222 (LAP)].

Mining for novel candidate clock genes in the circadian regulatory network

OpenAIRE

Bhargava, Anuprabha; Herzel, Hanspeter; Ananthasubramaniam, Bharath

2015-01-01

Background Most physiological processes in mammals are temporally regulated by means of a master circadian clock in the brain and peripheral oscillators in most other tissues. A transcriptional-translation feedback network of clock genes produces near 24 h oscillations in clock gene and protein expression. Here, we aim to identify novel additions to the clock network using a meta-analysis of public chromatin immunoprecipitation sequencing (ChIP-seq), proteomics and protein-protein interaction...

RNA Binding Proteins Posttranscriptionally Regulate Genes Involved In Oncogenesis

Science.gov (United States)

2010-06-01

HuR using RNA immunoprecipitations applied to m icroarray chip s ( RIP-Chip) in estrogen positiv e (ER+) and estrogen negative (ER-) breast ca ncer...CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull- down analysis. Conclusion: This is the first report of a side-by...labeled amplified cDNA) were quantitated using a Nanodrop™ (Thermo Fisher Scientific, Waltham, MA) spectrophotometer. RNA quality and integrity were

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

Science.gov (United States)

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

Science.gov (United States)

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Combinatorial Cis-regulation in Saccharomyces Species

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Aaron T. Spivak

2016-03-01

Full Text Available Transcriptional control of gene expression requires interactions between the cis-regulatory elements (CREs controlling gene promoters. We developed a sensitive computational method to identify CRE combinations with conserved spacing that does not require genome alignments. When applied to seven sensu stricto and sensu lato Saccharomyces species, 80% of the predicted interactions displayed some evidence of combinatorial transcriptional behavior in several existing datasets including: (1 chromatin immunoprecipitation data for colocalization of transcription factors, (2 gene expression data for coexpression of predicted regulatory targets, and (3 gene ontology databases for common pathway membership of predicted regulatory targets. We tested several predicted CRE interactions with chromatin immunoprecipitation experiments in a wild-type strain and strains in which a predicted cofactor was deleted. Our experiments confirmed that transcription factor (TF occupancy at the promoters of the CRE combination target genes depends on the predicted cofactor while occupancy of other promoters is independent of the predicted cofactor. Our method has the additional advantage of identifying regulatory differences between species. By analyzing the S. cerevisiae and S. bayanus genomes, we identified differences in combinatorial cis-regulation between the species and showed that the predicted changes in gene regulation explain several of the species-specific differences seen in gene expression datasets. In some instances, the same CRE combinations appear to regulate genes involved in distinct biological processes in the two different species. The results of this research demonstrate that (1 combinatorial cis-regulation can be inferred by multi-genome analysis and (2 combinatorial cis-regulation can explain differences in gene expression between species.

Comparative Analysis of Clinical Samples Showing Weak Serum Reaction on AutoVue System Causing ABO Blood Typing Discrepancies.

Science.gov (United States)

Jo, Su Yeon; Lee, Ju Mi; Kim, Hye Lim; Sin, Kyeong Hwa; Lee, Hyeon Ji; Chang, Chulhun Ludgerus; Kim, Hyung Hoi

2017-03-01

ABO blood typing in pre-transfusion testing is a major component of the high workload in blood banks that therefore requires automation. We often experienced discrepant results from an automated system, especially weak serum reactions. We evaluated the discrepant results by the reference manual method to confirm ABO blood typing. In total, 13,113 blood samples were tested with the AutoVue system; all samples were run in parallel with the reference manual method according to the laboratory protocol. The AutoVue system confirmed ABO blood typing of 12,816 samples (97.7%), and these results were concordant with those of the manual method. The remaining 297 samples (2.3%) showed discrepant results in the AutoVue system and were confirmed by the manual method. The discrepant results involved weak serum reactions (serum reactions, samples from patients who had received stem cell transplants, ABO subgroups, and specific system error messages. Among the 98 samples showing ≤1+ reaction grade in the AutoVue system, 70 samples (71.4%) showed a normal serum reaction (≥2+ reaction grade) with the manual method, and 28 samples (28.6%) showed weak serum reaction in both methods. ABO blood tying of 97.7% samples could be confirmed by the AutoVue system and a small proportion (2.3%) needed to be re-evaluated by the manual method. Samples with a 2+ reaction grade in serum typing do not need to be evaluated manually, while those with ≤1+ reaction grade do.

Quantitative expression patterns of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) protein in mice

International Nuclear Information System (INIS)

Girroir, Elizabeth E.; Hollingshead, Holly E.; He Pengfei; Zhu Bokai; Perdew, Gary H.; Peters, Jeffrey M.

2008-01-01

The expression patterns of PPARβ/δ have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPARβ/δ protein in mouse tissues. In the present study, a highly specific PPARβ/δ antibody was developed, characterized, and used to examine tissue expression patterns of PPARβ/δ. As compared to commercially available anti-PPARβ/δ antibodies, one of six polyclonal anti-PPARβ/δ antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPARβ/δ. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPARβ/δ was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPARβ/δ expression was localized in the nucleus and RXRα can be co-immunoprecipitated with nuclear PPARβ/δ. Results from these studies demonstrate that PPARβ/δ expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues

Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

Science.gov (United States)

Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

1987-02-01

Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

KAUST Repository

Ali, Amal J.

2017-05-03

Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

Downstream targets of WRKY33

DEFF Research Database (Denmark)

Petersen, Klaus; Fiil, Berthe Katrine; Mundy, John

2008-01-01

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. In a recent publication we show that MPK4 and its substrate MKS1 interact with WRKY33 in vivo, and that WRKY33 is released from complexes with MPK4 upon infection....... Transcriptome analysis of a wrky33 loss-of-function mutant identified a subset of defense-related genes as putative targets of WRKY33. These genes include PAD3 and CYP71A13, which encode cytochrome P450 monoxygenases required for synthesis of the antimicrobial phytoalexin camalexin. Chromatin...... immunoprecipitation confirmed that WRKY33 bound the promoter of PAD3 when plants were inoculated with pathogens. Here we further discuss the involvement of two other targets of WRKY33, NUDT6 and ROF2 in defense responses against invading pathogens....

Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

International Nuclear Information System (INIS)

Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin

2006-01-01

Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation

Exposure to teasing on popular television shows and associations with adolescent body satisfaction.

Science.gov (United States)

Eisenberg, Marla E; Ward, Ellen; Linde, Jennifer A; Gollust, Sarah E; Neumark-Sztainer, Dianne

2017-12-01

This study uses a novel mixed methods design to examine the relationship between incidents of teasing in popular television shows and body satisfaction of adolescent viewers. Survey data were used to identify 25 favorite television shows in a large population-based sample of Minnesota adolescents (N=2793, age=14.4years). Data from content analysis of teasing incidents in popular shows were linked to adolescent survey data. Linear regression models examined associations between exposure to on-screen teasing in adolescents' own favorite shows and their body satisfaction. Effect modification by adolescent weight status was tested using interaction terms. Teasing on TV was common, with 3.3 incidents per episode; over one-quarter of teasing was weight/shape-related. Exposure to weight/shape-related teasing (β=-0.43, p=0.008) and teasing with overweight targets (β=-0.03, p=0.02) was inversely associated with girls' body satisfaction; no associations were found for boys. Findings were similar regardless of the adolescent viewer's weight status. Families, health care providers, media literacy programs and the entertainment industry are encouraged to consider the negative effects exposure to weight stigmatization can have on adolescent girls. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-Wide DNA Methylation in Mixed Ancestry Individuals with Diabetes and Prediabetes from South Africa

Science.gov (United States)

Pheiffer, Carmen; Humphries, Stephen E.; Gamieldien, Junaid; Erasmus, Rajiv T.

2016-01-01

Aims. To conduct a genome-wide DNA methylation in individuals with type 2 diabetes, individuals with prediabetes, and control mixed ancestry individuals from South Africa. Methods. We used peripheral blood to perform genome-wide DNA methylation analysis in 3 individuals with screen detected diabetes, 3 individuals with prediabetes, and 3 individuals with normoglycaemia from the Bellville South Community, Cape Town, South Africa, who were age-, gender-, body mass index-, and duration of residency-matched. Methylated DNA immunoprecipitation (MeDIP) was performed by Arraystar Inc. (Rockville, MD, USA). Results. Hypermethylated DMRs were 1160 (81.97%) and 124 (43.20%), respectively, in individuals with diabetes and prediabetes when both were compared to subjects with normoglycaemia. Our data shows that genes related to the immune system, signal transduction, glucose transport, and pancreas development have altered DNA methylation in subjects with prediabetes and diabetes. Pathway analysis based on the functional analysis mapping of genes to KEGG pathways suggested that the linoleic acid metabolism and arachidonic acid metabolism pathways are hypomethylated in prediabetes and diabetes. Conclusions. Our study suggests that epigenetic changes are likely to be an early process that occurs before the onset of overt diabetes. Detailed analysis of DMRs that shows gradual methylation differences from control versus prediabetes to prediabetes versus diabetes in a larger sample size is required to confirm these findings. PMID:27555869

Combined chromatin and expression analysis reveals specific regulatory mechanisms within cytokine genes in the macrophage early immune response.

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Maria Jesus Iglesias

Full Text Available Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS.To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches--gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII, which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF, was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines, was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/-LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40% was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at

WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

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Bol John F

2011-05-01

Full Text Available Abstract Background Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR. The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA. An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3 plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. Results Expression studies with ICS1 promoter::β-glucuronidase (GUS genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. Conclusions The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress

CCAAT/Enhancer Binding Protein β Regulates Expression of Indian Hedgehog during Chondrocytes Differentiation

Science.gov (United States)

Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Ishihara, Kohei; Doi, Toshio; Iwamoto, Yukihide

2014-01-01

Background CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh) also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2) was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation. Methodology/Results Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between −214 and −210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression. Conclusions C

CCAAT/enhancer binding protein β regulates expression of Indian hedgehog during chondrocytes differentiation.

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Takahiro Ushijima

Full Text Available CCAAT/enhancer binding protein β (C/EBPβ is a transcription factor that promotes hypertrophic differentiation of chondrocytes. Indian hedgehog (Ihh also stimulates the hypertrophic transition of chondrocytes. Furthermore, runt-related transcription factor-2 (RUNX2 was reported to regulate chondrocyte maturation during skeletal development and to directly regulate transcriptional activity of Ihh. In this study, we investigated whether the interaction of C/EBPβ and RUNX2 regulates the expression of Ihh during chondrocyte differentiation.Immunohistochemistry of embryonic growth plate revealed that both C/EBPβ and Ihh were strongly expressed in pre-hypertrophic and hypertrophic chondrocytes. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked stimulation of Ihh and Runx2. Conversely, knockdown of C/EBPβ by lentivirus expressing shRNA significantly repressed Ihh and Runx2 in ATDC5 cells. A reporter assay revealed that C/EBPβ stimulated transcriptional activity of Ihh. Deletion and mutation analysis showed that the C/EBPβ responsive element was located between -214 and -210 bp in the Ihh promoter. An electrophoretic mobility shift assay (EMSA and a chromatin immunoprecipitation (ChIP assay also revealed the direct binding of C/EBPβ to this region. Moreover, reporter assays demonstrated that RUNX2 failed to stimulate the transcriptional activity of the Ihh promoter harboring a mutation at the C/EBPβ binding site. EMSA and ChIP assays showed that RUNX2 interacted to this element with C/EBPβ. Immunoprecipitation revealed that RUNX2 and C/EBPβ formed heterodimer complex with each other in the nuclei of chondrocytes. These data suggested that the C/EBPβ binding element is also important for RUNX2 to regulate the expression of Ihh. Ex vivo organ culture of mouse limbs transfected with C/EBPβ showed that the expression of Ihh and RUNX2 was increased upon ectopic C/EBPβ expression.C/EBPβ and RUNX2 cooperatively stimulate

A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

2008-01-01

RNAs, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly....

Migraineurs without aura show microstructural abnormalities in the cerebellum and frontal lobe.

Science.gov (United States)

Granziera, C; Romascano, D; Daducci, A; Roche, A; Vincent, M; Krueger, G; Hadjikhani, N

2013-12-01

The involvement of the cerebellum in migraine pathophysiology is not well understood. We used a biparametric approach at high-field MRI (3 T) to assess the structural integrity of the cerebellum in 15 migraineurs with aura (MWA), 23 migraineurs without aura (MWoA), and 20 healthy controls (HC). High-resolution T1 relaxation maps were acquired together with magnetization transfer images in order to probe microstructural and myelin integrity. Clusterwise analysis was performed on T1 and magnetization transfer ratio (MTR) maps of the cerebellum of MWA, MWoA, and HC using an ANOVA and a non-parametric clusterwise permutation F test, with age and gender as covariates and correction for familywise error rate. In addition, mean MTR and T1 in frontal regions known to be highly connected to the cerebellum were computed. Clusterwise comparison among groups showed a cluster of lower MTR in the right Crus I of MWoA patients vs. HC and MWA subjects (p = 0.04). Univariate and bivariate analysis on T1 and MTR contrasts showed that MWoA patients had longer T1 and lower MTR in the right and left pars orbitalis compared to MWA (p < 0.01 and 0.05, respectively), but no differences were found with HC. Lower MTR and longer T1 point at a loss of macromolecules and/or micro-edema in Crus I and pars orbitalis in MWoA patients vs. HC and vs. MWA. The pathophysiological implications of these findings are discussed in light of recent literature.

Molecular analysis of MLH1 variants in Chinese sporadic colorectal cancer patients.

Science.gov (United States)

Peng, H X; Xu, X; Yang, R; Chu, Y M; Yang, D M; Xu, Y; Zhou, F L; Ma, W Z; Zhang, X J; Guan, M; Yang, Z H; Jin, Z D

2016-04-26

Single nucleotide polymorphisms (SNPs) in mismatch repair genes, especially in the MLH1 gene, are closely associated with susceptibility to hereditary nonpolyposis colorectal cancer. However, few relevant findings are available regarding the association between sporadic colorectal cancer (SCRC) and SNPs of MLH1 in Chinese patients. Therefore, the present study aimed to describe the pathogenic association between three important MLH1 polymorphisms and SCRC in the Chinese population. Peripheral blood samples from 156 SCRC patients and 311 healthy controls were collected. DNA was purified from peripheral blood, and the V384D, R217C, and I219V polymorphisms were evaluated using high-resolution melting analysis and direct sequencing. The association between the three important MLH1 polymorphisms and clinical pathological features of the SCRC patients was analyzed. In addition, PMS2-MLH1 protein interactions were determined by co-immunoprecipitation (Co-IP) to determine the protein functional alteration induced by these SNPs. Among the three polymorphisms, V384D was significantly associated with the risk of SCRC (OR = 31.36, P MLH1 R217C, V384D, and I219V variants had relative binding abilities with PMS2 of 0.59, 0.70, and 0.80, respectively, compared with the wild-type. These findings suggest that MLH1 V384D could be a promising genetic marker for susceptibility to SCRC.

The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

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Herrmann Harald

2008-09-01

Full Text Available Abstract Background Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusion We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

Abrogation of the presenilin 1/beta-catenin interaction and preservation of the heterodimeric presenilin 1 complex following caspase activation.

Science.gov (United States)

Tesco, G; Kim, T W; Diehlmann, A; Beyreuther, K; Tanzi, R E

1998-12-18

beta-Catenin has previously been shown to interact with presenilin 1 (PS1) in transfected cells. Here we report that beta-catenin co-immunoprecipitates with the endogenous C-terminal fragment of presenilin 1 (PS1-CTF) but not with the endogenous CTF of presenilin 2 (PS2-CTF) in H4 human neuroglioma cells. During staurosporine (STS)-induced cell death, beta-catenin and PS1-CTF undergo a caspase-mediated cleavage. After 12 h of STS treatment, the beta-catenin.PS1-CTF interaction is abrogated. While PS1-CTF immunoprecipitated with all caspase-cleaved species of beta-catenin, beta-catenin holoprotein did not co-immunoprecipitate with the "alternative" caspase-derived PS1-CTF (PS1-aCTF). Thus, the abrogation of the beta-catenin.PS1-CTF complex was due to caspase cleavage of PS1-CTF. beta-Catenin co-immunoprecipitated with PS1-NTF, but only when PS1-NTF was associated with PS1-CTF. Even though PS1-NTF.CTF complex stability was not altered by caspase cleavage, its ability to bind beta-catenin was abolished. Thus, while the PS1-NTF.CTF complex is preserved after caspase cleavage, it may no longer be fully functional.

CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

Science.gov (United States)

Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

2011-01-01

The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

Association of mitochondrial lysyl-tRNA synthetase with HIV-1 GagPol involves catalytic domain of the synthetase and transframe and integrase domains of Pol

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Shalak V. F.

2011-10-01

Full Text Available Aim. Analyze the interaction between Lysyl-tRNA synthetase (LysRS and HIV-1 GagPol to know whether a particular N-terminal sequence of mitochondrial LysRS triggers a specific recognition with GagPol. Methods. Yeast two-hybrid analysis, immunoprecipitation. Results. We have shown that LysRS associates with the Pol domain of GagPol. Conclusions. A model of the assembly of the LysRS:tRNA3Lys:GagPol packaging complex is proposed.

Defining the Role of BTLA in Breast Cancer Immunosurveillance and Selective Targeting of the BTLA-HVEM-LIGHT Costimulatory System

Science.gov (United States)

2010-05-01

assay was conducted as reported by Jedema et al. (41), with alterations noted below. Briefly, allogeneic P815 (H-2Kd) or syngeneic EL4 (H-2Kb) target...cells lysed (CFSE7- AAD) was determined by FACS. CTL activity was not observed toward syngeneic (H-2Kb) EL4 cell targets (data not shown). The mean...could induce BTLA phosphor- ylation (Fig. 4c,d). We analyzed BTLA phosphorylation in EL4 cells using immunoprecipitation immunoblot analysis as

c-Fos downregulation positively regulates EphA5 expression in a congenital hypothyroidism rat model.

Science.gov (United States)

Song, Honghua; Zheng, Yuqin; Cai, Fuying; Ma, Yanyan; Yang, Jingyue; Wu, Youjia

2018-04-01

The EphA5 receptor is well established as an axon guidance molecule during neural system development and plays an important role in dendritic spine formation and synaptogenesis. Our previous study has showed that EphA5 is decreased in the developing brain of congenital hypothyroidism (CH) and the EphA5 promoter methylation modification participates in its decrease. c-Fos, a well-kown transcription factor, has been considered in association with brain development. Bioinformatics analysis showed that the EphA5 promoter region contained five putative c-fos binding sites. The chromatin immunoprecipitation (ChIP) assays were used to assess the direct binding of c-fos to the EphA5 promoter. Furthermore, dual-luciferase assays showed that these three c-fos protein binding sites were positive regulatory elements for EphA5 expression in PC12 cells. Moreover, We verified c-fos positively regulation for EphA5 expression in CH model. Q-PCR and Western blot showed that c-fos overexpression could upregulate EphA5 expression in hippocampal neurons of rats with CH. Our results suggest that c-fos positively regulates EphA5 expression in CH rat model.

Koncept "telling" a "showing" v románech The Bluest Eye a To Kill a Mockingbird

OpenAIRE

Felcmanová, Martina

2015-01-01

TITLE Telling and Showing in The Bluest Eye and To Kill a Mockingbird AUTOR Martina Felcmanová DEPARTMENT Department of English Language and Literature SUPERVISOR PhDr. Petr Chalupský, Ph.D. ABSTRACT This bachelor thesis focuses on similarities and differences in the narrative strategies in the novels To Kill a Mockingbird and The Bluest Eye. The main objective lies in the analysis of how, and for what purpose, the two modes of narration, telling and showing (also diegesis and mimesis) are us...

Identifying the heterotrimeric complex stoichiometry of AMPK in skeletal muscle by immunoprecipitation

DEFF Research Database (Denmark)

Birk, Jesper Bratz; Wojtaszewski, Jørgen

2018-01-01

The 5'-AMP-activated protein kinase is a complicated enzyme consisting of three different subunits, each of which is expressed as two or three isoforms. This gives the possibility of 12 different heterotrimeric complexes, which could have diverse functions within the cell. To map out which of the...

Polymorphism of CRISPR shows separated natural groupings of Shigella subtypes and evidence of horizontal transfer of CRISPR

Science.gov (United States)

Yang, Chaojie; Li, Peng; Su, Wenli; Li, Hao; Liu, Hongbo; Yang, Guang; Xie, Jing; Yi, Shengjie; Wang, Jian; Cui, Xianyan; Wu, Zhihao; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Qiu, Shaofu; Song, Hongbin

2015-01-01

Clustered, regularly interspaced, short palindromic repeats (CRISPR) act as an adaptive RNA-mediated immune mechanism in bacteria. They can also be used for identification and evolutionary studies based on polymorphisms within the CRISPR locus. We amplified and analyzed 6 CRISPR loci from 237 Shigella strains belonging to the 4 species groups, as well as 13 Escherichia coli strains. The CRISPR-associated (cas) gene sequence arrays of these strains were screened and compared. The CRISPR sequences from Shigella were conserved among subtypes, suggesting that CRISPR may represent a new identification tool for the detection and discrimination of Shigella species. Secondary structure analysis showed a different stem-loop structure at the terminal repeat, suggesting a distinct recognition mechanism in the formation of crRNA. In addition, the presence of “self-target” spacers and polymorphisms within CRISPR in Shigella indicated a selective pressure for inhibition of this system, which has the potential to damage “self DNA.” Homology analysis of spacers showed that CRISPR might be involved in the regulation of virulence transmission. Phylogenetic analysis based on CRISPR sequences from Shigella and E. coli indicated that although phenotypic properties maintain convergent evolution, the 4 Shigella species do not represent natural groupings. Surprisingly, comparative analysis of Shigella repeats with other species provided new evidence for CRISPR horizontal transfer. Our results suggested that CRISPR analysis is applicable for the detection of Shigella species and for investigation of evolutionary relationships. PMID:26327282

Polymorphism of CRISPR shows separated natural groupings of Shigella subtypes and evidence of horizontal transfer of CRISPR.

Science.gov (United States)

Yang, Chaojie; Li, Peng; Su, Wenli; Li, Hao; Liu, Hongbo; Yang, Guang; Xie, Jing; Yi, Shengjie; Wang, Jian; Cui, Xianyan; Wu, Zhihao; Wang, Ligui; Hao, Rongzhang; Jia, Leili; Qiu, Shaofu; Song, Hongbin

2015-01-01

Clustered, regularly interspaced, short palindromic repeats (CRISPR) act as an adaptive RNA-mediated immune mechanism in bacteria. They can also be used for identification and evolutionary studies based on polymorphisms within the CRISPR locus. We amplified and analyzed 6 CRISPR loci from 237 Shigella strains belonging to the 4 species groups, as well as 13 Escherichia coli strains. The CRISPR-associated (cas) gene sequence arrays of these strains were screened and compared. The CRISPR sequences from Shigella were conserved among subtypes, suggesting that CRISPR may represent a new identification tool for the detection and discrimination of Shigella species. Secondary structure analysis showed a different stem-loop structure at the terminal repeat, suggesting a distinct recognition mechanism in the formation of crRNA. In addition, the presence of "self-target" spacers and polymorphisms within CRISPR in Shigella indicated a selective pressure for inhibition of this system, which has the potential to damage "self DNA." Homology analysis of spacers showed that CRISPR might be involved in the regulation of virulence transmission. Phylogenetic analysis based on CRISPR sequences from Shigella and E. coli indicated that although phenotypic properties maintain convergent evolution, the 4 Shigella species do not represent natural groupings. Surprisingly, comparative analysis of Shigella repeats with other species provided new evidence for CRISPR horizontal transfer. Our results suggested that CRISPR analysis is applicable for the detection of Shigella species and for investigation of evolutionary relationships.

Myeloid cell leukemia-1 (Mc1-1 is a candidate target gene of hypoxia-inducible factor-1 (HIF-1 in the testis

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Palladino Michael A

2012-12-01

Full Text Available Abstract Background Spermatic cord torsion can lead to testis ischemia (I and subsequent ischemia-reperfusion (I/R causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1 transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’ identified myeloid cell leukemia-1 (Mcl-1 as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1. Methods The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA and electrophoretic mobility shift assays (EMSA. MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP analysis. Results The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter. Conclusions The results

Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction

DEFF Research Database (Denmark)

Markholt, Sara; Grøndahl, M L; Ernst, Erik

2012-01-01

The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood...... but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis......) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors...

Chronic myeloid leukemia patients sensitive and resistant to imatinib treatment show different metabolic responses.

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Jiye A

Full Text Available The BCR-ABL tyrosine kinase inhibitor imatinib is highly effective for chronic myeloid leukemia (CML. However, some patients gradually develop resistance to imatinib, resulting in therapeutic failure. Metabonomic and genomic profiling of patients' responses to drug interventions can provide novel information about the in vivo metabolism of low-molecular-weight compounds and extend our insight into the mechanism of drug resistance. Based on a multi-platform of high-throughput metabonomics, SNP array analysis, karyotype and mutation, the metabolic phenotypes and genomic polymorphisms of CML patients and their diverse responses to imatinib were characterized. The untreated CML patients (UCML showed different metabolic patterns from those of healthy controls, and the discriminatory metabolites suggested the perturbed metabolism of the urea cycle, tricarboxylic acid cycle, lipid metabolism, and amino acid turnover in UCML. After imatinib treatment, patients sensitive to imatinib (SCML and patients resistant to imatinib (RCML had similar metabolic phenotypes to those of healthy controls and UCML, respectively. SCML showed a significant metabolic response to imatinib, with marked restoration of the perturbed metabolism. Most of the metabolites characterizing CML were adjusted to normal levels, including the intermediates of the urea cycle and tricarboxylic acid cycle (TCA. In contrast, neither cytogenetic nor metabonomic analysis indicated any positive response to imatinib in RCML. We report for the first time the associated genetic and metabonomic responses of CML patients to imatinib and show that the perturbed in vivo metabolism of UCML is independent of imatinib treatment in resistant patients. Thus, metabonomics can potentially characterize patients' sensitivity or resistance to drug intervention.

Identification, characterization, and function analysis of the Cactus gene from Litopenaeus vannamei.

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Chaozheng Li

Full Text Available The nuclear factor-kappa B (NF-κB pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C, Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

Individual patient data meta-analysis shows no association between the SNP rs1800469 in TGFB and late radiotherapy toxicity

International Nuclear Information System (INIS)

Barnett, Gillian C.; Elliott, Rebecca M.; Alsner, Jan; Andreassen, Christian N.; Abdelhay, Osama; Burnet, Neil G.; Chang-Claude, Jenny; Coles, Charlotte E.; Gutiérrez-Enríquez, Sara; Fuentes-Raspall, Maria J.; Alonso-Muñoz, Maria C.; Kerns, Sarah; Raabe, Annette; Symonds, R. Paul; Seibold, Petra; Talbot, Chris J.; Wenz, Frederik; Wilkinson, Jennifer; Yarnold, John; Dunning, Alison M.

2012-01-01

Background and purpose: Reported associations between risk of radiation-induced normal tissue injury and single nucleotide polymorphisms (SNPs) in TGFB1, encoding the pro-fibrotic cytokine transforming growth factor-beta 1 (TGF-β1), remain controversial. To overcome publication bias, the international Radiogenomics Consortium collected and analysed individual patient level data from both published and unpublished studies. Materials and methods: TGFB1 SNP rs1800469 c.-1347T>C (previously known as C-509T) genotype, treatment-related data, and clinically-assessed fibrosis (measured at least 2 years after therapy) were available in 2782 participants from 11 cohorts. All received adjuvant breast radiotherapy. Associations between late fibrosis or overall toxicity, reported by STAT (Standardised Total Average Toxicity) score, and rs1800469 genotype were assessed. Results: No statistically significant associations between either fibrosis or overall toxicity and rs1800469 genotype were observed with univariate or multivariate regression analysis. The multivariate odds ratio (OR), obtained from meta-analysis, for an increase in late fibrosis grade with each additional rare allele of rs1800469 was 0.98 (95% Confidence Interval (CI) 0.85–1.11). This CI is sufficiently narrow to rule out any clinically relevant effect on toxicity risk in carriers vs. non-carriers with a high probability. Conclusion: This meta-analysis has not confirmed previous reports of association between fibrosis or overall toxicity and rs1800469 genotype in breast cancer patients. It has demonstrated successful collaboration within the Radiogenomics Consortium.

Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

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Jason M Shapiro

Full Text Available We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1, heat shock protein 90 (HSP90, and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

Cyclin D3 interacts with vitamin D receptor and regulates its transcription activity

International Nuclear Information System (INIS)

Jian Yongzhi; Yan Jun; Wang Hanzhou; Chen Chen; Sun Maoyun; Jiang Jianhai; Lu Jieqiong; Yang Yanzhong; Gu Jianxin

2005-01-01

D-type cyclins are essential for the progression through the G1 phase of the cell cycle. Besides serving as cell cycle regulators, D-type cyclins were recently reported to have transcription regulation functions. Here, we report that cyclin D3 is a new interacting partner of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and the fat-soluble vitamins A and D. The interaction was confirmed with methods of yeast two-hybrid system, in vitro binding analysis and in vivo co-immunoprecipitation. Cyclin D3 interacted with VDR in a ligand-independent manner, but treatment of the ligand, 1,25-dihydroxyvitamin D3, strengthened the interaction. Confocal microscopy analysis showed that ligand-activated VDR led to an accumulation of cyclin D3 in the nuclear region. Cyclin D3 up-regulated transcriptional activity of VDR and this effect was counteracted by overexpression of CDK4 and CDK6. These findings provide us a new clue to understand the transcription regulation functions of D-type cyclins

Perturbed desmosomal cadherin expression in grainy head-like 1-null mice.

Science.gov (United States)

Wilanowski, Tomasz; Caddy, Jacinta; Ting, Stephen B; Hislop, Nikki R; Cerruti, Loretta; Auden, Alana; Zhao, Lin-Lin; Asquith, Stephen; Ellis, Sarah; Sinclair, Rodney; Cunningham, John M; Jane, Stephen M

2008-03-19

In Drosophila, the grainy head (grh) gene plays a range of key developmental roles through the regulation of members of the cadherin gene family. We now report that mice lacking the grh homologue grainy head-like 1 (Grhl1) exhibit hair and skin phenotypes consistent with a reduction in expression of the genes encoding the desmosomal cadherin, desmoglein 1 (Dsg1). Grhl1-null mice show an initial delay in coat growth, and older mice exhibit hair loss as a result of poor anchoring of the hair shaft in the follicle. The mice also develop palmoplantar keratoderma, analogous to humans with DSG1 mutations. Sequence analysis, DNA binding, and chromatin immunoprecipitation experiments demonstrate that the human and mouse Dsg1 promoters are direct targets of GRHL1. Ultrastructural analysis reveals reduced numbers of abnormal desmosomes in the interfollicular epidermis. These findings establish GRHL1 as an important regulator of the Dsg1 genes in the context of hair anchorage and epidermal differentiation, and suggest that cadherin family genes are key targets of the grainy head-like genes across 700 million years of evolution.

Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12

International Nuclear Information System (INIS)

Fukui, Y.; Saito, I.; Shiroki, K.; Shimojo, H.

1984-01-01

The authors isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation

Screening and identification of host factors interacting with UL14 of herpes simplex virus 1.

Science.gov (United States)

Wu, Fuqing; Xing, Junji; Wang, Shuai; Li, Meili; Zheng, Chunfu

2011-08-01

The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

Mechanism of assembly of functional cytochrome C oxidase (CCO) in yeast Candida utilis

International Nuclear Information System (INIS)

Keyhani, J.

1987-01-01

Copper deficiency prevents the assembly of functional CCO; however it was found that both the apoprotein and porphyrin a are synthesized and integrated into the mitochondrial membrane. When copper-deficient cells are grown in copper-supplemented medium in the presence of [ 14 C]δ-aminolevulinic acid, precursor of hemes (Hms) and porphyrins (Porphs), and either chloramphenicol (Cm, inhibitor of mitochondrial protein synthesis) or cycloheximide (CHI, inhibitor of cytoplasmic protein synthesis), the amount of labeled Hms and Porphs is respectively 64% and 5.5% of the control value. When isolated mitochondria from cells grown in the presence of Cm or CHI are solubilized and incubated with antibodies against CCO, radioactivity is found to be associated with the immunoprecipitate. HCl fractionation of Hms and Porphs from the mitochondria and immunoprecipitate shows that a significant amount of radioactivity is extracted with 20% HCl which is the HCl number of porphyrin a. Data indicate that the formation of functional CCO depends on the assembly of holoenzyme, so that ferrochelatase incorporates iron into porphyrin a

Educational Outreach: The Space Science Road Show

Science.gov (United States)

Cox, N. L. J.

2002-01-01

The poster presented will give an overview of a study towards a "Space Road Show". The topic of this show is space science. The target group is adolescents, aged 12 to 15, at Dutch high schools. The show and its accompanying experiments would be supported with suitable educational material. Science teachers at schools can decide for themselves if they want to use this material in advance, afterwards or not at all. The aims of this outreach effort are: to motivate students for space science and engineering, to help them understand the importance of (space) research, to give them a positive feeling about the possibilities offered by space and in the process give them useful knowledge on space basics. The show revolves around three main themes: applications, science and society. First the students will get some historical background on the importance of space/astronomy to civilization. Secondly they will learn more about novel uses of space. On the one hand they will learn of "Views on Earth" involving technologies like Remote Sensing (or Spying), Communication, Broadcasting, GPS and Telemedicine. On the other hand they will experience "Views on Space" illustrated by past, present and future space research missions, like the space exploration missions (Cassini/Huygens, Mars Express and Rosetta) and the astronomy missions (Soho and XMM). Meanwhile, the students will learn more about the technology of launchers and satellites needed to accomplish these space missions. Throughout the show and especially towards the end attention will be paid to the third theme "Why go to space"? Other reasons for people to get into space will be explored. An important question in this is the commercial (manned) exploration of space. Thus, the questions of benefit of space to society are integrated in the entire show. It raises some fundamental questions about the effects of space travel on our environment, poverty and other moral issues. The show attempts to connect scientific with

Hanseniaspora uvarum from winemaking environments show spatial and temporal genetic clustering

Directory of Open Access Journals (Sweden)

Warren eAlbertin

2016-01-01

Full Text Available Hanseniaspora uvarum is one of the most abundant yeast species found on grapes and in grape must, at least before the onset of alcoholic fermentation which is usually performed by Saccharomyces species. The aim of this study was to characterise the genetic and phenotypic variability within the H. uvarum species. One hundred and fifteen strains isolated from winemaking environments in different geographical origins were analysed using 11 microsatellite markers and a subset of 47 strains were analysed by AFLP. H. uvarum isolates clustered mainly on the basis of their geographical localisation as revealed by microsatellites. In addition, a strong clustering based on year of isolation was evidenced, indicating that the genetic diversity of Hanseniaspora uvarum isolates was related to both spatial and temporal variations. Conversely, clustering analysis based on AFLP data provided a different picture with groups showing no particular characteristics, but provided higher strain discrimination. This result indicated that AFLP approaches are inadequate to establish the genetic relationship between individuals, but allowed good strain discrimination. At the phenotypic level, several extracellular enzymatic activities of enological relevance (pectinase, chitinase, protease, β-glucosidase were measured but showed low diversity. The impact of environmental factors of enological interest (temperature, anaerobia and copper addition on growth was also assessed and showed poor variation. Altogether, this work provided both new analytical tool (microsatellites and new insights into the genetic and phenotypic diversity of H. uvarum, a yeast species that has previously been identified as a potential candidate for co-inoculation in grape must, but whose intraspecific variability had never been fully assessed.

Human Bacterial Pathogens in Exported and Imported Foods and Evaluation of Methods of Analysis

Energy Technology Data Exchange (ETDEWEB)

Lustre, A.; Ramos, J.; Elano, R.; Co, C.; Manalastas, Z. [Food Development Center, National Food Authority, Metro Manila (Philippines)

2005-01-15

Phase I consisted of comparison of recovery of Salmonella in inoculated and uninoculated pond prawns, frozen prawns, and imported frozen fish fillet using six available rapid methods vs. conventional method. The best rapid method is S2 which is based on lateral immunoprecipitation. In Phase II, Salmonella was monitored in 120 samples of field prawns, catfish, milkfish and tilapia from ponds. S{sub 8} or Tecra Visual Immuno Assay (VIA) for Salmonella had 99.2 % agreement with BAM/AOAC method. During Phase III, Salmonella was monitored in a further 83 samples of aquaculture prawns and milkfish. Tecra VIA for Salmonella remained number 1 in accuracy with 99% agreement with BAM/AOAC method. (author)

The Construction of National Memory Through Popular Culture. The Case of the Argentinian TV Show,

Directory of Open Access Journals (Sweden)

Elizondo Oviedo, María Verónica

2010-07-01

Full Text Available This article addresses the borders of the complex television sign through the analysis of the Argentinian TV show "Peter Capusotto y sus videos". In it we find that humor is used as a mechanism to dismantle binary hierarchic political and social oppositions. The program is presented as a musical and comedy TV show where concepts such as popular culture and mass culture are questioned through different false musicians. We focus our attention in the character of Bombita Rodríguez, el Palito Ortega montonero due to the controversy generated in the cultural scene by the fact of bringing up recent aspects of Argentinian history.

Pigeons (Columba livia) show change blindness in a color-change detection task.

Science.gov (United States)

Herbranson, Walter T; Jeffers, Jacob S

2017-07-01

Change blindness is a phenomenon whereby changes to a stimulus are more likely go unnoticed under certain circumstances. Pigeons learned a change detection task, in which they observed sequential stimulus displays consisting of individual colors back-projected onto three response keys. The color of one response key changed during each sequence and pecks to the key that displayed the change were reinforced. Pigeons showed a change blindness effect, in that change detection accuracy was worse when there was an inter-stimulus interval interrupting the transition between consecutive stimulus displays. Birds successfully transferred to stimulus displays involving novel colors, indicating that pigeons learned a general change detection rule. Furthermore, analysis of responses to specific color combinations showed that pigeons could detect changes involving both spectral and non-spectral colors and that accuracy was better for changes involving greater differences in wavelength. These results build upon previous investigations of change blindness in both humans and pigeons and suggest that change blindness may be a general consequence of selective visual attention relevant to multiple species and stimulus dimensions.

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

Science.gov (United States)

May, Megan K.; Kevorkian, Richard T.; Steen, Andrew D.

2013-01-01

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly. PMID:24096423

Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

OpenAIRE

Almeida, Sandra; Zhou, Lijuan; Gao, Fen-Biao

2011-01-01

The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, an...

Overcoming Autophagy to Induce Apoptosis in Castration Resistant Prostate Cancer

Science.gov (United States)

2015-10-01

et al., Skp2 regulates androgen receptor through ubiquitin-mediated degradation independent of Akt /mTOR pathways in prostate cancer. Prostate, 2014...I remained fairly steady with only marginal production of LC-3 II, so did the p- Akt . Because of the relative stable AR expression and yet...show ubiquitination of AR via co- immunoprecipitation of AR and ubiquitin. LNCaP C4-2B cells were treated with metformin alone or in combinations

Gc, gc-ms analysis of lipophilic fractions of aerial parts of fagonia indica burm.f. showing growth inhibitory effect on ht 29 colorectal cancer cells

International Nuclear Information System (INIS)

Farheen, R.; Mahmood, I.

2016-01-01

Fagonia indica Burm.f. is a small genus of herbs and under shrubs. The plant contains potentially active substances and has been used traditionally for the treatment of many illnesses including cancer. Many polar compounds have been reported from this plant but its non-polar constituents have only been rarely studied. In the present studies these constituents of aerial parts of Fagonia indica Burm.f. and its sub fractions showing growth inhibitory effect on HT 29 colorectal cancer cells were analyzed using flame ionization detector (GC-FID) and GC-EIMS analysis. The present studies exhibited the presence of free fatty acids and their esters along with structurally diverse constituents including triterpene, heterocyclic organic compound, aromatics, hydrocarbons, alcohols, lactone and sterols which may be responsible for this activity. The results suggest that the non-polar constituents of F. indica bear a potential of further studies. (author)

Phosphorylation of chicken growth hormone

International Nuclear Information System (INIS)

Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

1990-01-01

The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.

Science.gov (United States)

Campbell, E; Pearson, R C; Parkinson, D

1999-11-15

A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

Science.gov (United States)

1996-08-01

that my statement of work (SOW) for the current project omitted many of the tasks that had to be carried out in order to get the lab up and running...that we knew that we could stabilize wild-type p53 in ML-1 cells along with the possibility of being able to get an excellent elutriation profile with...nuclear protein extract was immunoprecipitated with PAb421 cross-linked to ProteinA -Sepharose beads and analysed by SDS-PAGE Western blot analysis with

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity

International Nuclear Information System (INIS)

Hamada, Fumika N.; Koshiyama, Akiyo; Namekawa, Satoshi H.; Ishii, Satomi; Iwabata, Kazuki; Sugawara, Hiroko; Nara, Takayuki Y.; Sakaguchi, Kengo; Sawado, Tomoyuki

2007-01-01

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg 2+ ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis

Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity

Directory of Open Access Journals (Sweden)

Guevara David R

2012-10-01

Full Text Available Abstract Background Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. Results To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen

Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity.

Science.gov (United States)

Guevara, David R; Champigny, Marc J; Tattersall, Ashley; Dedrick, Jeff; Wong, Chui E; Li, Yong; Labbe, Aurelie; Ping, Chien-Lu; Wang, Yanxiang; Nuin, Paulo; Golding, G Brian; McCarry, Brian E; Summers, Peter S; Moffatt, Barbara A; Weretilnyk, Elizabeth A

2012-10-01

Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that Thellungiella shows

Transcriptomic and metabolomic analysis of Yukon Thellungiella plants grown in cabinets and their natural habitat show phenotypic plasticity

Science.gov (United States)

2012-01-01

Background Thellungiella salsuginea is an important model plant due to its natural tolerance to abiotic stresses including salt, cold, and water deficits. Microarray and metabolite profiling have shown that Thellungiella undergoes stress-responsive changes in transcript and organic solute abundance when grown under controlled environmental conditions. However, few reports assess the capacity of plants to display stress-responsive traits in natural habitats where concurrent stresses are the norm. Results To determine whether stress-responsive changes observed in cabinet-grown plants are recapitulated in the field, we analyzed leaf transcript and metabolic profiles of Thellungiella growing in its native Yukon habitat during two years of contrasting meteorological conditions. We found 673 genes showing differential expression between field and unstressed, chamber-grown plants. There were comparatively few overlaps between genes expressed under field and cabinet treatment-specific conditions. Only 20 of 99 drought-responsive genes were expressed both in the field during a year of low precipitation and in plants subjected to drought treatments in cabinets. There was also a general pattern of lower abundance among metabolites found in field plants relative to control or stress-treated plants in growth cabinets. Nutrient availability may explain some of the observed differences. For example, proline accumulated to high levels in cold and salt-stressed cabinet-grown plants but proline content was, by comparison, negligible in plants at a saline Yukon field site. We show that proline accumulated in a stress-responsive manner in Thellungiella plants salinized in growth cabinets and in salt-stressed seedlings when nitrogen was provided at 1.0 mM. In seedlings grown on 0.1 mM nitrogen medium, the proline content was low while carbohydrates increased. The relatively higher content of sugar-like compounds in field plants and seedlings on low nitrogen media suggests that

The regulatory effects of low-dose ionizing radiation on Ikaros-autotaxin interaction

Energy Technology Data Exchange (ETDEWEB)

Kang, Hana; Cho, Seong Jun; Kim, Sung Jin; Nam, Seon Young; Yang, Kwang Hee [KHNP Radiation Health Institute, Korea Hydro and Nuclear Power Co, Seoul (Korea, Republic of)

2016-11-15

Ikaros, a transcription factor containing zinc-finger motif, has known as a critical regulator of hematopoiesis in immune system. Ikaros protein modulates the transcription of target genes via binding to the regulatory elements of the genes promoters. However the regulatory function of Ikaros in other organelle except nuclear remains to be determined. This study explored radiation-induced modulatory function of Ikaros in cytoplasm. The results showed that Ikaros protein lost its DNA binding ability after LDIR (low-dose ionizing radiation) exposure. Cell fractionation and Western blot analysis showed that Ikaros protein was translocated into cytoplasm from nuclear by LDIR. This was confirmed by immunofluorescence assay. We identified Autotaxin as a novel protein which potentially interacts with Ikaros through in vitro protein-binding screening. Co-immunoprecipitation assay revealed that Ikaros and Autotaxin are able to bind each other. Autotaxin is a crucial enzyme generating lysophosphatidic acid (LPA), a phospholipid mediator, which has potential regulatory effects on immune cell growth and motility. Our results indicate that LDIR potentially regulates immune system via protein-protein interaction of Ikaros and Autotaxin.

Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene

International Nuclear Information System (INIS)

Kaneoka, Hidenori; Miyake, Katsuhide; Iijima, Shinji

2009-01-01

The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5 kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIP assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5 kb S/MAR-GRE) and the promoter to the inner nuclear matrix.

Interactions between the nuclear matrix and an enhancer of the tryptophan oxygenase gene

Energy Technology Data Exchange (ETDEWEB)

Kaneoka, Hidenori [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Miyake, Katsuhide, E-mail: miyake@nubio.nagoya-u.ac.jp [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Iijima, Shinji [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan)

2009-10-02

The gene for tryptophan oxygenase (TO) is expressed in adult hepatocytes in a tissue- and differentiation-specific manner. The TO promoter has two glucocorticoid-responsive elements (GREs), and its expression is regulated by glucocorticoid hormone in the liver. We found a novel GRE in close proximity to a scaffold/matrix attachment region (S/MAR) that was located around -8.5 kb from the transcriptional start site of the TO gene by electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays. A combination of nuclear fractionation and quantitative PCR analysis showed that the S/MAR was tethered to the nuclear matrix in both fetal and adult hepatocytes. ChIP assay showed that, in adult hepatocytes, the S/MAR-GRE and the promoter proximal regions interacted with lamin and heterogeneous nuclear ribonucleoprotein U in a dexamethasone dependent manner, but this was not the case in fetal cells, suggesting that developmental stage-specific expression of the TO gene might rely on the binding of the enhancer (the -8.5 kb S/MAR-GRE) and the promoter to the inner nuclear matrix.

Effect of gamma radiation on the leakage of substances from lymphocytes. In vitro study using immuno-precipitation techniques in gel medium

International Nuclear Information System (INIS)

Dubos, M.; Nguyen, T.-L.; Drouet, J.; Goujon, P.

In vitro effect of a 5000 R irradiation on rat blood lymphocytes was studied during several days in surviving cell suspensions, at various incubation temperature. Immunochemical analysis in agarose gel, using the simple diffusion technique, exhibited the leakage of two groups of antigenic compounds, from lymphocytes. The first group compounds seemed to be periodical renewal products of surface cell membrane elements, common to lymphocytes and erythrocytes; irradiation increased their release. A correlation was established between the second group compounds and cell metabolism and these compounds seemed to be enzymes of cytolitic origin [fr

Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

International Nuclear Information System (INIS)

Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

2011-01-01

Highlights: → Gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interact with the promoter region of IFNγ-associated genes along with transcription factor STAT1α. → We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. → The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The

Time dependent patient no-show predictive modelling development.

Science.gov (United States)

Huang, Yu-Li; Hanauer, David A

2016-05-09

Purpose - The purpose of this paper is to develop evident-based predictive no-show models considering patients' each past appointment status, a time-dependent component, as an independent predictor to improve predictability. Design/methodology/approach - A ten-year retrospective data set was extracted from a pediatric clinic. It consisted of 7,291 distinct patients who had at least two visits along with their appointment characteristics, patient demographics, and insurance information. Logistic regression was adopted to develop no-show models using two-thirds of the data for training and the remaining data for validation. The no-show threshold was then determined based on minimizing the misclassification of show/no-show assignments. There were a total of 26 predictive model developed based on the number of available past appointments. Simulation was employed to test the effective of each model on costs of patient wait time, physician idle time, and overtime. Findings - The results demonstrated the misclassification rate and the area under the curve of the receiver operating characteristic gradually improved as more appointment history was included until around the 20th predictive model. The overbooking method with no-show predictive models suggested incorporating up to the 16th model and outperformed other overbooking methods by as much as 9.4 per cent in the cost per patient while allowing two additional patients in a clinic day. Research limitations/implications - The challenge now is to actually implement the no-show predictive model systematically to further demonstrate its robustness and simplicity in various scheduling systems. Originality/value - This paper provides examples of how to build the no-show predictive models with time-dependent components to improve the overbooking policy. Accurately identifying scheduled patients' show/no-show status allows clinics to proactively schedule patients to reduce the negative impact of patient no-shows.

CLIP-seq analysis of multi-mapped reads discovers novel functional RNA regulatory sites in the human transcriptome.

Science.gov (United States)

Zhang, Zijun; Xing, Yi

2017-09-19

Crosslinking or RNA immunoprecipitation followed by sequencing (CLIP-seq or RIP-seq) allows transcriptome-wide discovery of RNA regulatory sites. As CLIP-seq/RIP-seq reads are short, existing computational tools focus on uniquely mapped reads, while reads mapped to multiple loci are discarded. We present CLAM (CLIP-seq Analysis of Multi-mapped reads). CLAM uses an expectation-maximization algorithm to assign multi-mapped reads and calls peaks combining uniquely and multi-mapped reads. To demonstrate the utility of CLAM, we applied it to a wide range of public CLIP-seq/RIP-seq datasets involving numerous splicing factors, microRNAs and m6A RNA methylation. CLAM recovered a large number of novel RNA regulatory sites inaccessible by uniquely mapped reads. The functional significance of these sites was demonstrated by consensus motif patterns and association with alternative splicing (splicing factors), transcript abundance (AGO2) and mRNA half-life (m6A). CLAM provides a useful tool to discover novel protein-RNA interactions and RNA modification sites from CLIP-seq and RIP-seq data, and reveals the significant contribution of repetitive elements to the RNA regulatory landscape of the human transcriptome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Immunological and biochemical studies of fascioliasis in goats and cattle

Energy Technology Data Exchange (ETDEWEB)

Reddington, J.J.

1985-01-01

Using the goat as a susceptible host and cattle as a resistant species to Fasciola hepatica infections, the humoral response of these animals to the surface of the newly excysted juvenile (NEJ) fluke was examined. Tegumental proteins of the NEJ were labeled with /sup 125/I by lactoperoxidase and analyzed after immunoprecipitation using a double antibody system. In addition, a comparison was made between the infected sera's capacity to immunoprecipitate surface antigens and their in vitro cytotoxic activity against the NEJ. In both goats and cattle the levels of NEJ surface antigens precipitated increased during the first 4 weeks PI. The peak immunoprecipitation of NEJ surface antigens by cattle sera (58%) was significantly higher than that of infected goat sera (33%). Immunoprecipitation of the available radiolabeled NEJ surface proteins by the infected cattle sera remained consistently higher than goat sera until the 16th week PI. The cytotoxic effects of these same caprine sera on NEJs in vitro was limited, while the cytotoxicity of the infected bovine sera closely approximated the sera's ability to precipitate NEJ surface antigens. There was also a qualitative difference between the species in their recognition of /sup 35/S and /sup 125/I radiolabeled NEJ surface antigens. Uninfected goat or cattle sera failed to precipitate any /sup 125/I or /sup 35/S-labeled surface proteins.

Immunological and biochemical studies of fascioliasis in goats and cattle

International Nuclear Information System (INIS)

Reddington, J.J.

1985-01-01

Using the goat as a susceptible host and cattle as a resistant species to Fasciola hepatica infections, the humoral response of these animals to the surface of the newly excysted juvenile (NEJ) fluke was examined. Tegumental proteins of the NEJ were labeled with 125 I by lactoperoxidase and analyzed after immunoprecipitation using a double antibody system. In addition, a comparison was made between the infected sera's capacity to immunoprecipitate surface antigens and their in vitro cytotoxic activity against the NEJ. In both goats and cattle the levels of NEJ surface antigens precipitated increased during the first 4 weeks PI. The peak immunoprecipitation of NEJ surface antigens by cattle sera (58%) was significantly higher than that of infected goat sera (33%). Immunoprecipitation of the available radiolabeled NEJ surface proteins by the infected cattle sera remained consistently higher than goat sera until the 16th week PI. The cytotoxic effects of these same caprine sera on NEJs in vitro was limited, while the cytotoxicity of the infected bovine sera closely approximated the sera's ability to precipitate NEJ surface antigens. There was also a qualitative difference between the species in their recognition of 35 S and 125 I radiolabeled NEJ surface antigens. Uninfected goat or cattle sera failed to precipitate any 125 I or 35 S-labeled surface proteins

Reality, ficción o show

Directory of Open Access Journals (Sweden)

Sandra Ruíz Moreno

2002-01-01

Full Text Available Para tener un punto de vista claro y objetivo frente a la polémica establecida en torno al programa “Protagonistas de novela” y la tendiente proliferación de los reality show en las parrillas de programación de la televisión colombiana, se realizó un análisis de texto y contenido de dicho programa, intentando definirlo desde sus posibilidades de realidad, ficción y show. Las unidades de análisis y el estudio de su tratamiento arrojaron un alto contenido que gira en torno a las emociones del ser humano relacionadas con la convivencia, tratadas a manera de show y con algunos aportes textuales de ficción, pero sin su elemento mediador básico, el actor, quitándole toda la posibilidad de tener un tratamiento con la profundidad, distancia y ética que requieren los temas de esta índole. El resultado es un formato que sólo busca altos índices de sintonía y que pertenece más a la denominada televisión “trash”, que a una búsqueda de realidad del hombre y mucho menos de sociedad.

Analysis of human articular chondrocyte CD44 isoform expression and function in health and disease.

Science.gov (United States)

Salter, D M; Godolphin, J L; Gourlay, M S; Lawson, M F; Hughes, D E; Dunne, E

1996-08-01

Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

Quantitative Analysis of the Contributing Factors Affecting Specialty Care No-Show Rates at Brooke Army Medical Center

Science.gov (United States)

2007-03-30

PHYSICAL THERAPY ,TMC 53 9 14.52% AUDIOLOGY,BAMC 133 22 14.19% OPHTH PEDS,BAMC 28 4 12.50% OCCUPATIONAL THERAPY ,BAMC 135 18 11.76% ALLERGY CLINIC,BAMC...for physical therapy and have a total of 4 appointments set up with that clinic. For this study, only the first referral was counted and not the follow...Nephrology. Some of the other low no-show rate clinics were: Pain Management (1.27%), Endocrinology (1.52%), General Surgery (1.83%), Rheumatology (1.89

Alcohol Content in the ‘Hyper-Reality’ MTV Show ‘Geordie Shore’

Science.gov (United States)

Lowe, Eden; Britton, John

2018-01-01

Abstract Aim To quantify the occurrence of alcohol content, including alcohol branding, in the popular primetime television UK Reality TV show ‘Geordie Shore’ Series 11. Methods A 1-min interval coding content analysis of alcohol content in the entire DVD Series 11 of ‘Geordie Shore’ (10 episodes). Occurrence of alcohol use, implied use, other alcohol reference/paraphernalia or branding was recorded. Results All categories of alcohol were present in all episodes. ‘Any alcohol’ content occurred in 78%, ‘actual alcohol use’ in 30%, ‘inferred alcohol use’ in 72%, and all ‘other’ alcohol references occurred in 59% of all coding intervals (ACIs), respectively. Brand appearances occurred in 23% of ACIs. The most frequently observed alcohol brand was Smirnoff which appeared in 43% of all brand appearances. Episodes categorized as suitable for viewing by adolescents below the legal drinking age of 18 years comprised of 61% of all brand appearances. Conclusions Alcohol content, including branding, is highly prevalent in the UK Reality TV show ‘Geordie Shore’ Series 11. Two-thirds of all alcohol branding occurred in episodes age-rated by the British Board of Film Classification (BBFC) as suitable for viewers aged 15 years. The organizations OfCom, Advertising Standards Authority (ASA) and the Portman Group should implement more effective policies to reduce adolescent exposure to on-screen drinking. The drinks industry should consider demanding the withdrawal of their brands from the show. Short Summary Alcohol content, including branding, is highly prevalent in the MTV reality TV show ‘Geordie Shore’ Series 11. Current alcohol regulation is failing to protect young viewers from exposure to such content. PMID:29365032

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

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Meilin Wu

Full Text Available Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS, which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K channels and downregulating nicotinic acetylcholine receptors (nAChRs in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

Science.gov (United States)

Wu, Meilin; Liu, Clifford Z; Joiner, William J

2016-01-01

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

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Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

2007-01-01

Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

Characterization and interactome study of white spot syndrome virus envelope protein VP11.

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Wang-Jing Liu

Full Text Available White spot syndrome virus (WSSV is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570, and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

Epigenetic regulation of the glucose transporter gene Slc2a1 by β-hydroxybutyrate underlies preferential glucose supply to the brain of fasted mice.

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Tanegashima, Kosuke; Sato-Miyata, Yukiko; Funakoshi, Masabumi; Nishito, Yasumasa; Aigaki, Toshiro; Hara, Takahiko

2017-01-01

We carried out liquid chromatography-tandem mass spectrometry analysis of metabolites in mice. Those metabolome data showed that hepatic glucose content is reduced, but that brain glucose content is unaffected, during fasting, consistent with the priority given to brain glucose consumption during fasting. The molecular mechanisms for this preferential glucose supply to the brain are not fully understood. We also showed that the fasting-induced production of the ketone body β-hydroxybutyrate (β-OHB) enhances expression of the glucose transporter gene Slc2a1 (Glut1) via histone modification. Upon β-OHB treatment, Slc2a1 expression was up-regulated, with a concomitant increase in H3K9 acetylation at the critical cis-regulatory region of the Slc2a1 gene in brain microvascular endothelial cells and NB2a neuronal cells, shown by quantitative PCR analysis and chromatin immunoprecipitation assay. CRISPR/Cas9-mediated disruption of the Hdac2 gene increased Slc2a1 expression, suggesting that it is one of the responsible histone deacetylases (HDACs). These results confirm that β-OHB is a HDAC inhibitor and show that β-OHB plays an important role in fasting-induced epigenetic activation of a glucose transporter gene in the brain. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

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Edgar E. Lara-Ramírez

2013-11-01

Full Text Available The past pandemic strain H1N1 (A (H1N1pdm09 has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI database were used for the analysis. The epitope compositions in A (H1N1pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB, were compared at genomic level. The result showed that A (H1N1 pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA, which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1pdm09 virus. This study may be helpful for better understanding of A (H1N1pdm09 and the production of new influenza vaccines.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

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Eric J Chater-Diehl

Full Text Available The molecular basis of Fetal Alcohol Spectrum Disorders (FASD is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

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Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

2016-01-01

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Genome-wide identification of Bcl11b gene targets reveals role in brain-derived neurotrophic factor signaling.

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Bin Tang

Full Text Available B-cell leukemia/lymphoma 11B (Bcl11b is a transcription factor showing predominant expression in the striatum. To date, there are no known gene targets of Bcl11b in the nervous system. Here, we define targets for Bcl11b in striatal cells by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq in combination with genome-wide expression profiling. Transcriptome-wide analysis revealed that 694 genes were significantly altered in striatal cells over-expressing Bcl11b, including genes showing striatal-enriched expression similar to Bcl11b. ChIP-seq analysis demonstrated that Bcl11b bound a mixture of coding and non-coding sequences that were within 10 kb of the transcription start site of an annotated gene. Integrating all ChIP-seq hits with the microarray expression data, 248 direct targets of Bcl11b were identified. Functional analysis on the integrated gene target list identified several zinc-finger encoding genes as Bcl11b targets, and further revealed a significant association of Bcl11b to brain-derived neurotrophic factor/neurotrophin signaling. Analysis of ChIP-seq binding regions revealed significant consensus DNA binding motifs for Bcl11b. These data implicate Bcl11b as a novel regulator of the BDNF signaling pathway, which is disrupted in many neurological disorders. Specific targeting of the Bcl11b-DNA interaction could represent a novel therapeutic approach to lowering BDNF signaling specifically in striatal cells.

The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3

International Nuclear Information System (INIS)

Alper, O.; Yamaguchi, K.; Hitomi, J.; Honda, S.; Matsushima, T.; Abe, K.

1990-01-01

The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein

Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

International Nuclear Information System (INIS)

Müller, Imke; Wischnewski, Frank; Pantel, Klaus; Schwarzenbach, Heidi

2010-01-01

The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs) and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3) at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. This study is one of the first to reveal the histone code and MBD profile

Candidate genetic pathways for attention-deficit/hyperactivity disorder (ADHD) show association to hyperactive/impulsive symptoms in children with ADHD.

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Bralten, Janita; Franke, Barbara; Waldman, Irwin; Rommelse, Nanda; Hartman, Catharina; Asherson, Philip; Banaschewski, Tobias; Ebstein, Richard P; Gill, Michael; Miranda, Ana; Oades, Robert D; Roeyers, Herbert; Rothenberger, Aribert; Sergeant, Joseph A; Oosterlaan, Jaap; Sonuga-Barke, Edmund; Steinhausen, Hans-Christoph; Faraone, Stephen V; Buitelaar, Jan K; Arias-Vásquez, Alejandro

2013-11-01

Because multiple genes with small effect sizes are assumed to play a role in attention-deficit/hyperactivity disorder (ADHD) etiology, considering multiple variants within the same analysis likely increases the total explained phenotypic variance, thereby boosting the power of genetic studies. This study investigated whether pathway-based analysis could bring scientists closer to unraveling the biology of ADHD. The pathway was described as a predefined gene selection based on a well-established database or literature data. Common genetic variants in pathways involved in dopamine/norepinephrine and serotonin neurotransmission and genes involved in neuritic outgrowth were investigated in cases from the International Multicentre ADHD Genetics (IMAGE) study. Multivariable analysis was performed to combine the effects of single genetic variants within the pathway genes. Phenotypes were DSM-IV symptom counts for inattention and hyperactivity/impulsivity (n = 871) and symptom severity measured with the Conners Parent (n = 930) and Teacher (n = 916) Rating Scales. Summing genetic effects of common genetic variants within the pathways showed a significant association with hyperactive/impulsive symptoms ((p)empirical = .007) but not with inattentive symptoms ((p)empirical = .73). Analysis of parent-rated Conners hyperactive/impulsive symptom scores validated this result ((p)empirical = .0018). Teacher-rated Conners scores were not associated. Post hoc analyses showed a significant contribution of all pathways to the hyperactive/impulsive symptom domain (dopamine/norepinephrine, (p)empirical = .0004; serotonin, (p)empirical = .0149; neuritic outgrowth, (p)empirical = .0452). The present analysis shows an association between common variants in 3 genetic pathways and the hyperactive/impulsive component of ADHD. This study demonstrates that pathway-based association analyses, using quantitative measurements of ADHD symptom domains, can increase the power of genetic analyses to

Laser entertainment and light shows in education

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Sabaratnam, Andrew T.; Symons, Charles

2002-05-01

Laser shows and beam effects have been a source of entertainment since its first public performance May 9, 1969, at Mills College in Oakland, California. Since 1997, the Photonics Center, NgeeAnn Polytechnic, Singapore, has been using laser shows as a teaching tool. Students are able to exhibit their creative skills and learn at the same time how lasers are used in the entertainment industry. Students will acquire a number of skills including handling three- phase power supply, operation of cooling system, and laser alignment. Students also acquire an appreciation of the arts, learning about shapes and contours as they develop graphics for the shows. After holography, laser show animation provides a combination of the arts and technology. This paper aims to briefly describe how a krypton-argon laser, galvanometer scanners, a polychromatic acousto-optic modulator and related electronics are put together to develop a laser projector. The paper also describes how students are trained to make their own laser animation and beam effects with music, and at the same time have an appreciation of the operation of a Class IV laser and the handling of optical components.

Men and women show similar survival outcome in stage IV breast cancer.

Science.gov (United States)

Wu, San-Gang; Zhang, Wen-Wen; Liao, Xu-Lin; Sun, Jia-Yuan; Li, Feng-Yan; Su, Jing-Jun; He, Zhen-Yu

2017-08-01

To evaluate the clinicopathological features, patterns of distant metastases, and survival outcome between stage IV male breast cancer (MBC) and female breast cancer (FBC). Patients diagnosed with stage IV MBC and FBC between 2010 and 2013 were included using the Surveillance, Epidemiology, and End Results program. Univariate and multivariate Cox regression analyses were used to analyze risk factors for overall survival (OS). A total of 4997 patients were identified, including 60 MBC and 4937 FBC. Compared with FBC, patients with MBC were associated with a significantly higher rate of estrogen receptor-positive, progesterone receptor-positive, unmarried, lung metastases, and a lower frequency of liver metastases. Univariate and multivariate analyses showed no significant difference in OS between MBC and FBC. In the propensity score-matched population, there was also no difference in survival between MBC and FBC. Multivariate analysis of MBC showed that OS was longer for patients aged 50-69 years and with estrogen receptor-positive disease. There was no significant difference in survival outcome between stage IV MBC and FBC, but significant differences in clinicopathological features and patterns of metastases between the genders. Copyright © 2017 Elsevier Ltd. All rights reserved.

CXCR7 is induced by hypoxia and mediates glioma cell migration towards SDF-1α

International Nuclear Information System (INIS)

Esencay, Mine; Sarfraz, Yasmeen; Zagzag, David

2013-01-01

Glioblastomas, the most common and malignant brain tumors of the central nervous system, exhibit high invasive capacity, which hinders effective therapy. Therefore, intense efforts aimed at improved therapeutics are ongoing to delineate the molecular mechanisms governing glioma cell migration and invasion. In order to perform the studies, we employed optimal cell culture methods and hypoxic conditions, lentivirus-mediated knockdown of protein expression, Western Blot analysis, migration assays and immunoprecipitation. We determined statistical significance by unpaired t-test. In this report, we show that U87MG, LN229 and LN308 glioma cells express CXCR7 and that exposure to hypoxia upregulates CXCR7 protein expression in these cell lines. CXCR7-expressing U87MG, LN229 and LN308 glioma cells migrated towards stromal-derived factor (SDF)-1α/CXCL12 in hypoxic conditions in the Boyden chamber assays. While shRNA-mediated knockdown of CXCR7 expression did not affect the migration of any of the three cell lines in normoxic conditions, we observed a reduction in the migration of LN229 and LN308, but not U87MG, glioma cells towards SDF-1α in hypoxic conditions. In addition, knockdown of CXCR7 expression in LN229 and LN308 glioma cells decreased levels of SDF-1α-induced phosphorylation of ERK1/2 and Akt. Inhibiting CXCR4 in LN229 and LN308 glioma cells that were knocked down for CXCR7 did not further reduce migration towards SDF-1α in hypoxic conditions and did not affect the levels of phosphorylated ERK1/2 and Akt. Analysis of immunoprecipitated CXCR4 from LN229 and LN308 glioma cells revealed co-precipitated CXCR7. Taken together, our findings indicate that both CXCR4 and CXCR7 mediate glioma cell migration towards SDF-1α in hypoxic conditions and support the development of therapeutic agents targeting these receptors

Up-Regulation of Follistatin-Like 1 By the Androgen Receptor and Melanoma Antigen-A11 in Prostate Cancer.

Science.gov (United States)

Su, Shifeng; Parris, Amanda B; Grossman, Gail; Mohler, James L; Wang, Zengjun; Wilson, Elizabeth M

2017-04-01

High affinity androgen binding to the androgen receptor (AR) activates genes required for male sex differentiation and promotes the development and progression of prostate cancer. Human AR transcriptional activity involves interactions with coregulatory proteins that include primate-specific melanoma antigen-A11 (MAGE-A11), a coactivator that increases AR transcriptional activity during prostate cancer progression to castration-resistant/recurrent prostate cancer (CRPC). Microarray analysis and quantitative RT-PCR were performed to identify androgen-regulated MAGE-A11-dependent genes in LAPC-4 prostate cancer cells after lentivirus shRNA knockdown of MAGE-A11. Chromatin immunoprecipitation was used to assess androgen-dependent AR recruitment, and immunocytochemistry to localize an androgen-dependent protein in prostate cancer cells and tissue and in the CWR22 human prostate cancer xenograft. Microarray analysis of androgen-treated LAPC-4 prostate cancer cells indicated follistatin-like 1 (FSTL1) is up-regulated by MAGE-A11. Androgen-dependent up-regulation of FSTL1 was inhibited in LAPC-4 cells by lentivirus shRNA knockdown of AR or MAGE-A11. Chromatin immunoprecipitation demonstrated AR recruitment to intron 10 of the FSTL1 gene that contains a classical consensus androgen response element. Increased levels of FSTL1 protein in LAPC-4 cells correlated with higher levels of MAGE-A11 relative to other prostate cancer cells. FSTL1 mRNA levels increased in CRPC and castration-recurrent CWR22 xenografts in association with predominantly nuclear FSTL1. Increased nuclear localization of FSTL1 in prostate cancer was suggested by predominantly cytoplasmic FSTL1 in benign prostate epithelial cells and predominantly nuclear FSTL1 in epithelial cells in CRPC tissue and the castration-recurrent CWR22 xenograft. AR expression studies showed nuclear colocalization of AR and endogenous FSTL1 in response to androgen. AR and MAGE-A11 cooperate in the up-regulation of FSTL1 to

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

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Veronika N Bade

Full Text Available The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls, ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation. ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no

Alterations of retinol-binding protein 4 species in patients with different stages of chronic kidney disease and their relation to lipid parameters

DEFF Research Database (Denmark)

Henze, Andrea; Frey, Simone K; Raila, Jens

2010-01-01

) was assessed in serum of 45 healthy controls and 52 patients with stage 2-5 of CKD using ELISA and RBP4 immunoprecipitation with subsequent MALDI-TOF-MS analysis. A reduction of glomerular filtration rate was accompanied by a gradual elevation of RBP4 serum levels and relative amounts of RBP4-LL. Correlation...... analysis revealed a strong association of the RBP4-TTR ratio with parameters of lipid metabolism and with diabetes-related factors. In conclusion, RBP4 serum concentration and the appearance of RBP4-LL seem to be influenced by kidney function. Furthermore, the RBP4-TTR ratio may provide diagnostic...

Davedan Show Di Amphi Theatre Nusa Dua Bali

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Ni Made Ruastiti

2018-05-01

Full Text Available Artikel ini disusun dari hasil penelitian yang bertujuan untuk dapat memahami pertunjukan Davedan Show di Amphi Theatre Nusa Dua Bali. Penelitian ini dilakukan karena adanya ketimpangan antara asumsi dan kenyataan di lapangan. Pada umumnya wisatawan yang datang ke Bali hanya senang dan antusias menonton seni pertunjukan pariwisata berbasis seni budaya lokal saja. Tetapi kenyataan ini berbeda. Walaupun Davedan Show tidak dibangun dari seni budaya lokal saja, tetapi kenyataannya wisatawan sangat senang menonton pertunjukan tersebut. Pertanyaannya: bagaimanakah bentuk pertunjukan Davedan Show tersebut?; mengapa wisatawan senang menonton pertunjukan itu?; apa implikasinya bagi pelaku, masyarakat, dan industri pariwisata di Nusa Dua, Bali?. Penelitian ini menggunakan metode penelitian kualitatif, khususnya implementatif partisipatoris yang mengutamakan kerjasama antara periset dengan para informan terkait. Sumber data penelitian ini adalah pertunjukan Davedan itu sendiri, pihak manajemen, para penari, penonton, hasil-hasil penelitian yang telah ada sebelumnya. Seluruh data yang telah dikumpulkan dengan teknik observasi, wawancara, FGD, dan studi kepustakaan itu dianalisis secara kritis dengan menggunakan teori estetika postmodern, teori praktik, dan teori relasi kuasa pengetahuan. Hasil penelitian menunjukan bahwa: (1 Davedan Show disajikan dalam bentuk oratorium. Hal itu dapat dilihat dari cara penyajian, koreografi, dan iringan pertunjukannya. Davedan Show yang menampilkan tema Treasure of The Archipelago, membuka gerbang petualangan baru itu diiringi musik rekaman etnik Nusantara secara medley, berkelanjutan dengan struktur pertunjukan: seni budaya Bali, Sumatra, Sunda, Solo, Kalimantan, dan seni budaya Papua; (2 Davedan Show banyak diminati wisatawan manca negara karena penciptaan pertunjukan itu dilatari oleh ideologi pasar, ideologi estetika, dan ideologi budaya Nusantara; (3 Hingga kini Davedan Show berkembang secara berkelanjutan di Nusa Dua

A novel missense mutation (G43S) in the switch I region of Rab27A causing Griscelli syndrome

DEFF Research Database (Denmark)

Westbroek, W.; Tuchman, M.; Tinloy, B.

2008-01-01

The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking......-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins Udgivelsesdato: 2008/6...

Interactions of Polyhomeotic with Polycomb Group Genes of Drosophila Melanogaster

OpenAIRE

Cheng, N. N.; Sinclair, DAR.; Campbell, R. B.; Brock, H. W.

1994-01-01

The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting ...

UV Photography Shows Hidden Sun Damage

Science.gov (United States)

... mcat1=de12", ]; for (var c = 0; c UV photography shows hidden sun damage A UV photograph gives ... developing skin cancer and prematurely aged skin. Normal photography UV photography 18 months of age: This boy's ...

The NRP1 migraine risk variant shows evidence of association with menstrual migraine.

Science.gov (United States)

Pollock, Charmaine E; Sutherland, Heidi G; Maher, Bridget H; Lea, Rodney A; Haupt, Larisa M; Frith, Alison; Anne MacGregor, E; Griffiths, Lyn R

2018-04-18

In 2016, a large meta-analysis brought the number of susceptibility loci for migraine to 38. While sub-type analysis for migraine without aura (MO) and migraine with aura (MA) found some loci showed specificity to MO, the study did not test the loci with respect to other subtypes of migraine. This study aimed to test the hypothesis that single nucleotide polymorphisms (SNPs) robustly associated with migraine are individually or collectively associated with menstrual migraine (MM). Genotyping of migraine susceptibility SNPs was conducted using the Agena MassARRAY platform on DNA samples from 235 women diagnosed with menstrual migraine as per International Classification for Headache Disorders II (ICHD-II) criteria and 140 controls. Alternative genotyping methods including restriction fragment length polymorphism, pyrosequencing and Sanger sequencing were used for validation. Statistical analysis was performed using PLINK and SPSS. Genotypes of 34 SNPs were obtained and investigated for their potential association with menstrual migraine. Of these SNPs, rs2506142 located near the neuropilin 1 gene (NRP1), was found to be significantly associated with menstrual migraine (p = 0.003). Genomic risk scores were calculated for all 34 SNPs as well as a subset of 7 SNPs that were nearing individual significance. Overall, this analysis suggested these SNPs to be weakly predictive of MM, but of no prognostic or diagnostic value. Our results suggest that NRP1 may be important in the etiology of MM. It also suggests some genetic commonality between common migraine subtypes (MA and MO) and MM. The identification of associated SNPs may be the starting point to a better understanding of how genetic factors may contribute to the menstrual migraine sub-type.

Effects of gamma radiation on Crotalus durissus terrificus venom

International Nuclear Information System (INIS)

Murata, Y.

1988-01-01

A poll of crotomine positive Crotalus durissus terrificus venom was dissolved in 0.15 M NaCl and the supernatant irradiated using 60 CO. Doses of 100,250,750,1000,1500 and 2000 Cy were used at a dose rate of 1.190Gy/h. The presence of free SH, casein hydrolytic degradation, SDS-PAGE and molecular exclusion chromatography analysis together with LD 50 determination in mice were studied. The antigenic properties of samples were investigated by immunodiffision and immunoprecipitation. (M.A.C.) [pt

The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

OpenAIRE

Mansour, Marc R.; Sanda, Takaomi; Lawton, Lee N.; Li, Xiaoyu; Kreslavsky, Taras; Novina, Carl D.; Brand, Marjorie; Gutierrez, Alejandro; Kelliher, Michelle A.; Jamieson, Catriona H.M.; von Boehmer, Harald; Young, Richard A.; Look, A. Thomas

2013-01-01

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GAT...

The three catalases in Deinococcus radiodurans: Only two show catalase activity

Energy Technology Data Exchange (ETDEWEB)

Jeong, Sun-Wook [Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, 580-185 (Korea, Republic of); Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Jung, Jong-Hyun; Kim, Min-Kyu; Seo, Ho Seong [Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, 580-185 (Korea, Republic of); Lim, Heon-Man [Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, 305-764 (Korea, Republic of); Lim, Sangyong, E-mail: saylim@kaeri.re.kr [Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, 580-185 (Korea, Republic of)

2016-01-15

Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H{sub 2}O{sub 2}) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H{sub 2}O{sub 2} treatments, whereas ΔdrA0146 showed no change in its H{sub 2}O{sub 2} resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H{sub 2}O{sub 2}, but DRA0146 does not have catalase activity and is not involved in the resistance to H{sub 2}O{sub 2} stress. - Highlights: • The dr1998 mutant strain lost 90% of its total catalase activity. • Increased ROS levels and decreased H{sub 2}O{sub 2} resistance were observed in dr1998 mutants. • Lack of drA0146 did not affect any oxidative stress-related phenotypes. • The purified DRA0146 did not show catalase activity.

The three catalases in Deinococcus radiodurans: Only two show catalase activity

International Nuclear Information System (INIS)

Jeong, Sun-Wook; Jung, Jong-Hyun; Kim, Min-Kyu; Seo, Ho Seong; Lim, Heon-Man; Lim, Sangyong

2016-01-01

Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H_2O_2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H_2O_2 treatments, whereas ΔdrA0146 showed no change in its H_2O_2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H_2O_2, but DRA0146 does not have catalase activity and is not involved in the resistance to H_2O_2 stress. - Highlights: • The dr1998 mutant strain lost 90% of its total catalase activity. • Increased ROS levels and decreased H_2O_2 resistance were observed in dr1998 mutants. • Lack of drA0146 did not affect any oxidative stress-related phenotypes. • The purified DRA0146 did not show catalase activity.

Cell cloning-based transcriptome analysis in Rett patients: relevance to the pathogenesis of Rett syndrome of new human MeCP2 target genes.

Science.gov (United States)

Nectoux, J; Fichou, Y; Rosas-Vargas, H; Cagnard, N; Bahi-Buisson, N; Nusbaum, P; Letourneur, F; Chelly, J; Bienvenu, T

2010-07-01

More than 90% of Rett syndrome (RTT) patients have heterozygous mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene that encodes the methyl-CpG-binding protein 2, a transcriptional modulator. Because MECP2 is subjected to X chromosome inactivation (XCI), girls with RTT either express the wild-type or mutant allele in each individual cell. To test the consequences of MECP2 mutations resulting from a genome-wide transcriptional dysregulation and to identify its target genes in a system that circumvents the functional mosaicism resulting from XCI, we carried out gene expression profiling of clonal populations derived from fibroblast primary cultures expressing exclusively either the wild-type or the mutant MECP2 allele. Clonal cultures were obtained from skin biopsy of three RTT patients carrying either a non-sense or a frameshift MECP2 mutation. For each patient, gene expression profiles of wild-type and mutant clones were compared by oligonucleotide expression microarray analysis. Firstly, clustering analysis classified the RTT patients according to their genetic background and MECP2 mutation. Secondly, expression profiling by microarray analysis and quantitative RT-PCR indicated four up-regulated genes and five down-regulated genes significantly dysregulated in all our statistical analysis, including excellent potential candidate genes for the understanding of the pathophysiology of this neurodevelopmental disease. Thirdly, chromatin immunoprecipitation analysis confirmed MeCP2 binding to respective CpG islands in three out of four up-regulated candidate genes and sequencing of bisulphite-converted DNA indicated that MeCP2 preferentially binds to methylated-DNA sequences. Most importantly, the finding that at least two of these genes (BMCC1 and RNF182) were shown to be involved in cell survival and/or apoptosis may suggest that impaired MeCP2 function could alter the survival of neurons thus compromising brain function without inducing cell death.

Remembering Operación Triunfo: a Latin Music Reality Show in the Era of Talent Shows

NARCIS (Netherlands)

Savini, Paola

2016-01-01

abstractThe music format Operación Triunfo (2001–2011), which aired on RTVE for the first time in 2001, started as a television (TV) and musical success in Spain and today is one of the most famous shows around the world as well as an incredible socio-economic phenomenon in Spanish TV. This paper

Show me the money: incorporating financial motives into the gambling motives questionnaire.

Science.gov (United States)

Dechant, Kristianne

2014-12-01

Although research has only recently begun to measure what motivates all levels of gambling involvement, motives could offer a theoretically interesting and practical way to subtype gamblers in research and for responsible gambling initiatives. The Gambling Motives Questionnaire (GMQ) is one measure that weaves together much of the gambling motives literature, but it has been criticized for neglecting financial reasons for gambling. This study uses a series of factor analyses to explore the effect of adding nine financial motives to the GMQ in a heterogeneous sample of 1,014 adult past-year gamblers. After trimming trivial financial motives, the penultimate factor analysis of the 15 GMQ items and four financial motives led to a four-factor solution, with factors tapping enhancement, social, coping and financial motives, as predicted. A final factor analysis performed on a modified GMQ-F (i.e., 16 items, including a financial subscale) revealed the same four factors, and hierarchical regression showed that the financial motives improve the GMQ-F's prediction of gambling frequency. This study provides evidence that omitting financial motives is a clear gap in the GMQ, yet suggests that the GMQ is a promising tool that can be conceptually and empirically strengthened with the simple addition of financial items.

Influence of training and competitive sessions on peripheral β-endorphin levels in training show jumping horses

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Cristina Cravana

2017-01-01

Full Text Available Aim: To investigate the effects of training sessions on circulating β-endorphin changes in sport horses before and after competition and to ascertain whether competition would affect this response. Materials and Methods: A total of 24 trained jumping horses were randomly assigned to one of two training groups: Group A (competing and Group B (not competing. To determined plasma β-endorphin concentrations, two pre- and postcompetition training weeks at aerobic workout and two competitive show jumping event days at anaerobic workout were measured before, 5 and 30 min after exercise. Exercise intensity is described using lactate concentrations and heart rate. The circuit design, intensity, and duration of training sessions were the same for both groups. Results: In Group A, one-way analysis of variance for repeated measures (RM-ANOVA showed significant effects of exercise on β-endorphin changes (F=14.41; p<0.001, only in the post-competition training sessions, while in Group B showed no significant effects. Two-way RM-ANOVA showed, after post-competition training sessions, a significant difference between Group A and Group B (F=6.235; p=0.023, with higher β-endorphin changes in Group A, compared to Group B. During the competitive show jumping sessions, one-way RM ANOVA showed significant effects of exercise on β-endorphin changes (F=51.10; p<0.001. The statistical analysis, in Group A, showed a significant difference between postcompetition training and competitive exercise (F=6.32; p=0.024 with higher β-endorphin values in competitive sessions compared to those of post-competition training. Conclusion: Lactate concentrations seem to be the main factors being correlated with the raise of β-endorphin during anaerobic exercise of competitive events. Exercise of low intensity, as well as that one of training sessions, does not appear to stimulate a significant increased release of β-endorphin and it may depend on the duration of the exercise

The PRR11-SKA2 Bidirectional Transcription Unit Is Negatively Regulated by p53 through NF-Y in Lung Cancer Cells

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Yitao Wang

2017-03-01

Full Text Available We previously identified proline-rich protein 11 (PRR11 as a novel cancer-related gene that is implicated in the regulation of cell cycle and tumorigenesis. Our recent study demonstrated that PRR11 and its adjacent gene, kinetochore associated 2 (SKA2, constitute a classic head-to-head gene pair that is coordinately regulated by nuclear factor Y (NF-Y. In the present study, we further show that the PRR11-SKA2 bidirectional transcription unit is an indirect target of the tumor suppressor p53. A luciferase reporter assay revealed that overexpression of wild type p53, but not mutant p53, significantly represses the basal activity and NF-Y mediated transactivation of the PRR11-SKA2 bidirectional promoter. Deletion and mutation analysis of the PRR11-SKA2 promoter revealed that p53-mediated PRR11-SKA2 repression is dependent on the presence of functional NF-Y binding sites. Furthermore, a co-immunoprecipitation assay revealed that p53 associates with NF-Y in lung cancer cells, and a chromatin immunoprecipitation assay showed that p53 represses PRR11-SKA2 transcription by reducing the binding amount of NF-Y in the PRR11-SKA2 promoter region. Consistently, the ability of p53 to downregulate PRR11-SKA2 transcription was significantly attenuated upon siRNA-mediated depletion of nuclear factor Y subunit beta (NF-YB. Notably, lung cancer patients with lower expression of either PRR11 or SKA2 along with wild type p53 exhibited the best overall survival compared with others with p53 mutation and/or higher expression of either PRR11 or SKA2. Taken together, our results demonstrate that p53 negatively regulates the expression of the PRR11-SKA2 bidirectional transcription unit through NF-Y, suggesting that the inability to repress the PRR11-SKA2 bidirectional transcription unit after loss of p53 might contribute to tumorigenesis.

Rad51-Rad52 mediated maintenance of centromeric chromatin in Candida albicans.

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Sreyoshi Mitra

2014-04-01

Full Text Available Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI proximal to an early replicating centromere (CEN7 in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52

Robotic Assisted Radical Cystectomy with Extracorporeal Urinary Diversion Does Not Show a Benefit over Open Radical Cystectomy: A Systematic Review and Meta-Analysis of Randomised Controlled Trials.

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Wei Shen Tan

Full Text Available The number of robotic assisted radical cystectomy (RARC procedures is increasing despite the lack of Level I evidence showing any advantages over open radical cystectomy (ORC. However, several systematic reviews with meta-analyses including non-randomised studies, suggest an overall benefit for RARC compared to ORC. We performed a systematic review with meta-analysis of randomised controlled trials (RCTs to evaluate the perioperative morbidity and efficacy of RARC compared to ORC in patients with bladder cancer.Literature searches of Medline/Pubmed, Embase, Web of Science and clinicaltrials.gov databases up to 10th March 2016 were performed. The inclusion criteria for eligible studies were RCTs which compared perioperative outcomes of ORC and RARC for bladder cancer. Primary objective was perioperative and histopathological outcomes of RARC versus ORC while the secondary objective was quality of life assessment (QoL, oncological outcomes and cost analysis.Four RCTs (from 5 articles met the inclusion criteria, with a total of 239 patients all with extracorporeal urinary diversion. Patient demographics and clinical characteristics of RARC and ORC patients were evenly matched. There was no significant difference between groups in perioperative morbidity, length of stay, positive surgical margin, lymph node yield and positive lymph node status. RARC group had significantly lower estimated blood loss (p<0.001 and wound complications (p = 0.03 but required significantly longer operating time (p<0.001. QoL was not measured uniformly across trials and cost analysis was reported in one RCTs. A test for heterogeneity did highlight differences across operating time of trials suggesting that surgeon experience may influence outcomes.This study does not provide evidence to support a benefit for RARC compared to ORC. These results may not have inference for RARC with intracorporeal urinary diversion. Well-designed trials with appropriate endpoints conducted

2008 LHC Open Days Physics: the show

CERN Multimedia

2008-01-01

A host of events and activities await visitors to the LHC Open Days on 5 and 6 April. A highlight will be the physics shows funded by the European Physical Society (EPS), which are set to surprise and challenge children and adults alike! School children use their experience of riding a bicycle to understand how planets move around the sun (Copyright : Circus Naturally) Participating in the Circus Naturally show could leave a strange taste in your mouth! (Copyright : Circus Naturally) The Rino Foundation’s experiments with liquid nitrogen can be pretty exciting! (Copyright: The Rino Foundation)What does a bicycle have in common with the solar system? Have you ever tried to weigh air or visualise sound? Ever heard of a vacuum bazooka? If you want to discover the answers to these questions and more then come to the Physics Shows taking place at the CERN O...

Analysis of the initiating events in HIV-1 particle assembly and genome packaging.

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Sebla B Kutluay

2010-11-01

Full Text Available HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD of capsid (CA, which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

International Nuclear Information System (INIS)

Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

2014-01-01

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ( 258 RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP 289 ). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved

Igs expressed by chronic lymphocytic leukemia B cells show limited binding-site structure variability.

Science.gov (United States)

Marcatili, Paolo; Ghiotto, Fabio; Tenca, Claudya; Chailyan, Anna; Mazzarello, Andrea N; Yan, Xiao-Jie; Colombo, Monica; Albesiano, Emilia; Bagnara, Davide; Cutrona, Giovanna; Morabito, Fortunato; Bruno, Silvia; Ferrarini, Manlio; Chiorazzi, Nicholas; Tramontano, Anna; Fais, Franco

2013-06-01

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated.

BnNHL18A shows a localization change by stress-inducing chemical treatments

International Nuclear Information System (INIS)

Lee, Suk-Bae; Ham, Byung-Kook; Park, Jeong Mee; Kim, Young Jin; Paek, Kyung-Hee

2006-01-01

The two genes, named BnNHL18A and BnNHL18B, showing sequence homology with Arabidopsis NDR1/HIN1-like (NHL) genes, were isolated from cDNA library prepared with oilseed rape (Brassica napus) seedlings treated with NaCl. The transcript level of BnNHL18A was increased by sodium chloride, ethephon, hydrogen peroxide, methyl jasmonate, or salicylic acid treatment. The coding regions of BnNHL18A and BnNHL18B contain a sarcolipin (SLN)-like sequence. Analysis of the localization of smGFP fusion proteins showed that BnNHL18A is mainly localized to endoplasmic reticulum (ER). This result suggests that the SLN-like sequence plays a role in retaining proteins in ER membrane in plants. In response to NaCl, hydrogen peroxide, ethephon, and salicylic acid treatments, the protein localization of BnNHL18A was changed. Our findings suggest a common function of BnNHL18A in biotic and abiotic stresses, and demonstrate the presence of the shared mechanism of protein translocalization between the responses to plant pathogen and to osmotic stress

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Institute of Scientific and Technical Information of China (English)

ZHOU; Bin(周斌); PENG; Kaiman(彭开蔓); CHU; Zhaohui(储昭晖); WANG; Shiping(王石平); ZHANG; Qifa(张启发)

2002-01-01

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with genes in databases which encode or putatively encode transcription regulating proteins, translation regulating proteins, transport proteins, kinases, metabolic enzymes, and proteins involved in other functions. Most of the genes have not been previously reported as being involved in the disease resistance response in rice. The results from cDNA arrays, reverse transcription-polymerase chain reaction, and RNA gel blot analysis suggest that activation or repression of most of these genes might occur commonly in the defense response.

The three catalases in Deinococcus radiodurans: Only two show catalase activity.

Science.gov (United States)

Jeong, Sun-Wook; Jung, Jong-Hyun; Kim, Min-Kyu; Seo, Ho Seong; Lim, Heon-Man; Lim, Sangyong

2016-01-15

Deinococcus radiodurans, which is extremely resistant to ionizing radiation and oxidative stress, is known to have three catalases (DR1998, DRA0146, and DRA0259). In this study, to investigate the role of each catalase, we constructed catalase mutants (Δdr1998, ΔdrA0146, and ΔdrA0259) of D. radiodurans. Of the three mutants, Δdr1998 exhibited the greatest decrease in hydrogen peroxide (H2O2) resistance and the highest increase in intracellular reactive oxygen species (ROS) levels following H2O2 treatments, whereas ΔdrA0146 showed no change in its H2O2 resistance or ROS level. Catalase activity was not attenuated in ΔdrA0146, and none of the three bands detected in an in-gel catalase activity assay disappeared in ΔdrA0146. The purified His-tagged recombinant DRA0146 did not show catalase activity. In addition, the phylogenetic analysis of the deinococcal catalases revealed that the DR1998-type catalase is common in the genus Deinococcus, but the DRA0146-type catalase was found in only 4 of 23 Deinococcus species. Taken together, these results indicate that DR1998 plays a critical role in the anti-oxidative system of D. radiodurans by detoxifying H2O2, but DRA0146 does not have catalase activity and is not involved in the resistance to H2O2 stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Next-Generation Sequencing of Genomic DNA Fragments Bound to a Transcription Factor in Vitro Reveals Its Regulatory Potential

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Yukio Kurihara

2014-12-01

Full Text Available Several transcription factors (TFs coordinate to regulate expression of specific genes at the transcriptional level. In Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant’s transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5 transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq instead of the in vivo chromatin immunoprecipitation (ChIP-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function.

Functional analysis of bipartite begomovirus coat protein promoter sequences

International Nuclear Information System (INIS)

Lacatus, Gabriela; Sunter, Garry

2008-01-01

We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters

Inference of RNA polymerase II transcription dynamics from chromatin immunoprecipitation time course data.

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Ciira wa Maina

2014-05-01

Full Text Available Gene transcription mediated by RNA polymerase II (pol-II is a key step in gene expression. The dynamics of pol-II moving along the transcribed region influence the rate and timing of gene expression. In this work, we present a probabilistic model of transcription dynamics which is fitted to pol-II occupancy time course data measured using ChIP-Seq. The model can be used to estimate transcription speed and to infer the temporal pol-II activity profile at the gene promoter. Model parameters are estimated using either maximum likelihood estimation or via Bayesian inference using Markov chain Monte Carlo sampling. The Bayesian approach provides confidence intervals for parameter estimates and allows the use of priors that capture domain knowledge, e.g. the expected range of transcription speeds, based on previous experiments. The model describes the movement of pol-II down the gene body and can be used to identify the time of induction for transcriptionally engaged genes. By clustering the inferred promoter activity time profiles, we are able to determine which genes respond quickly to stimuli and group genes that share activity profiles and may therefore be co-regulated. We apply our methodology to biological data obtained using ChIP-seq to measure pol-II occupancy genome-wide when MCF-7 human breast cancer cells are treated with estradiol (E2. The transcription speeds we obtain agree with those obtained previously for smaller numbers of genes with the advantage that our approach can be applied genome-wide. We validate the biological significance of the pol-II promoter activity clusters by investigating cluster-specific transcription factor binding patterns and determining canonical pathway enrichment. We find that rapidly induced genes are enriched for both estrogen receptor alpha (ERα and FOXA1 binding in their proximal promoter regions.

Insights into the polerovirus-plant interactome revealed by co-immunoprecipitation and mass spectrometry

Science.gov (United States)

The identification of host proteins that interact with virus proteins is a major challenge for the field of virology. Phloem-limited viruses pose extraordinary challenges for in vivo protein interaction experiments because these viruses are localized in very few and highly specialized host cells. ...

A Talk Show from the Past.

Science.gov (United States)

Gallagher, Arlene F.

1991-01-01

Describes a two-day activity in which elementary students examine voting rights, the right to assemble, and women's suffrage. Explains the game, "Assemble, Reassemble," and a student-produced talk show with five students playing the roles of leaders of the women's suffrage movement. Profiles Elizabeth Cady Stanton, Lucretia Mott, Susan…

The Last Great American Picture Show

NARCIS (Netherlands)

Elsaesser, Thomas; King, Noel; Horwath, Alexander

2004-01-01

The Last Great American Picture Show brings together essays by scholars and writers who chart the changing evaluations of the American cinema of the 1970s, sometimes referred to as the decade of the lost generation, but now more and more recognized as the first New Hollywood, without which the

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Haruhiko [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Nakashima, Tomoki [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, PRESTO, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Mikihito [Department of Cell Signaling, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549 (Japan); Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Izawa, Naohiro; Yasui, Tetsuro [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Aburatani, Hiroyuki [Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1, Komaba, Meguro-ku, Tokyo 153-8904 (Japan); Tanaka, Sakae [Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan); Takayanagi, Hiroshi, E-mail: takayana@m.u-tokyo.ac.jp [Japan Science and Technology Agency, ERATO, Takayanagi Osteonetwork Project, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

2014-12-12

Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion.

Global epigenomic analysis indicates protocadherin-7 activates osteoclastogenesis by promoting cell–cell fusion

International Nuclear Information System (INIS)

Nakamura, Haruhiko; Nakashima, Tomoki; Hayashi, Mikihito; Izawa, Naohiro; Yasui, Tetsuro; Aburatani, Hiroyuki; Tanaka, Sakae; Takayanagi, Hiroshi

2014-01-01

Highlights: • Identification of epigenetically regulated genes during osteoclastogenesis. • Pcdh7 is regulated by H3K4me3 and H3K27me3 during osteoclastogenesis. • Pcdh7 expression is increased by RANKL during osteoclastogenesis. • Establishment of novel cell fusion analysis for osteoclasts by imaging cytometer. • Pcdh7 regulates osteoclastogenesis by promoting cell fusion related gene expressions. - Abstract: Gene expression is dependent not only on genomic sequences, but also epigenetic control, in which the regulation of chromatin by histone modification plays a crucial role. Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) are related to transcriptionally activated and silenced sequences, respectively. Osteoclasts, the multinucleated cells that resorb bone, are generated by the fusion of precursor cells of monocyte/macrophage lineage. To elucidate the molecular and epigenetic regulation of osteoclast differentiation, we performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis for H3K4me3 and H3K27me3 in combination with RNA sequencing. We focused on the histone modification change from H3K4me3(+)H3K27me3(+) to H3K4me3(+)H3K27me3(–) and identified the protocadherin-7 gene (Pcdh7) to be among the genes epigenetically regulated during osteoclastogenesis. Pcdh7 was induced by RANKL stimulation in an NFAT-dependent manner. The knockdown of Pcdh7 inhibited RANKL-induced osteoclast differentiation due to the impairment of cell–cell fusion, accompanied by a decreased expression of the fusion-related genes Dcstamp, Ocstamp and Atp6v0d2. This study demonstrates that Pcdh7 plays a key role in osteoclastogenesis by promoting cell–cell fusion

Exploring the Possible Role of Lysine Acetylation on Entamoeba histolytica Virulence: A Focus on the Dynamics of the Actin Cytoskeleton

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L. López-Contreras

2013-01-01

Full Text Available Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

BFV activates the NF-κB pathway through its transactivator (BTas) to enhance viral transcription

International Nuclear Information System (INIS)

Wang Jian; Tan Juan; Zhang Xihui; Guo Hongyan; Zhang Qicheng; Guo Tingting; Geng Yunqi; Qiao Wentao

2010-01-01

Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-κB (NF-κB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-κB pathway through the action of its transactivator, BTas. Both cellular IKKβ and IκBα also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-κB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKα and IKKβ), which may be responsible for regulation of IKK kinase activity and persistent NF-κB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-κB. Together, this study suggests that BFV activates the NF-κB pathway through BTas to enhance viral transcription.

cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

International Nuclear Information System (INIS)

D'Arpa, P.; Machlin, P.S.; Ratrie, H. III; Rothfield, N.F.; Cleveland, D.W.; Earnshaw, W.C.

1988-01-01

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

Rice homeobox transcription factor HOX1a positively regulates gibberellin responses by directly suppressing EL1.

Science.gov (United States)

Wen, Bi-Qing; Xing, Mei-Qing; Zhang, Hua; Dai, Cheng; Xue, Hong-Wei

2011-11-01

Homeobox transcription factors are involved in various aspects of plant development, including maintenance of the biosynthesis and signaling pathways of different hormones. However, few direct targets of homeobox proteins have been identified. We here show that overexpression of rice homeobox gene HOX1a resulted in enhanced gibberellin (GA) response, indicating a positive effect of HOX1a in GA signaling. HOX1a is induced by GA and encodes a homeobox transcription factor with transcription repression activity. In addition, HOX1a suppresses the transcription of early flowering1 (EL1), a negative regulator of GA signaling, and further electrophoretic mobility shift assay and chromatin immunoprecipitation analysis revealed that HOX1a directly bound to the promoter region of EL1 to suppress its expression and stimulate GA signaling. These results demonstrate that HOX1a functions as a positive regulator of GA signaling by suppressing EL1, providing informative hints on the study of GA signaling. © 2011 Institute of Botany, Chinese Academy of Sciences.

Rab39a interacts with phosphatidylinositol 3-kinase and negatively regulates autophagy induced by lipopolysaccharide stimulation in macrophages.

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Shintaro Seto

Full Text Available Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K. Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34(th to 41(st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

Science.gov (United States)

Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

1998-01-01

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

MHC class I phenotype and function of human beta 2-microglobulin transgenic murine lymphocytes

DEFF Research Database (Denmark)

Bjerager, L; Pedersen, L O; Bregenholt, S

1996-01-01

. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous h beta 2m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/h beta 2m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS......-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous h beta 2m to MHC-I, in particular, to the H-2Db molecule through an exchange with endogenous mouse beta 2m. In contrast to normal H-2Db molecules, hybrid H-2Db expressed on the surface of transgenic lymphocytes...... binds radiolabelled peptide in the absence of exogenous added h beta 2m suggesting that a stable fraction of hybrid H-2Db molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than...

Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

Science.gov (United States)

Shono, Takumi; Ishikawa, Naoki; Toume, Kazufumi; Arai, Midori A; Ahmed, Firoj; Sadhu, Samir K; Ishibashi, Masami

2015-07-15

The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 μM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear β-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing β-catenin levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

SOD1 aggregation in ALS mice shows simplistic test tube behavior.

Science.gov (United States)

Lang, Lisa; Zetterström, Per; Brännström, Thomas; Marklund, Stefan L; Danielsson, Jens; Oliveberg, Mikael

2015-08-11

A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.

Plasmodium falciparum: characterization of toxin-associated proteins and identification of a hemoglobin containing parasite cytokine stimulator

DEFF Research Database (Denmark)

Kristensen, G; Jakobsen, P H

1996-01-01

]-methionine and immunoprecipitated the labeled antigens with an antiserum against IMP which blocks malaria parasite-induced TNF production. We detected four proteins associated with IMP when the immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography. To evaluate the capacity of different P. falciparum......Previous studies have indicated the inositol monophosphate (IMP) is a component of the malaria parasite toxin that induces cytokines such as tumour necrosis factor (TNF). To further characterize the toxin we have labeled Plasmodium falciparum in vitro cultures with [14C]inositol or [35S...

Comprehensive Protein Interactome Analysis of a Key RNA Helicase: Detection of Novel Stress Granule Proteins

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Rebecca Bish

2015-07-01

Full Text Available DDX6 (p54/RCK is a human RNA helicase with central roles in mRNA decay and translation repression. To help our understanding of how DDX6 performs these multiple functions, we conducted the first unbiased, large-scale study to map the DDX6-centric protein-protein interactome using immunoprecipitation and mass spectrometry. Using DDX6 as bait, we identify a high-confidence and high-quality set of protein interaction partners which are enriched for functions in RNA metabolism and ribosomal proteins. The screen is highly specific, maximizing the number of true positives, as demonstrated by the validation of 81% (47/58 of the RNA-independent interactors through known functions and interactions. Importantly, we minimize the number of indirect interaction partners through use of a nuclease-based digestion to eliminate RNA. We describe eleven new interactors, including proteins involved in splicing which is an as-yet unknown role for DDX6. We validated and characterized in more detail the interaction of DDX6 with Nuclear fragile X mental retardation-interacting protein 2 (NUFIP2 and with two previously uncharacterized proteins, FAM195A and FAM195B (here referred to as granulin-1 and granulin-2, or GRAN1 and GRAN2. We show that NUFIP2, GRAN1, and GRAN2 are not P-body components, but re-localize to stress granules upon exposure to stress, suggesting a function in translation repression in the cellular stress response. Using a complementary analysis that resolved DDX6’s multiple complex memberships, we further validated these interaction partners and the presence of splicing factors. As DDX6 also interacts with the E3 SUMO ligase TIF1β, we tested for and observed a significant enrichment of sumoylation amongst DDX6’s interaction partners. Our results represent the most comprehensive screen for direct interaction partners of a key regulator of RNA life cycle and localization, highlighting new stress granule components and possible DDX6 functions�

TELEVISÃO E ESPETÁCULO: O TALK SHOW CASOS DE FAMÍLIA

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Juliana Cristina da Silva

2016-01-01

Full Text Available Este artigo tem como objetivo discutir, a partir do referencial teórico e metodológico na Análise do Discurso pêcheutiana, como a visibilidade que o talk show Casos de Família dá aos seus convidados converte seus depoimentos em espetáculo, pelo qual a exposição se justifica por si mesma. Através da análise do programa com tema “Gravidez na adolescência”, evidencia-se como os participantes produzem discursos identificados com formações discursivas dominantes, mas que em diversos momentos – quando a irrupção do real de suas experiências de vida surge como acontecimento em seus depoimentos – o discurso se desloca para formações discursivas outras, abrindo-se para a polifonia. No entanto, as reações da apresentadora e da plateia controlam a dispersão, indicando quais são os sentidos possíveis de serem produzidos naquele contexto, e que estão de acordo com uma ideologia dominante e de senso comum. O resultado é um fechamento dos sentidos, numa produção contínua do mesmo, da repetição. Palavras-chave: Análise do discurso. Mídia. Espetáculo. Abstract This article aims to discuss, from the theoretical and methodological perspective of pecheutian Discourse Analysis, how the visibility that the talk show “Casos de Família” (“Family Cases” provides its guests converts their statements into spectacles, by which the exposure is justified in itself. By means of analysis of the episode themed as “Teenage pregnancy”, it is evidenced how participants produce discourses that are identified with dominant discursive formations, but which at several occasions - when the eruption of the real experiences in their lives emerges as a happening in their statements - shift into other discursive formations, opening up to polyphony. However, the reactions of both the host and audience control the dispersion, indicating which meanings are allowed to be produced within that context, these

Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

DEFF Research Database (Denmark)

Couchman, J R; Ljubimov, A V

1989-01-01

muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...

WNT7A/?-catenin signaling induces FGF1 and influences sensitivity to niclosamide in ovarian cancer

OpenAIRE

King, Mandy L.; Lindberg, Mallory E.; Stodden, Genna R.; Okuda, Hiroshi; Ebers, Steven D.; Johnson, Alyssa; Montag, Anthony; Lengyel, Ernst; MacLean, James A.; Hayashi, Kanako

2014-01-01

We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/?-catenin...

Isolation and expression analysis of EcbZIP17 from different finger millet genotypes shows conserved nature of the gene.

Science.gov (United States)

Chopperla, Ramakrishna; Singh, Sonam; Mohanty, Sasmita; Reddy, Nanja; Padaria, Jasdeep C; Solanke, Amolkumar U

2017-10-01

Basic leucine zipper (bZIP) transcription factors comprise one of the largest gene families in plants. They play a key role in almost every aspect of plant growth and development and also in biotic and abiotic stress tolerance. In this study, we report isolation and characterization of EcbZIP17 , a group B bZIP transcription factor from a climate smart cereal, finger millet ( Eleusine coracana L.). The genomic sequence of EcbZIP17 is 2662 bp long encompassing two exons and one intron with ORF of 1722 bp and peptide length of 573 aa. This gene is homologous to AtbZIP17 ( Arabidopsis ), ZmbZIP17 (maize) and OsbZIP60 (rice) which play a key role in endoplasmic reticulum (ER) stress pathway. In silico analysis confirmed the presence of basic leucine zipper (bZIP) and transmembrane (TM) domains in the EcbZIP17 protein. Allele mining of this gene in 16 different genotypes by Sanger sequencing revealed no variation in nucleotide sequence, including the 618 bp long intron. Expression analysis of EcbZIP17 under heat stress exhibited similar pattern of expression in all the genotypes across time intervals with highest upregulation after 4 h. The present study established the conserved nature of EcbZIP17 at nucleotide and expression level.

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

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Nan Zhong

Full Text Available We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP protocols.

Comprehensive Analysis of Oculocutaneous Albinism among Non-Hispanic Caucasians Shows that OCA1 Is the Most Prevalent OCA Type

OpenAIRE

Hutton, Saunie M.; Spritz, Richard A.

2008-01-01

Oculocutaneous albinism (OCA) is a genetically heterogeneous group of disorders characterized by absent or reduced pigmentation of the skin, hair, and eyes. In humans, four genes have been associated with “classical” OCA and another 12 genes with syndromic forms of OCA. To assess the prevalence of different forms of OCA and different gene mutations among non-Hispanic Caucasian patients, we performed DNA sequence analysis of the four genes associated with “classical” OCA (TYR, OCA2, TYRP1, SLC...

HIV-1 Vif promotes the formation of high molecular mass APOBEC3G complexes

OpenAIRE

Goila-Gaur, Ritu; Khan, Mohammad A.; Miyagi, Eri; Kao, Sandra; Opi, Sandrine; Takeuchi, Hiroaki; Strebel, Klaus

2007-01-01

HIV-1 Vif inhibits the antiviral activity of APOBEC3G (APO3G) by inducing proteasomal degradation. Here, we studied the effects of Vif on APO3G in vitro. In this system, Vif did not cause APO3G degradation. Instead, Vif induced changes in APO3G that affected immunoprecipitation of the native protein. This effect required wt Vif and was reversed by heat-denaturation of APO3G. Sucrose gradient analysis demonstrated that wt Vif induced the gradual transition of APO3G translated in vitro or expre...

FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer

International Nuclear Information System (INIS)

Qiu, Meiting; Bao, Wei; Wang, Jingyun; Yang, Tingting; He, Xiaoying; Liao, Yun; Wan, Xiaoping

2014-01-01

Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear. FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays. We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth. These

High Glucose-Induced Reactive Oxygen Species Stimulates Human Mesenchymal Stem Cell Migration Through Snail and EZH2-Dependent E-Cadherin Repression

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Ji Young Oh

2018-04-01

Full Text Available Background/Aims: Glucose plays an important role in stem cell fate determination and behaviors. However, it is still not known how glucose contributes to the precise molecular mechanisms responsible for stem cell migration. Thus, we investigate the effect of glucose on the regulation of the human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC migration, and analyze the mechanism accompanied by this effect. Methods: Western blot analysis, wound healing migration assays, immunoprecipitation, and chromatin immunoprecipitation assay were performed to investigate the effect of high glucose on hUCB-MSC migration. Additionally, hUCB-MSC transplantation was performed in the mouse excisional wound splinting model. Results: High concentration glucose (25 mM elicits hUCB-MSC migration compared to normal glucose and high glucose-pretreated hUCB-MSC transplantation into the wound sites in mice also accelerates skin wound repair. We therefore elucidated the detailed mechanisms how high glucose induces hUCB-MSC migration. We showed that high glucose regulates E-cadherin repression through increased Snail and EZH2 expressions. And, we found high glucose-induced reactive oxygen species (ROS promotes two signaling; JNK which regulates γ–secretase leading to the cleavage of Notch proteins and PI3K/Akt signaling which enhances GSK-3β phosphorylation. High glucose-mediated JNK/Notch pathway regulates the expression of EZH2, and PI3K/Akt/GSK-3β pathway stimulates Snail stabilization, respectively. High glucose enhances the formation of EZH2/Snail/HDAC1 complex in the nucleus, which in turn causes E-cadherin repression. Conclusion: This study reveals that high glucose-induced ROS stimulates the migration of hUCB-MSC through E-cadherin repression via Snail and EZH2 signaling pathways.

The Daily Show with Jon Stewart: Part 2

Science.gov (United States)

Trier, James

2008-01-01

"The Daily Show With Jon Stewart" is one of the best critical literacy programs on television, and in this Media Literacy column the author suggests ways that teachers can use video clips from the show in their classrooms. (For Part 1, see EJ784683.)

Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

Directory of Open Access Journals (Sweden)

Soufi Muhidien

2008-11-01

Full Text Available Abstract Background Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH with those from healthy individuals. Methods Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p Results Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells. Conclusion Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

16 CFR 5.57 - Order to show cause.

Science.gov (United States)

2010-01-01

... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Order to show cause. 5.57 Section 5.57 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE STANDARDS OF CONDUCT Disciplinary Actions Concerning Postemployment Conflict of Interest § 5.57 Order to show cause. (a...

Igs Expressed by Chronic Lymphocytic Leukemia B Cells Show Limited Binding-Site Structure Variability

KAUST Repository

Marcatili, P.

2013-05-01

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated. Copyright © 2013 by The American Association of Immunologists, Inc.

Igs Expressed by Chronic Lymphocytic Leukemia B Cells Show Limited Binding-Site Structure Variability

KAUST Repository

Marcatili, P.; Ghiotto, F.; Tenca, C.; Chailyan, A.; Mazzarello, A. N.; Yan, X.-J.; Colombo, M.; Albesiano, E.; Bagnara, D.; Cutrona, G.; Morabito, F.; Bruno, S.; Ferrarini, M.; Chiorazzi, N.; Tramontano, A.; Fais, F.

2013-01-01

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated. Copyright © 2013 by The American Association of Immunologists, Inc.

Deal or No Deal? Decision Making under Risk in a Large-Stake TV Game Show and Related Experiments

NARCIS (Netherlands)

M.J. van den Assem (Martijn)

2008-01-01

textabstractThe central theme of this dissertation is the analysis of risky choice. The first two chapters analyze the choice behavior of contestants in a TV game show named “Deal or No Deal” (DOND). DOND provides a unique opportunity to study risk behavior, because it is characterized by very large

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

An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein

International Nuclear Information System (INIS)

Janaki Ramaiah, M.; Parnaik, Veena K.

2006-01-01

Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif

DINUCLEAR NICKEL(II PIVALATE WITH µ-AQUA AND DI-µ-PIVALATO BRIDGES SHOWING A FERROMAGNETIC INTERACTION

Directory of Open Access Journals (Sweden)

Masahiro Mikuriya

2014-12-01

Full Text Available Dinuclear nickel(II complex, [Ni2{O2CC(CH33}4(OH2{HO2CC(CH33}4] (1, was synthesized and characterized by elemental analysis, IR and UV-Vis-NIR spectroscopy, and temperature dependence of magnetic susceptibilities (4.5—300 K. Single-crystal X-ray crystallography revealed a dinuclear core with µ-aqua and di-µ-pivalato bridges having monodentate pivalato and monodentate pivalic acid molecules. Magnetic data analysis showed a ferromagnetic interactions between the two nickel atoms with g = 2.251, J = 2.78 cm−1, D = 3.75 cm–1, and tip = 184 x 10–6 cm3 mol–1; g = 2.253, J = 2.73 cm−1, D = –3.26 cm–1, and tip = 176 x 10–6 cm3 mol–1.

Endoglin expression in blood and endothelium is differentially regulated by modular assembly of the Ets/Gata hemangioblast code.

Science.gov (United States)

Pimanda, John E; Chan, Wan Y I; Wilson, Nicola K; Smith, Aileen M; Kinston, Sarah; Knezevic, Kathy; Janes, Mary E; Landry, Josette-Renée; Kolb-Kokocinski, Anja; Frampton, Jonathan; Tannahill, David; Ottersbach, Katrin; Follows, George A; Lacaud, Georges; Kouskoff, Valerie; Göttgens, Berthold

2008-12-01

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast, early hematopoietic, and vascular development. We have previously shown that an upstream enhancer, Eng -8, together with the promoter region, mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements, we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8, +7+9 enhancers in both blood and endothelial cells. By contrast Pu.1, an Ets factor specific to the blood lineage, and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence.

Environmental drivers of phototrophic biofilms in an Alpine show cave (SW-Italian Alps)

International Nuclear Information System (INIS)

Piano, E.; Bona, F.; Falasco, E.; La Morgia, V.; Badino, G.; Isaia, M.

2015-01-01

The proliferation of lampenflora is a major threat for the conservation of show caves, since phototrophic organisms cause physical, chemical and aesthetic damage to speleothems. In this paper we examine the environmental factors influencing the presence and the growth of the three main photosynthetic groups composing phototrophic biofilms in the Bossea show cave (SW-Italian Alps). The presence and the primary production of cyanobacteria, diatoms and green algae were detected with BenthoTorch®, an instrument for in situ measurement of chlorophyll a concentration that has never been used before in caves. By means of different techniques of regression analysis, we highlighted the response of the three photosynthetic groups to different environmental factors. Illuminance proved to be the main factor influencing positively both the probability of the presence and the productivity of the three groups. The presence of seeping water on the substrate and the distance from the cave entrance proved to play an important role in determining patterns of colonization. By means of GIS techniques, we provide thematic maps of the cave, providing a representation of pattern of the density of the three examined photosynthetic groups within different areas of the cave. The same approach may apply to other show caves, aiming at providing suggestions for the cave management (i.e. cleaning of the cave walls and positioning of artificial lights) and reduce impact caused by tourism. - Highlights: • We used a PAM fluorimeter on autotrophic biofilms in a show cave for the first time. • We modelled the environmental factors influencing phototrophic biofilms. • Illuminance, moisture and distance from the entrance proved to be significant. • We produced thematic maps illustrating our results. • We provide suggestions for cave management

Environmental drivers of phototrophic biofilms in an Alpine show cave (SW-Italian Alps)

Energy Technology Data Exchange (ETDEWEB)

Piano, E., E-mail: elena.piano@unito.it [Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin (Italy); Bona, F.; Falasco, E. [Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin (Italy); La Morgia, V. [ISPRA, via Ca' Fornacetta, 9, 40064 Ozzano dell' Emilia (Italy); Badino, G. [Department of Physics, University of Turin, Via P. Giuria 1, 10125 Turin (Italy); Isaia, M. [Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin (Italy)

2015-12-01

The proliferation of lampenflora is a major threat for the conservation of show caves, since phototrophic organisms cause physical, chemical and aesthetic damage to speleothems. In this paper we examine the environmental factors influencing the presence and the growth of the three main photosynthetic groups composing phototrophic biofilms in the Bossea show cave (SW-Italian Alps). The presence and the primary production of cyanobacteria, diatoms and green algae were detected with BenthoTorch®, an instrument for in situ measurement of chlorophyll a concentration that has never been used before in caves. By means of different techniques of regression analysis, we highlighted the response of the three photosynthetic groups to different environmental factors. Illuminance proved to be the main factor influencing positively both the probability of the presence and the productivity of the three groups. The presence of seeping water on the substrate and the distance from the cave entrance proved to play an important role in determining patterns of colonization. By means of GIS techniques, we provide thematic maps of the cave, providing a representation of pattern of the density of the three examined photosynthetic groups within different areas of the cave. The same approach may apply to other show caves, aiming at providing suggestions for the cave management (i.e. cleaning of the cave walls and positioning of artificial lights) and reduce impact caused by tourism. - Highlights: • We used a PAM fluorimeter on autotrophic biofilms in a show cave for the first time. • We modelled the environmental factors influencing phototrophic biofilms. • Illuminance, moisture and distance from the entrance proved to be significant. • We produced thematic maps illustrating our results. • We provide suggestions for cave management.

Major depressive disorder and generalized anxiety disorder show different autonomic dysregulations revealed by heart-rate variability analysis in first-onset drug-naïve patients without comorbidity.

Science.gov (United States)

Shinba, Toshikazu

2017-02-01

The aim of the present study was to examine whether depression and anxiety disorder manifest different autonomic dysregulations using heart-rate variability (HRV) and heart rate (HR) measurements. HRV and HR were recorded both at rest and during task execution (random-number generation) in first-onset drug-naïve patients with major depressive disorder (MDD, n = 14) and generalized anxiety disorder (GAD, n = 11) as well as in healthy controls (n = 41). The patients showed no comorbidity of depression and anxiety disorder. GAD patients did not exhibit panic or phobic symptoms at the time of measurement. Following power spectrum analysis of HR trend, the high- (HF) and low-frequency (LF) components, the sum (LF + HF), and the LF/HF ratio were compared among the groups. In the MDD patients, as previously reported, HF was low and the LF/HF ratio was high during the initial-rest condition, and HF was less reactive to the task. In contrast, GAD patients showed significantly high HF, although autonomic reactivity was not impaired. The results indicate that baseline autonomic activity and its reactivity to behavioral changes are different between MDD and GAD in the early stage of illness. High parasympathetic tone in GAD may reflect responses of the parasympathetic system to anxiety. MDD is accompanied by an autonomic shift toward sympathetic activation and a reduced reactivity to task. © 2016 The Authors. Psychiatry and Clinical Neurosciences © 2016 Japanese Society of Psychiatry and Neurology.

Radio Show on Books and Libraries as a Means of Promoting the Public Library: Marking the 200th Edition of the Show Tvoja,

Directory of Open Access Journals (Sweden)

Peter Pavletič

2014-04-01

Full Text Available ABSTRACTSince 2004, the Ciril Kosmač Library of Tolmin has been continuously preparing a radio show on books and libraries on the local radio station Alpski val. The show has been used as an advertising platform or as a means of primary library promotion. The radio show is regarded as one of the tools of what is termed market communication, and it is intended to provide broad public information on the services of a public library, to promote the library’s activities and holdings and to foster a reading culture. The aim of the show is to stimulate the potential user interest in visiting and using the services of a public library, to expand the circle of users and consequently raise the number of visits and library loans. The title of the show, its audio image, target audience, metapromotion and substantive concept are all key to planning the radio show on books and libraries. The radio show is an example of an effective tool that helps to establish the library’s ties with the local community. This example of best practices shows that it is worth pursuing this activity in Slovenia’s public libraries and highlighting it on their websites.

Molecular characterization of Marek's disease herpesvirus B antigen

International Nuclear Information System (INIS)

Isfort, R.J.; Sithole, I.; Kung, H.J.; Velicer, L.F.

1986-01-01

The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [ 35 S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (RαB), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [ 35 S] methionine- and 14 C-labeled infected cells into two other sera shown to have anti-B activity. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well-absorbed RαPM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell. These preliminary data point to the future membrane biochemistry and membrane immunology experiments needed to understand the MDHV system, and they may explain the high level of immunogenicity of MDHV-B in the infected chicken, as shown by its immunoprecipitation with immune chicken serum

Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2)

DEFF Research Database (Denmark)

Boyd, Mette; Hansen, Morten; Jensen, Tine G K

2010-01-01

The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice......, revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing...... resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels...

Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

Science.gov (United States)

Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

2005-08-01

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

Production of C-reactive protein by human lymphocytes

International Nuclear Information System (INIS)

Kuta, A.E.; Baum, L.L.

1986-01-01

C-reactive protein (CRP) is a major acute phase serum protein in humans; it is detectable at very high concentrations during infection and tissue trauma. This protein is a pentame composed of five identical, 21,500 MW subunits. CRP is detectable on the surface of approximately 4% of normal peripheral blood lymphocytes (PBL). CRP binds its physiological ligands in a Ca ++ dependent manner; removal of Ca ++ does not alter the expression of CRP on the lymphocyte surface. Recently, investigators in this laboratory reported substantial inhibition of natural killer cell (NK) activity with anti-CRP antibodies. The following studies were undertaken to determine the origin of surface-CRP (S-CRP) found on normal PBL. Cells were incubated in methionine-free DMEM supplemented with 35 S-methionine. Cells were lysed and subjected to immunoprecipitation with anti-CRP and Staphylococcus aureus; immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Data presented here suggested that lymphocytes, in particular, LGL produce small amounts of CRP and express it on their surface. Lymphocytes do not appear to secrete CRP since no CRP could be detected in culture supernatants. In addition, preliminary evidence indicates that peripheral blood monocytes produce no detectable CRP. Present studies utilizing Northern blot analysis are underway in order to detect CRP-mRNA

Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

Science.gov (United States)

Demangeat, G; Greif, C; Hemmer, O; Fritsch, C

1990-08-01

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

DEFF Research Database (Denmark)

Beavan, L A; Davies, M; Couchman, J R

1989-01-01

The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently......-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane...

A hidden Ising model for ChIP-chip data analysis

KAUST Repository

Mo, Q.

2010-01-28

Motivation: Chromatin immunoprecipitation (ChIP) coupled with tiling microarray (chip) experiments have been used in a wide range of biological studies such as identification of transcription factor binding sites and investigation of DNA methylation and histone modification. Hidden Markov models are widely used to model the spatial dependency of ChIP-chip data. However, parameter estimation for these models is typically either heuristic or suboptimal, leading to inconsistencies in their applications. To overcome this limitation and to develop an efficient software, we propose a hidden ferromagnetic Ising model for ChIP-chip data analysis. Results: We have developed a simple, but powerful Bayesian hierarchical model for ChIP-chip data via a hidden Ising model. Metropolis within Gibbs sampling algorithm is used to simulate from the posterior distribution of the model parameters. The proposed model naturally incorporates the spatial dependency of the data, and can be used to analyze data with various genomic resolutions and sample sizes. We illustrate the method using three publicly available datasets and various simulated datasets, and compare it with three closely related methods, namely TileMap HMM, tileHMM and BAC. We find that our method performs as well as TileMap HMM and BAC for the high-resolution data from Affymetrix platform, but significantly outperforms the other three methods for the low-resolution data from Agilent platform. Compared with the BAC method which also involves MCMC simulations, our method is computationally much more efficient. Availability: A software called iChip is freely available at http://www.bioconductor.org/. Contact: moq@mskcc.org. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org.

"The Daily Show with Jon Stewart": Part 1

Science.gov (United States)

Trier, James

2008-01-01

Comedy Central's popular program "The Daily Show With Jon Stewart" is the best critical media literacy program on television, and it can be used in valuable ways in the classroom as part of a media literacy pedagogy. This Media Literacy column provides an overview of the show and its accompanying website and considers ways it might be used in the…

Dopamine signaling leads to loss of Polycomb repression and aberrant gene activation in experimental parkinsonism

DEFF Research Database (Denmark)

Södersten, Erik; Feyder, Michael; Lerdrup, Mads

2014-01-01

. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation....... The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p...

Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

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Wang Pu

2010-10-01

Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions

Science.gov (United States)

Ma, Zhaoxue; Hu, Xupeng; Cai, Wenjuan; Huang, Weihua; Zhou, Xin; Luo, Qian; Yang, Hongquan; Wang, Jiawei; Huang, Jirong

2014-01-01

An extraordinarily precise regulation of chlorophyll biosynthesis is essential for plant growth and development. However, our knowledge on the complex regulatory mechanisms of chlorophyll biosynthesis is very limited. Previous studies have demonstrated that miR171-targeted scarecrow-like proteins (SCL6/22/27) negatively regulate chlorophyll biosynthesis via an unknown mechanism. Here we showed that SCLs inhibit the expression of the key gene encoding protochlorophyllide oxidoreductase (POR) in light-grown plants, but have no significant effect on protochlorophyllide biosynthesis in etiolated seedlings. Histochemical analysis of β-glucuronidase (GUS) activity in transgenic plants expressing pSCL27::rSCL27-GUS revealed that SCL27-GUS accumulates at high levels and suppresses chlorophyll biosynthesis at the leaf basal proliferation region during leaf development. Transient gene expression assays showed that the promoter activity of PORC is indeed regulated by SCL27. Consistently, chromatin immunoprecipitation and quantitative PCR assays showed that SCL27 binds to the promoter region of PORC in vivo. An electrophoretic mobility shift assay revealed that SCL27 is directly interacted with G(A/G)(A/T)AA(A/T)GT cis-elements of the PORC promoter. Furthermore, genetic analysis showed that gibberellin (GA)-regulated chlorophyll biosynthesis is mediated, at least in part, by SCLs. We demonstrated that SCL27 interacts with DELLA proteins in vitro and in vivo by yeast-two-hybrid and coimmunoprecipitation analysis and found that their interaction reduces the binding activity of SCL27 to the PORC promoter. Additionally, we showed that SCL27 activates MIR171 gene expression, forming a feedback regulatory loop. Taken together, our data suggest that the miR171-SCL module is critical for mediating GA-DELLA signaling in the coordinate regulation of chlorophyll biosynthesis and leaf growth in light. PMID:25101599

Hepatitis C virus E2 protein involve in insulin resistance through an impairment of Akt/PKB and GSK3β signaling in hepatocytes

Directory of Open Access Journals (Sweden)

Hsieh Ming-Ju

2012-06-01

Full Text Available Abstract Background Hepatitis C virus (HCV infection may cause liver diseases of various severities ranging from primary acute infection to life-threatening diseases, such as cirrhosis or hepatocellular carcinoma with poor prognosis. According to clinical findings, HCV infection may also lead to some extra-hepatic symptoms, including type 2 diabetes mellitus (DM. Since insulin resistance is the major etiology for type 2 DM and numerous evidences showed that HCV infection associated with insulin resistance, the involvement of E2 in the pathogenesis of type 2 DM and underlying mechanisms were investigated in this study. Methods Reverse transcription and real-time PCR, Western blot assay, Immunoprecipitation, Glucose uptake assay and analysis of cellular glycogen content. Results Results showed that E2 influenced on protein levels of insulin receptor substrate-1 (IRS-1 and impaired insulin-induced Ser308 phosphorylation of Akt/PKB and Ser9 phosphorylation of GSK3β in Huh7 cells, leading to an inhibition of glucose uptake and glycogen synthesis, respectively, and eventually insulin resistance. Conclusions Therefore, HCV E2 protein indeed involved in the pathogenesis of type 2 DM by inducing insulin resistance.

A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

Science.gov (United States)

Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

2010-10-01

Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

SP140L, an Evolutionarily Recent Member of the SP100 Family, Is an Autoantigen in Primary Biliary Cirrhosis

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Mario Saare

2015-01-01

Full Text Available The SP100 family members comprise a set of closely related genes on chromosome 2q37.1. The widely expressed SP100 and the leukocyte-specific proteins SP110 and SP140 have been associated with transcriptional regulation and various human diseases. Here, we have characterized the SP100 family member SP140L. The genome sequence analysis showed the formation of SP140L gene through rearrangements of the two neighboring genes, SP100 and SP140, during the evolution of higher primates. The SP140L expression is interferon-inducible with high transcript levels in B cells and other peripheral blood mononuclear cells. Subcellularly, SP140L colocalizes with SP100 and SP140 in nuclear structures that are devoid of SP110, PML, or p300 proteins. Similarly to SP100 and SP140 protein, we detected serum autoantibodies to SP140L in patients with primary biliary cirrhosis using luciferase immunoprecipitation system and immunoblotting assays. In conclusion, our results show that SP140L is phylogenetically recent member of SP100 proteins and acts as an autoantigen in primary biliary cirrhosis patients.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Science.gov (United States)

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-05-19

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope

International Nuclear Information System (INIS)

Zon, Arend van; Mossink, Marieke H.; Houtsmuller, Adriaan B.; Schoester, Martijn; Scheffer, George L.; Scheper, Rik J.; Sonneveld, Pieter; Wiemer, Erik A.C.

2006-01-01

Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and β-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP +/+ and MVP -/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus

Global Mapping of Transcription Factor Binding Sites by Sequencing Chromatin Surrogates: a Perspective on Experimental Design, Data Analysis, and Open Problems.

Science.gov (United States)

Wei, Yingying; Wu, George; Ji, Hongkai

2013-05-01

Mapping genome-wide binding sites of all transcription factors (TFs) in all biological contexts is a critical step toward understanding gene regulation. The state-of-the-art technologies for mapping transcription factor binding sites (TFBSs) couple chromatin immunoprecipitation (ChIP) with high-throughput sequencing (ChIP-seq) or tiling array hybridization (ChIP-chip). These technologies have limitations: they are low-throughput with respect to surveying many TFs. Recent advances in genome-wide chromatin profiling, including development of technologies such as DNase-seq, FAIRE-seq and ChIP-seq for histone modifications, make it possible to predict in vivo TFBSs by analyzing chromatin features at computationally determined DNA motif sites. This promising new approach may allow researchers to monitor the genome-wide binding sites of many TFs simultaneously. In this article, we discuss various experimental design and data analysis issues that arise when applying this approach. Through a systematic analysis of the data from the Encyclopedia Of DNA Elements (ENCODE) project, we compare the predictive power of individual and combinations of chromatin marks using supervised and unsupervised learning methods, and evaluate the value of integrating information from public ChIP and gene expression data. We also highlight the challenges and opportunities for developing novel analytical methods, such as resolving the one-motif-multiple-TF ambiguity and distinguishing functional and non-functional TF binding targets from the predicted binding sites. The online version of this article (doi:10.1007/s12561-012-9066-5) contains supplementary material, which is available to authorized users.

MANIFESTATION OF MANIPULATION IN POLITICAL TALK-SHOWS: COGNITIVE AND MULTIMODAL ASPECTS

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Petrova Anna Aleksandrovna

2014-11-01

Full Text Available The article deals with the problems of the manipulation manifestation in political television talk-shows. The suggestive processes of interaction in the analyzed genre of the media political discourse are studied in two aspects: а monomodal – as speech manipulation by verbal means at the level of emotional suggestion; b multimodal – as counter-suggestion, that restricts the effect of suggestion with visual and kinetic resources. The foundation of the cognitive analysis is a modeling method with a linguistic model which contains components of the cognitive and emotional processing of meaning, conclusions and reasoning. According to this three-component model, the speech manipulation consists in activation of dominant scripts of an addressee and is assured by the verbal resources of suggestion which associate with these scripts. The foundation of the multimodal research of the situations with counter-suggestion in the mass-media discourse is an ethnomethodological method with a reconstruction device. With this scientific attitude the authors have divided the resources of protection from the activating manipulation into two groups: 1 passive interactive communication of a suggestee in a verbal pause 2 active interactive communication of a suggestee aimed at changing the status and role domination. The empiric study of two isolated modalities and their correlations in specific situations of political talk shows allowed to develop the hypothesis on the existence of the fourth visual and kinetic component which represents space and corporal constellations with other models (or modalities of communication and their configurations. This study emphasizes the need to extend the research frames for the complex interactive processes of communication through their study in the multimodal aspect.

The first sequenced carnivore genome shows complex host-endogenous retrovirus relationships.

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Álvaro Martínez Barrio

Full Text Available Host-retrovirus interactions influence the genomic landscape and have contributed substantially to mammalian genome evolution. To gain further insights, we analyzed a female boxer (Canis familiaris genome for complexity and integration pattern of canine endogenous retroviruses (CfERV. Intriguingly, the first such in-depth analysis of a carnivore species identified 407 CfERV proviruses that represent only 0.15% of the dog genome. In comparison, the same detection criteria identified about six times more HERV proviruses in the human genome that has been estimated to contain a total of 8% retroviral DNA including solitary LTRs. These observed differences in man and dog are likely due to different mechanisms to purge, restrict and protect their genomes against retroviruses. A novel group of gammaretrovirus-like CfERV with high similarity to HERV-Fc1 was found to have potential for active retrotransposition and possibly lateral transmissions between dog and human as a result of close interactions during at least 10.000 years. The CfERV integration landscape showed a non-uniform intra- and inter-chromosomal distribution. Like in other species, different densities of ERVs were observed. Some chromosomal regions were essentially devoid of CfERVs whereas other regions had large numbers of integrations in agreement with distinct selective pressures at different loci. Most CfERVs were integrated in antisense orientation within 100 kb from annotated protein-coding genes. This integration pattern provides evidence for selection against CfERVs in sense orientation relative to chromosomal genes. In conclusion, this ERV analysis of the first carnivorous species supports the notion that different mammals interact distinctively with endogenous retroviruses and suggests that retroviral lateral transmissions between dog and human may have occurred.

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

OpenAIRE

Zielke, Norman; Querings, Silvia; Grosskortenhaus, Ruth; Reis, Tânia; Sprenger, Frank

2006-01-01

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the ...

Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

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Schwarzenbach Heidi

2010-06-01

Full Text Available Abstract Background The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. Methods In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Results Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3 at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. Conclusions This study is one

Bacillus subtilis from Soybean Food Shows Antimicrobial Activity for Multidrug-Resistant Acinetobacter baumannii by Affecting the adeS Gene.

Science.gov (United States)

Wang, Tieshan; Su, Jianrong

2016-12-28

Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii . Nineteen multidrug-resistant A. baumannii strains were clinifcally isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii . The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis . Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.

Rescuing Alu: recovery of new inserts shows LINE-1 preserves Alu activity through A-tail expansion.

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Bradley J Wagstaff

Full Text Available Alu elements are trans-mobilized by the autonomous non-LTR retroelement, LINE-1 (L1. Alu-induced insertion mutagenesis contributes to about 0.1% human genetic disease and is responsible for the majority of the documented instances of human retroelement insertion-induced disease. Here we introduce a SINE recovery method that provides a complementary approach for comprehensive analysis of the impact and biological mechanisms of Alu retrotransposition. Using this approach, we recovered 226 de novo tagged Alu inserts in HeLa cells. Our analysis reveals that in human cells marked Alu inserts driven by either exogenously supplied full length L1 or ORF2 protein are indistinguishable. Four percent of de novo Alu inserts were associated with genomic deletions and rearrangements and lacked the hallmarks of retrotransposition. In contrast to L1 inserts, 5' truncations of Alu inserts are rare, as most of the recovered inserts (96.5% are full length. De novo Alus show a random pattern of insertion across chromosomes, but further characterization revealed an Alu insertion bias exists favoring insertion near other SINEs, highly conserved elements, with almost 60% landing within genes. De novo Alu inserts show no evidence of RNA editing. Priming for reverse transcription rarely occurred within the first 20 bp (most 5' of the A-tail. The A-tails of recovered inserts show significant expansion, with many at least doubling in length. Sequence manipulation of the construct led to the demonstration that the A-tail expansion likely occurs during insertion due to slippage by the L1 ORF2 protein. We postulate that the A-tail expansion directly impacts Alu evolution by reintroducing new active source elements to counteract the natural loss of active Alus and minimizing Alu extinction.

Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa

International Nuclear Information System (INIS)

Klinefelter, G.R.; Hamilton, D.W.

1985-01-01

We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis

Dolphin shows and interaction programs: benefits for conservation education?

Science.gov (United States)

Miller, L J; Zeigler-Hill, V; Mellen, J; Koeppel, J; Greer, T; Kuczaj, S

2013-01-01

Dolphin shows and dolphin interaction programs are two types of education programs within zoological institutions used to educate visitors about dolphins and the marine environment. The current study examined the short- and long-term effects of these programs on visitors' conservation-related knowledge, attitude, and behavior. Participants of both dolphin shows and interaction programs demonstrated a significant short-term increase in knowledge, attitudes, and behavioral intentions. Three months following the experience, participants of both dolphin shows and interaction programs retained the knowledge learned during their experience and reported engaging in more conservation-related behaviors. Additionally, the number of dolphin shows attended in the past was a significant predictor of recent conservation-related behavior suggesting that repetition of these types of experiences may be important in inspiring people to conservation action. These results suggest that both dolphin shows and dolphin interaction programs can be an important part of a conservation education program for visitors of zoological facilities. © 2012 Wiley Periodicals, Inc.

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

Energy Technology Data Exchange (ETDEWEB)

Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D. [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Mymryk, Joe S., E-mail: jmymryk@uwo.ca [Department of Microbiology and Immunology, The University of Western Ontario, A4-833 London Regional Cancer Centre, 800 Commissioners Road E., London, Ontario, N6A 4L6 Canada (Canada); Department of Oncology, The University of Western Ontario, London Regional Cancer Centre, Ontario (Canada)

2014-11-15

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

Health Information Technology Continues to Show Positive Effect on Medical Outcomes: Systematic Review.

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Kruse, Clemens Scott; Beane, Amanda

2018-02-05

Health information technology (HIT) has been introduced into the health care industry since the 1960s when mainframes assisted with financial transactions, but questions remained about HIT's contribution to medical outcomes. Several systematic reviews since the 1990s have focused on this relationship. This review updates the literature. The purpose of this review was to analyze the current literature for the impact of HIT on medical outcomes. We hypothesized that there is a positive association between the adoption of HIT and medical outcomes. We queried the Cumulative Index of Nursing and Allied Health Literature (CINAHL) and Medical Literature Analysis and Retrieval System Online (MEDLINE) by PubMed databases for peer-reviewed publications in the last 5 years that defined an HIT intervention and an effect on medical outcomes in terms of efficiency or effectiveness. We structured the review from the Primary Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA), and we conducted the review in accordance with the Assessment for Multiple Systematic Reviews (AMSTAR). We narrowed our search from 3636 papers to 37 for final analysis. At least one improved medical outcome as a result of HIT adoption was identified in 81% (25/37) of research studies that met inclusion criteria, thus strongly supporting our hypothesis. No statistical difference in outcomes was identified as a result of HIT in 19% of included studies. Twelve categories of HIT and three categories of outcomes occurred 38 and 65 times, respectively. A strong majority of the literature shows positive effects of HIT on the effectiveness of medical outcomes, which positively supports efforts that prepare for stage 3 of meaningful use. This aligns with previous reviews in other time frames. ©Clemens Scott Kruse, Amanda Beane. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 05.02.2018.

Army Study Shows Decline In Behavioral Health Stigma

Science.gov (United States)

2012-01-01

Army Study Shows Decline in Behavioral Health Stigma By Rob McIlvaine Army News Service WASHINGTON, Jan. 20, 2012 - A newly released Army study on...conference yesterday. The three-year study outlines the problem of suicide in the Army and related issues of substance abuse, spouse abuse and child abuse...REPORT TYPE 3. DATES COVERED 00-00-2012 to 00-00-2012 4. TITLE AND SUBTITLE Army Study Shows Decline In Behavioral Health Stigma 5a. CONTRACT

Dietary fiber showed no preventive effect against colon and rectal cancers in Japanese with low fat intake: an analysis from the results of nutrition surveys from 23 Japanese prefectures

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Sugawara Kazuo

2001-10-01

Full Text Available Abstract Background Since Fuchs' report in 1999, the reported protective effect of dietary fiber from colorectal carcinogenesis has led many researchers to question its real benefit. The aim of this study is to evaluate the association between diet, especially dietary fiber and fat and colorectal cancer in Japan. Methods A multiple regression analysis (using the stepwise variable selection method was performed using the standardized mortality ratios (SMRs of colon and rectal cancer in 23 Japanese prefectures as objective variables and dietary fiber, nutrients and food groups as explanatory variables. Results As for colon cancer, the standardized partial correlation coefficients were positively significant for fat (1,13, P = 0.000, seaweeds (0.41, P = 0.026 and beans (0.45, P = 0.017 and were negatively significant for vitamin A (-0.63, P = 0.003, vitamin C (-0.42, P = 0.019 and yellow-green vegetables (-0.37, P = 0.046. For rectal cancer, the standardized partial correlation coefficient in fat (0.60, P = 0.002 was positively significant. Dietary fiber was not found to have a significant relationship with either colon or rectal cancers. Conclusions This study failed to show any protective effect of dietary fiber in subjects with a low fat intake (Japanese in this analysis, which supports Fuchs' findings in subjects with a high fat intake (US Americans.

Study shows women fail to land top grants

Science.gov (United States)

Jenner, Nicola

2014-05-01

Women in the UK are less successful than men at securing research council funding at almost every stage of their careers, according to an analysis published by Research Councils UK (RCUK) - the umbrella organization for the country's seven research councils.

Interleukin-1beta-induced release of matrix proteins into culture media causes inhibition of mineralization of nodules formed by periodontal ligament cells in vitro.

Science.gov (United States)

Chien, H H; Lin, W L; Cho, M I

1999-05-01

The mechanism by which interleukin-1beta (IL-1) inhibits the formation of mineralized tissue nodules by periodontal ligament (PDL) cells in vitro was investigated through the processes of morphological analysis, immunoprecipitation, and Northern blot analysis. PDL cells were obtained from a 2-day-old coagulum in tooth socket and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bone serum (FBS) and antibiotics. Confluent cells were grown for up to 3 weeks in the presence of ascorbic acid (AA), beta-glycerophosphate (GP), and dexamethasone (Dex), or IL-1. PDL cells cultured in the presence of GP and AA did not differentiate, but those treated with Dex, GP, and AA (Dex group) underwent differentiation, showing four stages (confluent, multilayer, nodule, and mineralization) of disparate morphological characteristics. In contrast, the cells treated with IL-1, Dex, GP, and AA (IL-1 group) did form multilayers but failed to form mineralized nodules. Electron microscopy demonstrated that the Dex-induced mineralized nodules contain multilayers of fibroblastic cells, numerous collagen fibrils, and dense globular as well as fused electron dense patches that are associated with numerous apatite crystals. The nodule-like structures in the IL-1 group were also comprised of multilayered fibroblastic cells, but they contained only a small number of collagen fibrils, and no dense globular or fused patches. Von Kossa staining confirmed the presence of numerous mineralized nodules in the Dex group and their scarceness in the IL-1 group. Northern blot analysis of IL-1-treated cells, however, revealed the presence of mRNAs for type I collagen (Col I), secreted protein, acidic and rich in cysteine (SPARC), osteopontin (OPN), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC), whose expression patterns and levels were comparable to those of the Dex group. Immunoprecipitation analysis of OPN and BSP in the cell/matrix layers and the culture

Anatomical Network Analysis Shows Decoupling of Modular Lability and Complexity in the Evolution of the Primate Skull

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Esteve-Altava, Borja; Boughner, Julia C.; Diogo, Rui; Villmoare, Brian A.; Rasskin-Gutman, Diego

2015-01-01

Modularity and complexity go hand in hand in the evolution of the skull of primates. Because analyses of these two parameters often use different approaches, we do not know yet how modularity evolves within, or as a consequence of, an also-evolving complex organization. Here we use a novel network theory-based approach (Anatomical Network Analysis) to assess how the organization of skull bones constrains the co-evolution of modularity and complexity among primates. We used the pattern of bone contacts modeled as networks to identify connectivity modules and quantify morphological complexity. We analyzed whether modularity and complexity evolved coordinately in the skull of primates. Specifically, we tested Herbert Simon’s general theory of near-decomposability, which states that modularity promotes the evolution of complexity. We found that the skulls of extant primates divide into one conserved cranial module and up to three labile facial modules, whose composition varies among primates. Despite changes in modularity, statistical analyses reject a positive feedback between modularity and complexity. Our results suggest a decoupling of complexity and modularity that translates to varying levels of constraint on the morphological evolvability of the primate skull. This study has methodological and conceptual implications for grasping the constraints that underlie the developmental and functional integration of the skull of humans and other primates. PMID:25992690

Polypyridyl iron(II) complexes showing remarkable photocytotoxicity ...

Indian Academy of Sciences (India)

aditya

Polypyridyl iron(II) complexes showing remarkable photocytotoxicity in visible light. ADITYA GARAI a. , UTTARA BASU a. , ILA PANT b. , PATURU KONDAIAH*. ,b. AND. AKHIL R. CHAKRAVARTY*. ,a a. Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore. 560012, India. E-mail: ...

Microbial Communities Show Parallels at Sites with Distinct

Czech Academy of Sciences Publication Activity Database

Ságová-Marečková, M.; Omelka, M.; Čermák, L.; Kameník, Zdeněk; Olšovská, J.; Hackl, E.; Kopecký, J.; Hadacek, F.

2011-01-01

Roč. 77, č. 21 (2011), s. 7560-7567 ISSN 0099-2240 Institutional research plan: CEZ:AV0Z50200510 Keywords : 16S RIBOSOMAL-RNA * LENGTH-POLYMORPHISM ANALYSIS * MULTIPLE SEQUENCE ALIGNMENT Subject RIV: EE - Microbiology, Virology Impact factor: 3.829, year: 2011

BDNF-Deficient Mice Show Reduced Psychosis-Related Behaviors Following Chronic Methamphetamine.

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Manning, Elizabeth E; Halberstadt, Adam L; van den Buuse, Maarten

2016-04-01

One of the most devastating consequences of methamphetamine abuse is increased risk of psychosis. Brain-derived neurotrophic factor has been implicated in both psychosis and neuronal responses to methamphetamine. We therefore examined persistent psychosis-like behavioral effects of methamphetamine in brain-derived neurotrophic factor heterozygous mice. Mice were chronically treated with methamphetamine from 6 to 9 weeks of age, and locomotor hyperactivity to an acute D-amphetamine challenge was tested in photocell cages after a 2-week withdrawal period. Methamphetamine-treated wild-type mice, but not brain-derived neurotrophic factor heterozygous mice, showed locomotor sensitization to acute 3mg/kg D-amphetamine. Qualitative analysis of exploration revealed tolerance to D-amphetamine effects on entropy in methamphetamine-treated brain-derived neurotrophic factor heterozygous mice, but not wild-type mice. Chronic methamphetamine exposure induces contrasting profiles of behavioral changes in wild-type and brain-derived neurotrophic factor heterozygous mice, with attenuation of behaviors relevant to psychosis in methamphetamine-treated brain-derived neurotrophic factor heterozygous mice. This suggests that brain-derived neurotrophic factor signalling changes may contribute to development of psychosis in methamphetamine users. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

DEFF Research Database (Denmark)

Wewer, U M; Albrechtsen, R; Hassell, J R

1985-01-01

in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeled glycosaminoglycans, the mouse EHS tumor cells were found to produce...... proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000 daltons from 35S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to be distributed in the extracellular matrix and also...

Dioxin increases the interaction between aryl hydrocarbon receptor and estrogen receptor alpha at human promoters

DEFF Research Database (Denmark)

Ahmed, Shaaima; Valen, Eivind; Sandelin, Albin Gustav

2009-01-01

genes with little knowledge of what was occurring at other genomic regions. In this study, we showed using chromatin immunoprecipitation followed by hybridization to promoter focused microarrays (ChIP-chip) that 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment significantly increased the overlap of genomic...... , suggesting that AHR was the important factor determining the recruitment of ER to these regions. RNA interference-mediated knockdown of AHR confirmed its requirement for the recruitment of ER to some, but not all, of the shared regions. Our findings demonstrate not only that dioxin induces the recruitment...

Integrated genetic and epigenetic analysis identifies haplotype-specific methylation in the FTO type 2 diabetes and obesity susceptibility locus.

Directory of Open Access Journals (Sweden)

Christopher G Bell

2010-11-01

Full Text Available Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D, focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip and absolute methylation values were estimated using a Bayesian algorithm (BATMAN. Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10(-4, permutation p = 1.0×10(-3. Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10(-7. Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM, encapsulates a Highly Conserved Non-Coding Element (HCNE that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

CERN cars drive by the Geneva Motor Show

CERN Multimedia

CERN Bulletin

2011-01-01

One of CERN's new gas-fuelled cars was a special guest at the press days of the Geneva motor show this year. The car enjoyed a prominent position on the Gazmobil stand, right next to the latest Mazeratis and Ferraris. Journalists previewing the motor show could discover CERN's support for green technologies and also find out more about the lab - home to the fastest racetrack on the planet, with protons in the LHC running at 99.9999991% of the speed of light.    

Reducing no-show behavior at a community mental health center

NARCIS (Netherlands)

Dieren, Q van; Rijckmans, M.J.N.; Mathijssen, J.J.P.; Lobbestael, J.; Arntz, A.R.

2013-01-01

The objective of this study was to examine whether an easy to apply no-show policy can substantially reduce no-show behavior of 16–25-year-old clients undergoing individual outpatient treatment at a community mental health center. After introduction of the new no-show policy, the no-show percentage

Actinobacteria from Termite Mounds Show Antiviral Activity against Bovine Viral Diarrhea Virus, a Surrogate Model for Hepatitis C Virus

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Marina Aiello Padilla

2015-01-01

Full Text Available Extracts from termite-associated bacteria were evaluated for in vitro antiviral activity against bovine viral diarrhea virus (BVDV. Two bacterial strains were identified as active, with percentages of inhibition (IP equal to 98%. Both strains were subjected to functional analysis via the addition of virus and extract at different time points in cell culture; the results showed that they were effective as posttreatments. Moreover, we performed MTT colorimetric assays to identify the CC50, IC50, and SI values of these strains, and strain CDPA27 was considered the most promising. In parallel, the isolates were identified as Streptomyces through 16S rRNA gene sequencing analysis. Specifically, CDPA27 was identified as S. chartreusis. The CDPA27 extract was fractionated on a C18-E SPE cartridge, and the fractions were reevaluated. A 100% methanol fraction was identified to contain the compound(s responsible for antiviral activity, which had an SI of 262.41. GC-MS analysis showed that this activity was likely associated with the compound(s that had a peak retention time of 5 min. Taken together, the results of the present study provide new information for antiviral research using natural sources, demonstrate the antiviral potential of Streptomyces chartreusis compounds isolated from termite mounds against BVDV, and lay the foundation for further studies on the treatment of HCV infection.

»According to Common Sense«: Gender, Nationality and Class in Cooking Show Ljubezen skozi želodec

Directory of Open Access Journals (Sweden)

Kristina Meršak

2014-03-01

Full Text Available »Ljubezen skozi želodec« is the first Slovenian lifestyle cooking show hosted by spouses, publishers and foodies Valentina Smej Novak and Luka Novak. The aim of the article is to determine what gender, class and national representations are reproduced by the show. The analysis of four seasons of the cooking show demonstrates that, in regard to social class and gender, »Ljubezen skozi želodec« can be compared to similar shows abroad, such as those hosted by Jamie Oliver and Nigella. Masculinity and femininity are constructed through traditional roles where the ideology of family and care for the children is vital. All this corresponds to the Slovenian socio-cultural environment characterized by Catholicism. Class identities are the reflection of Bourdieu’s taste of luxury and a contemporary omnivorous taste of an environmentally conscious consumer. As for the national angle, the Central-European and partially also the French and Italian identity are stressed rather than the Slovenian one, which puts Slovenia on the socio-cultural map of the Central Europe and at the same time negates the culinary influence of the former Yugoslav republics.

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...

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

International Nuclear Information System (INIS)

Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

1989-01-01

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

Identification of proteins specific for human herpesvirus 6-infected human T cells

International Nuclear Information System (INIS)

Balachandran, N.; Amelse, R.E.; Zhou, W.W.; Chang, C.K.

1989-01-01

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [ 35 S]methionine- and [ 3 H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [ 35 S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-M r polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k

Identification of proteins specific for human herpesvirus 6-infected human T cells

International Nuclear Information System (INIS)

Balachandran, N.; Amelse, R.E.; Zhou, W.W.; Chang, C.K.

1989-01-01

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [ 35 S]methionine- and [ 3 H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [ 35 S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpes viruses reacted with few HHV-6-infected cell proteins, and only a 135,000-M/sub r/ polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105K and gp92k, gp116k, gp64k, and gp54k, and gp102k

GenoGAM: genome-wide generalized additive models for ChIP-Seq analysis.

Science.gov (United States)

Stricker, Georg; Engelhardt, Alexander; Schulz, Daniel; Schmid, Matthias; Tresch, Achim; Gagneur, Julien

2017-08-01

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a widely used approach to study protein-DNA interactions. Often, the quantities of interest are the differential occupancies relative to controls, between genetic backgrounds, treatments, or combinations thereof. Current methods for differential occupancy of ChIP-Seq data rely however on binning or sliding window techniques, for which the choice of the window and bin sizes are subjective. Here, we present GenoGAM (Genome-wide Generalized Additive Model), which brings the well-established and flexible generalized additive models framework to genomic applications using a data parallelism strategy. We model ChIP-Seq read count frequencies as products of smooth functions along chromosomes. Smoothing parameters are objectively estimated from the data by cross-validation, eliminating ad hoc binning and windowing needed by current approaches. GenoGAM provides base-level and region-level significance testing for full factorial designs. Application to a ChIP-Seq dataset in yeast showed increased sensitivity over existing differential occupancy methods while controlling for type I error rate. By analyzing a set of DNA methylation data and illustrating an extension to a peak caller, we further demonstrate the potential of GenoGAM as a generic statistical modeling tool for genome-wide assays. Software is available from Bioconductor: https://www.bioconductor.org/packages/release/bioc/html/GenoGAM.html . gagneur@in.tum.de. Supplementary information is available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Biosynthesis of human sialophorins and analysis of the polypeptide core

International Nuclear Information System (INIS)

Remold-O'Donnell, E.; Kenney, D.; Rosen, F.S.

1987-01-01

Biosynthesis was examined of sialophorin (formerly called gpL115) which is altered in the inherited immunodeficiency Wiskott-Aldrich syndrome. Sialophorin is greater than 50% carbohydrate, primarily O-linked units of sialic acid, galactose, and galactosamine. Pulse-labeling with [ 35 S]methionine and chase incubation established that sialophorin is synthesized in CEM lymphoblastoid cells as an Mr 62,000 precursor which is converted within 45 min to mature glycosylated sialophorin, a long-lived molecule. Experiments with tunicamycin and endoglycosidase H demonstrated that sialophorin contains N-linked carbohydrate (approximately two units per molecule) and is therefore an N,O-glycoprotein. Pulse-labeling of tunicamycin-treated CEM cells together with immunoprecipitation provided the means to isolate the [ 35 S]-methionine-labeled polypeptide core of sialophorin and determine its molecular weight (58,000). This datum allowed us to express the previously established composition on a per molecule basis and determine that sialophorin molecules contain approximately 520 amino acid residues and greater than or equal to 100 O-linked carbohydrate units. A recent study showed that various blood cells express sialophorin and that there are two molecular forms: lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin. Biosynthesis of the two forms was compared by using sialophorin of CEM cells and sialophorin of MOLT-4 cells (another lymphoblastoid line) as models for lymphocyte/monocyte sialophorin and platelet/neutrophil sialophorin, respectively. The time course of biosynthesis and the content of N units were found to be identical for the two sialophorin species. [ 35 S]Methionine-labeled polypeptide cores of CEM sialophorin and MOLT sialophorin were isolated and compared by electrophoresis, isoelectrofocusing, and a newly developed peptide mapping technique

Human-directed social behaviour in dogs shows significant heritability.

Science.gov (United States)

Persson, M E; Roth, L S V; Johnsson, M; Wright, D; Jensen, P

2015-04-01

Through domestication and co-evolution with humans, dogs have developed abilities to attract human attention, e.g. in a manner of seeking assistance when faced with a problem solving task. The aims of this study were to investigate within breed variation in human-directed contact seeking in dogs and to estimate its genetic basis. To do this, 498 research beagles, bred and kept under standardized conditions, were tested in an unsolvable problem task. Contact seeking behaviours recorded included both eye contact and physical interactions. Behavioural data was summarized through a principal component analysis, resulting in four components: test interactions, social interactions, eye contact and physical contact. Females scored significantly higher on social interactions and physical contact and age had an effect on eye contact scores. Narrow sense heritabilities (h(2) ) of the two largest components were estimated at 0.32 and 0.23 but were not significant for the last two components. These results show that within the studied dog population, behavioural variation in human-directed social behaviours was sex dependent and that the utilization of eye contact seeking increased with age and experience. Hence, heritability estimates indicate a significant genetic contribution to the variation found in human-directed social interactions, suggesting that social skills in dogs have a genetic basis, but can also be shaped and enhanced through individual experiences. This research gives the opportunity to further investigate the genetics behind dogs' social skills, which could also play a significant part into research on human social disorders such as autism. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

QLab 3 show control projects for live performances & installations

CERN Document Server

Hopgood, Jeromy

2013-01-01

Used from Broadway to Britain's West End, QLab software is the tool of choice for many of the world's most prominent sound, projection, and integrated media designers. QLab 3 Show Control: Projects for Live Performances & Installations is a project-based book on QLab software covering sound, video, and show control. With information on both sound and video system basics and the more advanced functions of QLab such as MIDI show control, new OSC capabilities, networking, video effects, and microphone integration, each chapter's specific projects will allow you to learn the software's capabilitie

Fungal diversity in oil palm leaves showing symptoms of Fatal Yellowing disease.

Science.gov (United States)

de Assis Costa, Ohana Yonara; Tupinambá, Daiva Domenech; Bergmann, Jessica Carvalho; Barreto, Cristine Chaves; Quirino, Betania Ferraz

2018-01-01

Oil palm (Elaeis guineensis Jacq.) is an excellent source of vegetable oil for biodiesel production; however, there are still some limitations for its cultivation in Brazil such as Fatal Yellowing (FY) disease. FY has been studied for many years, but its causal agent has never been determined. In Colombia and nearby countries, it was reported that the causal agent of Fatal Yellowing (Pudrición del Cogollo) is the oomycete Phytophthora palmivora, however, several authors claim that Fatal Yellowing and Pudrición del Cogollo (PC) are different diseases. The major aims of this work were to test, using molecular biology tools, Brazilian oil palm trees for the co-occurrence of the oomycete Phytophthora and FY symptoms, and to characterize the fungal diversity in FY diseased and healthy leaves by next generation sequencing. Investigation with specific primers for the genus Phytophthora showed amplification in only one of the samples. Analysis of the fungal ITS region demonstrated that, at the genus level, different groups predominated in all symptomatic samples, while Pyrenochaetopsis and unclassified fungi predominated in all asymptomatic samples. Our results show that fungal communities were not the same between samples at the same stage of the disease or among all the symptomatic samples. This is the first study that describes the evolution of the microbial community in the course of plant disease and also the first work to use high throughput next generation sequencing to evaluate the fungal community associated with leaves of oil palm trees with and without symptoms of FY.

Fungal diversity in oil palm leaves showing symptoms of Fatal Yellowing disease.

Directory of Open Access Journals (Sweden)

Ohana Yonara de Assis Costa

Full Text Available Oil palm (Elaeis guineensis Jacq. is an excellent source of vegetable oil for biodiesel production; however, there are still some limitations for its cultivation in Brazil such as Fatal Yellowing (FY disease. FY has been studied for many years, but its causal agent has never been determined. In Colombia and nearby countries, it was reported that the causal agent of Fatal Yellowing (Pudrición del Cogollo is the oomycete Phytophthora palmivora, however, several authors claim that Fatal Yellowing and Pudrición del Cogollo (PC are different diseases. The major aims of this work were to test, using molecular biology tools, Brazilian oil palm trees for the co-occurrence of the oomycete Phytophthora and FY symptoms, and to characterize the fungal diversity in FY diseased and healthy leaves by next generation sequencing. Investigation with specific primers for the genus Phytophthora showed amplification in only one of the samples. Analysis of the fungal ITS region demonstrated that, at the genus level, different groups predominated in all symptomatic samples, while Pyrenochaetopsis and unclassified fungi predominated in all asymptomatic samples. Our results show that fungal communities were not the same between samples at the same stage of the disease or among all the symptomatic samples. This is the first study that describes the evolution of the microbial community in the course of plant disease and also the first work to use high throughput next generation sequencing to evaluate the fungal community associated with leaves of oil palm trees with and without symptoms of FY.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

Directory of Open Access Journals (Sweden)

Laura Graf

2013-12-01

Full Text Available The human cytomegalovirus (HCMV-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins.

HIC1 interacts with a specific subunit of SWI/SNF complexes, ARID1A/BAF250A

International Nuclear Information System (INIS)

Van Rechem, Capucine; Boulay, Gaylor; Leprince, Dominique

2009-01-01

HIC1, a tumor suppressor gene epigenetically silenced in many human cancers encodes a transcriptional repressor involved in regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. HIC1 is also implicated in growth control since it recruits BRG1, one of the two alternative ATPases (BRM or BRG1) of SWI/SNF chromatin-remodeling complexes to repress transcription of E2F1 in quiescent fibroblasts. Here, through yeast two-hybrid screening, we identify ARID1A/BAF250A, as a new HIC1 partner. ARID1A/BAF250A is one of the two mutually exclusive ARID1-containing subunits of SWI/SNF complexes which define subsets of complexes endowed with anti-proliferative properties. Co-immunoprecipitation assays in WI38 fibroblasts and in BRG1-/- SW13 cells showed that endogenous HIC1 and ARID1A proteins interact in a BRG1-dependent manner. Furthermore, we demonstrate that HIC1 does not interact with BRM. Finally, sequential chromatin immunoprecipitation (ChIP-reChIP) experiments demonstrated that HIC1 represses E2F1 through the recruitment of anti-proliferative SWI/SNF complexes containing ARID1A.

Functional interrelationship between TFII-I and E2F transcription factors at specific cell cycle gene loci.

Science.gov (United States)

Shen, Yong; Nar, Rukiye; Fan, Alex X; Aryan, Mahmoud; Hossain, Mir A; Gurumurthy, Aishwarya; Wassel, Paul C; Tang, Ming; Lu, Jianrong; Strouboulis, John; Bungert, Jörg

2018-01-01

Transcription factor TFII-I is a multifunctional protein implicated in the regulation of cell cycle and stress-response genes. Previous studies have shown that a subset of TFII-I associated genomic sites contained DNA-binding motifs for E2F family transcription factors. We analyzed the co-association of TFII-I and E2Fs in more detail using bioinformatics, chromatin immunoprecipitation, and co-immunoprecipitation experiments. The data show that TFII-I interacts with E2F transcription factors. Furthermore, TFII-I, E2F4, and E2F6 interact with DNA-regulatory elements of several genes implicated in the regulation of the cell cycle, including DNMT1, HDAC1, CDKN1C, and CDC27. Inhibition of TFII-I expression led to a decrease in gene expression and in the association of E2F4 and E2F6 with these gene loci in human erythroleukemia K562 cells. Finally, TFII-I deficiency reduced the proliferation of K562 cells and increased the sensitivity toward doxorubicin toxicity. The results uncover novel interactions between TFII-I and E2Fs and suggest that TFII-I mediates E2F function at specific cell cycle genes. © 2017 Wiley Periodicals, Inc.

AirShow 1.0 CFD Software Users' Guide

Science.gov (United States)

Mohler, Stanley R., Jr.

2005-01-01

AirShow is visualization post-processing software for Computational Fluid Dynamics (CFD). Upon reading binary PLOT3D grid and solution files into AirShow, the engineer can quickly see how hundreds of complex 3-D structured blocks are arranged and numbered. Additionally, chosen grid planes can be displayed and colored according to various aerodynamic flow quantities such as Mach number and pressure. The user may interactively rotate and translate the graphical objects using the mouse. The software source code was written in cross-platform Java, C++, and OpenGL, and runs on Unix, Linux, and Windows. The graphical user interface (GUI) was written using Java Swing. Java also provides multiple synchronized threads. The Java Native Interface (JNI) provides a bridge between the Java code and the C++ code where the PLOT3D files are read, the OpenGL graphics are rendered, and numerical calculations are performed. AirShow is easy to learn and simple to use. The source code is available for free from the NASA Technology Transfer and Partnership Office.

Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

International Nuclear Information System (INIS)

Bullock, S.; Hayward, C.; Manson, J.; Brock, D.J.H.; Raeburn, J.A.

1982-01-01

Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis. (author)

Synthesis of erythrocyte membrane proteins in dispersed cells from fetal rat liver

International Nuclear Information System (INIS)

Kitagawa, Yasuo; Murakami, Akihiko; Sugimoto, Etsuro

1984-01-01

Protein synthesis in dispersed cells from fetal liver was studied by fluorography of SDS-polyacrylamide gel electrophoresis of a [ 35 S] methionine labeled cell lysate. Synthesis of several proteins with molecular weights ranging from 45,000 to 220,000 was observed during erythropoiesis in fetal liver. Some of these proteins were demonstrated to be erythrocyte membrane proteins because they were immunoprecipitated with antiserum against rat red blood cells and the immunoprecipitation was competitive with non-radioactive proteins solubilized from erythrocyte ghosts. The same antiserum caused agglutination of dispered cells from fetal liver. This supported the possibility that these proteins are translocated onto plasma membranes of the dispersed cells. (author)