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Sample records for analysis reveals phosphorylation

  1. Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

    Science.gov (United States)

    Kohansal-Nodehi, Mahdokht; Chua, John Je; Urlaub, Henning; Jahn, Reinhard; Czernik, Dominika

    2016-01-01

    Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity. PMID:27115346

  2. Phosphoproteome analysis of streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation

    DEFF Research Database (Denmark)

    Manteca, Angel; Ye, Juanying; Sánchez, Jesús;

    2011-01-01

    bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation...

  3. Comparative analysis reveals conserved protein phosphorylation networks implicated in multiple diseases

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Bodenmiller, Bernd; Pasculescu, Adrian;

    2009-01-01

    Protein kinases enable cellular information processing. Although numerous human phosphorylation sites and their dynamics have been characterized, the evolutionary history and physiological importance of many signaling events remain unknown. Using target phosphoproteomes determined with a similar ...

  4. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene;

    2008-01-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes...

  5. Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

    Directory of Open Access Journals (Sweden)

    Patrick M McDonough

    Full Text Available Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL. Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5 and serine 522 (PKA-site 6. To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK and L-γ-melanocyte stimulating hormone (L-γ-MSH. In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

  6. Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

    Science.gov (United States)

    McDonough, Patrick M; Maciejewski-Lenoir, Dominique; Hartig, Sean M; Hanna, Rita A; Whittaker, Ross; Heisel, Andrew; Nicoll, James B; Buehrer, Benjamin M; Christensen, Kurt; Mancini, Maureen G; Mancini, Michael A; Edwards, Dean P; Price, Jeffrey H

    2013-01-01

    Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3',5'-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

  7. Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

    Directory of Open Access Journals (Sweden)

    Nespoulous Claude

    2012-10-01

    Full Text Available Abstract Background Genome-wide statistics established that long intrinsically disordered regions (over 30 residues are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale. Results Using a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation. It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane. Conclusions The new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.

  8. Phosphoproteome analysis of functional mitochondria isolated from resting human muscle reveals extensive phosphorylation of inner membrane protein complexes and enzymes

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Leon, Ileana R; Bak, Steffen;

    2011-01-01

    for protein kinase A, protein kinase C, casein kinase II and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria...

  9. Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity.

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    Hao, Nan; Yildirim, Necmettin; Nagiec, Michal J; Parnell, Stephen C; Errede, Beverly; Dohlman, Henrik G; Elston, Timothy C

    2012-10-01

    Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity. PMID:22875986

  10. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone;

    2016-01-01

    . New insights into phosphorylation-dependent signaling networks that impact on nuclear proteins and controls adipocyte differentiation and cell fate. Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency...

  11. Proteomic analysis of the oil palm fruit mesocarp reveals elevated oxidative phosphorylation activity is critical for increased storage oil production.

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    Loei, Hendrick; Lim, Justin; Tan, Melvin; Lim, Teck Kwang; Lin, Qing Song; Chew, Fook Tim; Kulaveerasingam, Harikrishna; Chung, Maxey C M

    2013-11-01

    Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages--12, 16, 18, and 22 weeks after pollination--by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the β subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees. PMID:24083564

  12. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

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    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  13. Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration.

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    Kuropka, Benno; Witte, Amelie; Sticht, Jana; Waldt, Natalie; Majkut, Paul; Hackenberger, Christian P R; Schraven, Burkhart; Krause, Eberhard; Kliche, Stefanie; Freund, Christian

    2015-11-01

    Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.

  14. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV;

    2010-01-01

    ) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells....

  15. Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

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    Gregorich, Zachery R; Peng, Ying; Cai, Wenxuan; Jin, Yutong; Wei, Liming; Chen, Albert J; McKiernan, Susan H; Aiken, Judd M; Moss, Richard L; Diffee, Gary M; Ge, Ying

    2016-08-01

    Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia. PMID:27362462

  16. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

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    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  17. Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Wrzesinski, Krzysztof; Larsen, Peter Mose;

    2003-01-01

    Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can...... state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle...

  18. Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

    Directory of Open Access Journals (Sweden)

    Raabe Timothy D

    2003-04-01

    Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

  19. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

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    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  20. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    DEFF Research Database (Denmark)

    Mann, Matthias; Ong, Shao En; Grønborg, Mads;

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling...

  1. Network Analysis of Epidermal Growth Factor Signaling Using Integrated Genomic, Proteomic and Phosphorylation Data

    OpenAIRE

    Waters, Katrina M.; Tao Liu,; Quesenberry, Ryan D.; Willse, Alan R.; Somnath Bandyopadhyay; Kathmann, Loel E.; Thomas J. Weber; Smith, Richard D.; H Steven Wiley; Thrall, Brian D.

    2012-01-01

    To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by...

  2. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.

    Science.gov (United States)

    Huguenin-Dezot, Nicolas; De Cesare, Virginia; Peltier, Julien; Knebel, Axel; Kristaryianto, Yosua Adi; Rogerson, Daniel T; Kulathu, Yogesh; Trost, Matthias; Chin, Jason W

    2016-07-26

    Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages. PMID:27425610

  3. Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max

    2012-01-01

    in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins....

  4. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    Energy Technology Data Exchange (ETDEWEB)

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  5. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

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    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. PMID:27017623

  6. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    Science.gov (United States)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  7. Novel Tyrosine Phosphorylation Sites in Rat Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    OpenAIRE

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun; Meyer, Christian; Thangiah, Geetha; Yi, Zhengping

    2012-01-01

    Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (

  8. Single cell analysis of G1 check points-the relationship between the restriction point and phosphorylation of pRb

    International Nuclear Information System (INIS)

    Single cell analysis allows high resolution investigation of temporal relationships between transition events in G1. It has been suggested that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is the molecular mechanism behind passage through the restriction point (R). We performed a detailed single cell study of the temporal relationship between R and pRb phosphorylation in human fibroblasts using time lapse video-microscopy combined with immunocytochemistry. Four principally different criteria for pRb phosphorylation were used, namely (i) phosphorylation of residues Ser795 and Ser780 (ii) degree of pRb-association with the nuclear structure, a property that is closely related with pRb phosphorylation status, (iii) release of the transcription factor E2F-1 from pRb, and (iv) accumulation of cyclin E, which is dependent on phosphorylation of pRb. The analyses of individual cells revealed that passage through R preceded phosphorylation of pRb, which occurs in a gradually increasing proportion of cells in late G1. Our data clearly suggest that pRb phosphorylation is not the molecular mechanism behind the passage through R. The restriction point and phosphorylation of pRb thus seem to represent two separate check point in G1

  9. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    Science.gov (United States)

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  10. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

    Science.gov (United States)

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  11. Construction and Analysis of N-phosphoryl Peptide Libraries

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Ming Yu NIU; Kui LU; Xin Cheng LIAO; Yu Fen ZHAO

    2004-01-01

    N-Phosphoryl peptide libraries were constructed by transformation from homo-oligopeptide libraries, which was synthesized by self-assembly of amino acids with the assistance of phosphorus oxychloride. Electrospray ionization mass spectrometry (ESI-MS) was used to monitor the reaction.

  12. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  13. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Science.gov (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  14. Identification and functional analysis of phosphorylation in Newcastle disease virus phosphoprotein.

    Science.gov (United States)

    Qiu, Xusheng; Zhan, Yuan; Meng, Chunchun; Wang, Junqing; Dong, LuNa; Sun, Yingjie; Tan, Lei; Song, Cuiping; Yu, Shengqing; Ding, Chan

    2016-08-01

    Newcastle disease virus (NDV) encodes a highly phosphorylated P protein; however, the phosphorylation sites have not been identified, and the relationship between phosphorylation and protein function is still unclear. In this study, we bioinformatically predicted 26 amino acid residues in the P protein as potential phosphorylation sites. Furthermore, we treated infected cells with kinase inhibitors to investigate NDV propagation and found that protein kinase C (PKC) is involved in the NDV life cycle and that PKC-activated phosphorylation functions in NDV replication. Using an NDV minigenome assay, we found that expression of a reporter protein decreased when the minigenome system contained P mutants lacking T44, S48, T271, S373 and especially T111. The phosphorylation status of S48, T111, S125 and T271 was determined by Phos-tag SDS-PAGE analysis. Coimmunoprecipitation assays showed that the binding activity of NP and the P-T111A mutant was stronger than that of NP and the wild-type P, suggesting that P-T111 is involved in NP-P interaction. This study sheds light on the mechanism by which P protein phosphorylation affects NDV replication and transcription. PMID:27160999

  15. Analysis of phosphorylation of human heat shock factor 1 in cells experiencing a stress

    Directory of Open Access Journals (Sweden)

    Lane William S

    2005-03-01

    Full Text Available Abstract Background Heat shock factor (HSF/HSF1 not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs. HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser303, Ser307 and Ser363, which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser230 contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells. Results An alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444. Phosphorylation of Ser326 but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser326 increased rapidly during heat stress as shown by experiments using a pSer326 phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type

  16. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F;

    2008-01-01

    Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently...... sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info)....

  17. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Sebastian König

    Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

  18. Network Analysis of Epidermal Growth Factor Signaling using Integrated Genomic, Proteomic and Phosphorylation Data

    Energy Technology Data Exchange (ETDEWEB)

    Waters, Katrina M.; Liu, Tao; Quesenberry, Ryan D.; Willse, Alan R.; Bandyopadhyay, Somnath; Kathmann, Loel E.; Weber, Thomas J.; Smith, Richard D.; Wiley, H. S.; Thrall, Brian D.

    2012-03-29

    To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

  19. Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data.

    Directory of Open Access Journals (Sweden)

    Katrina M Waters

    Full Text Available To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

  20. Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

    Science.gov (United States)

    Zhang, Hao; Sun, Jun; Ye, Jing; Ashraf, Usama; Chen, Zheng; Zhu, Bibo; He, Wen; Xu, Qiuping; Wei, Yanming; Chen, Huanchun; Fu, Zhen F; Liu, Rong; Cao, Shengbo

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

  1. Monoclonal Antibodies Specific for STAT3β Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bharadwaj, Uddalak; Kasembeli, Moses M.; Eckols, T. Kris; Kolosov, Mikhail; Lang, Paul [Section of Infectious Disease, Department of Medicine, Baylor College of Medicine, Houston, TX 77030 (United States); Christensen, Kurt [Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Edwards, Dean P. [Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 77030 (United States); Tweardy, David J., E-mail: dtweardy@bcm.edu [Section of Infectious Disease, Department of Medicine, Baylor College of Medicine, Houston, TX 77030 (United States); Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Department of Biochemistry & Molecular Biology, BCM 286, Room N-1319, Baylor College of Medicine, Houston, TX 77030 (United States)

    2014-09-29

    Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3β, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3β homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3β acted as a dominant-negative of Stat3α in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3β proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3β (CT7) and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3β protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3β may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3β from proteolytic Stat3β and Stat3α will provide new insights into the contribution of Stat3β vs. Stat3α to oncogenesis, as well as other biological and pathological processes.

  2. Monoclonal Antibodies Specific for STAT3β Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Uddalak Bharadwaj

    2014-09-01

    Full Text Available Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3β, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p Stat3β homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3β acted as a dominant-negative of Stat3α in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3β proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3β (CT7 and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3β protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3β may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3β from proteolytic Stat3β and Stat3α will provide new insights into the contribution of Stat3β vs. Stat3α to oncogenesis, as well as other biological and pathological processes.

  3. Top-down control analysis of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes.

    Science.gov (United States)

    Ainscow, E K; Brand, M D

    1999-08-01

    Control analysis was used to analyse the internal control of rat hepatocyte metabolism. The reactions of the cell were grouped into nine metabolic blocks linked by five key intermediates. The blocks were glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption. The linking intermediates were intracellular glucose-6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential. The steady-state fluxes through the blocks and the levels of the intermediates were measured in the absence and presence of specific effectors of hepatocyte metabolism. Application of the multiple modulation approach gave the kinetic responses of each block to each intermediate (the elasticities). These were then used to calculate all of the control coefficients, which describe the degree of control each block had over the level of each intermediate, and over the rate of each process. Within this full description of control, many different interactions could be identified. One key finding was that the processes that consumed ATP had only 35% of the control over the rate of ATP consumption. Instead, the reactions that produced ATP exerted the most control over ATP consumption rate; particularly important were mitochondrial phosphorylation (30% of control) and glycolysis (19%). The rate of glycolysis was positively controlled by the glycolytic enzymes themselves (66% of control) and by ATP consumption (47%). Mitochondrial production of ATP, including oxidative, proton leak and phosphorylation processes, had negative control over glycolysis (-26%; the Pasteur effect). In contrast, glycolysis had little control over the rate of ATP production by the mitochondria (-10%; the Crabtree effect). Control over the flux through the mitochondrial phosphorylation block was shared between pyruvate oxidation (23%), ATP consumption (28%) and the

  4. Is Utility Transferable? A Revealed Preference Analysis

    NARCIS (Netherlands)

    Cherchye, L.J.H.; Demuynck, T.; de Rock, B.

    2011-01-01

    We provide a revealed preference analysis of the transferable utility hypothesis, which is widely used in economic models. First, we establish revealed preference conditions that must be satisfied for observed group behavior to be consistent with Pareto efficiency under transferable utility. Next, w

  5. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    OpenAIRE

    Sheng-Bao Suo; Jian-Ding Qiu; Shao-Ping Shi; Xiang Chen; Ru-Ping Liang

    2014-01-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by...

  6. A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.

    Science.gov (United States)

    Meistermann, Hélène; Gao, Junjun; Golling, Sabrina; Lamerz, Jens; Le Pogam, Sophie; Tzouros, Manuel; Sankabathula, Sailaja; Gruenbaum, Lore; Nájera, Isabel; Langen, Hanno; Klumpp, Klaus; Augustin, Angélique

    2014-11-01

    Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.

  7. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Seon Sook; Lee, Eun Hye [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Lee, Kooyeon [Department of Bio-Health Technology, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Jo, Su-Hyun, E-mail: suhyunjo@kangwon.ac.kr [Department of Physiology, BK21 Plus Graduate Program, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of); Seo, Su Ryeon, E-mail: suryeonseo@kangwon.ac.kr [Department of Molecular Bioscience, College of Biomedical Science, Institute of Bioscience & Biotechnology, Kangwon National University, Chuncheon 200-701 (Korea, Republic of)

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  8. Analysis of tyrosine phosphorylation sites in signaling molecules by a phosphotyrosine-specific immonium ion scanning method

    DEFF Research Database (Denmark)

    Steen, Hanno; Pandey, Akhilesh; Andersen, Jens S;

    2002-01-01

    Signal transduction pathways involve cascades of events, such as formation of second messengers and protein complexes that alter the activities of proteins. This can ultimately lead to changes in gene expression in response to the stimuli. Reversible phosphorylation of proteins is an important me...... mechanism for activating or inhibiting enzymes and for the assembly of multiprotein complexes. Here, we describe a mass spectrometry-based phosphotyrosine-specific immonium ion scanning (PSI scanning) method for selective detection of tyrosine-phosphorylated peptides. Once the tyrosine......-phosphorylated peptides are identified, they can be directly sequenced in the same experiment to localize the phosphorylation site. We provide protocols for isolation and preparation of samples for analysis, and detailed instructions for operation of a quadrupole time-of-flight (TOF) mass spectrometer for this method...

  9. Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine.

    Science.gov (United States)

    Perazzolli, Michele; Palmieri, Maria Cristina; Matafora, Vittoria; Bachi, Angela; Pertot, Ilaria

    2016-05-20

    Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew. PMID:27010348

  10. Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes.

    Directory of Open Access Journals (Sweden)

    Justin D Blethrow

    Full Text Available The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI and electrospray ionization (ESI ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.

  11. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    Science.gov (United States)

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  12. Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β.

    Science.gov (United States)

    Wang, Shuxin; Li, Zheyi; Shen, Hongyan; Zhang, Zhong; Yin, Yuxin; Wang, Qingsong; Zhao, Xuyang; Ji, Jianguo

    2016-08-01

    Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3β (Gsk3β). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3β, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101. PMID:27097102

  13. Proteomic analysis of ERK1/2-mediated human sickle red blood cell membrane protein phosphorylation

    Directory of Open Access Journals (Sweden)

    Soderblom Erik J

    2013-01-01

    Full Text Available Abstract Background In sickle cell disease (SCD, the mitogen-activated protein kinase (MAPK ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs. ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. Results To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2

  14. High-accuracy identification and bioinformatic analysis of in vivo protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Gnad, Florian; de Godoy, Lyris M F; Cox, Jürgen;

    2009-01-01

    Protein phosphorylation is a fundamental regulatory mechanism that affects many cell signaling processes. Using high-accuracy MS and stable isotope labeling in cell culture-labeling, we provide a global view of the Saccharomyces cerevisiae phosphoproteome, containing 3620 phosphorylation sites ma...

  15. IN VITRO ANALYSIS OF τ PHOSPHORYLATION SITES AND ITS BIOLOGICAL ACTIVITY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective.To explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) τ and the inhibition of its biological activity. Methods.Ultracentrifugation,chromatography,manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant τ ,isolate and purify 32P τ peptides and determine phosphorylation sites. Results.Phosphorylation of τ by casein kinase 1 (CK 1),cyclic AMP dependent protein kinase (PKA) and glycogen synthetase kinase 3 (GSK 3) separately inhibited its biological activity and the inhibition of this activity by GSK 3 was significantly increased if τ was prephosphorylated by CK 1 or PKA.The most potent inhibition was seen by a combined phosphorylation of τ with PKA and GSK 3.The treatment of τ by PKA and GSK 3 combination induced phosphorylation of τ at Ser 195,Ser 198,Ser 199,Ser 202,Thr 205,Thr 231,Ser 235,Ser 262,Ser 356,Ser 404,whereas Thr 181,Ser 184,Ser 262,Ser 356 and Ser 400 were phosphorylated by GSK 3 alone under the same condition. Conclusion.Phosphorylation of τ by PKA plus GSK 3 at Thr 205 might play a key role in τ pathology in AD.

  16. Quantitative phosphoproteomic analysis of early alterations in protein phosphorylation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

    DEFF Research Database (Denmark)

    Schulz, Melanie; Brandner, Stefanie; Eberhagen, Carola;

    2013-01-01

    A comprehensive quantitative analysis of changes in protein phosphorylation preceding or accompanying transcriptional activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in 5L rat hepatoma cells was performed using the SILAC approach. Following exposure of the cells to DMSO or 1 nM TCDD for 0...

  17. Label-free quantitative phosphoproteomic analysis reveals differentially regulated proteins and pathway in PRRSV-infected pulmonary alveolar macrophages.

    Science.gov (United States)

    Luo, Rui; Fang, Liurong; Jin, Hui; Wang, Dang; An, Kang; Xu, Ningzhi; Chen, Huanchun; Xiao, Shaobo

    2014-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen of swine worldwide and causes significant economic losses. Through regulating the host proteins phosphorylation, PRRSV was found to manipulate the activities of several signaling molecules to regulate innate immune responses. However, the role of protein phosphorylation during PRRSV infection and the signal pathways responsible for it are relatively unknown. Here liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global phosphorylation events in PRRSV-infected pulmonary alveolar macrophages. In total, we identified 2125 unique phosphosites, of which the phosphorylation level of 292 phosphosites on 242 proteins and 373 phosphosites on 249 proteins was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics data were analyzed using ingenuity pathways analysis to identify defined canonical pathways and functional networks. Pathway analysis revealed that PRRSV-induced inflammatory cytokines production was probably due to the activation of mitogen-activated protein kinase and NF-κB signal pathway, which were regulated by several protein kinases during virus infection. Interacting network analysis indicated that altered phosphoproteins were involved in cellular assembly and organization, protein synthesis, molecular transport, and signal transduction in PRRSV infected cells. These pathways and functional networks analysis could provide direct insights into the biological significance of phosphorylation events modulated by PRRSV and may help us elucidate the pathogenic mechanisms of PRRSV infection. PMID:24533505

  18. Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Wang Yan

    2009-10-01

    Full Text Available Abstract Background- Aberrant activity of tyrosine-phosphorylated proteins is commonly associated with HCC metastasis. Cell signaling events driven by these proteins are implicated in numerous processes that alter cancer cell behavior. Exploring the activities and signaling pathways of these proteins in HCC metastasis may help in identifying new candidate molecules for HCC-targeted therapy. Methods- Hep3B (a nonmetastatic HCC cell line and MHCC97H (a highly metastatic HCC cell line were used in this study, and the tyrosine-phosphorylated proteins expressed in these cell lines were profiled by a phosphoproteomics technique based on LC-MS/MS. Protein-protein interaction and functional clustering analyses were performed to determine the activities of the identified proteins and the signaling pathways closely related to HCC metastasis. Results- In both cell lines, a total of 247 phosphotyrosine (pTyr proteins containing 281 pTyr sites were identified without any stimulation. The involvement of almost 30% of these in liver or liver cancer has not been reported previously. Biological process clustering analysis indicated that pTyr proteins involved in cell motility, migration, protein autophosphorylation, cell-cell communication, and antiapoptosis functions were overexpressed during metastasis. Pathway clustering analysis revealed that signaling pathways such as those involved in EGFR signaling, cytokine- and chemokine-mediated signal transduction, and the PI3K and JAK-STAT cascades were significantly activated during HCC metastasis. Moreover, noncanonical regulation of the JNK cascade might also provide new targets for HCC metastasis. After comparing the pTyr proteins that were differentially expressed during HCC cell metastasis, we selected FER, a nonreceptor tyrosine kinase, and validated its role in terms of both expression and function. The data confirmed that FER might play a critical role in the invasion and metastasis of HCC. Conclusion- The

  19. Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato

    NARCIS (Netherlands)

    Bultema, Jelle B.; Braun, Hans-Peter; Boekema, Egbert J.; Kouril, Roman; Kouřil, Roman

    2009-01-01

    The individual protein complexes of the oxidative phosphorylation system (OXPHOS complexes 1 to V) specifically interact and form defined supramolecular structures, the so-called "respiratory supercomplexes". Some supercomplexes appear to associate into larger structures, or megacomplexes, such as a

  20. Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Nicolay, Brandon N; Danielian, Paul S; Kottakis, Filippos; Lapek, John D; Sanidas, Ioannis; Miles, Wayne O; Dehnad, Mantre; Tschöp, Katrin; Gierut, Jessica J; Manning, Amity L; Morris, Robert; Haigis, Kevin; Bardeesy, Nabeel; Lees, Jacqueline A; Haas, Wilhelm; Dyson, Nicholas J

    2015-09-01

    The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

  1. Bioinformatics Study of Cancer-Related Mutations within p53 Phosphorylation Site Motifs

    Directory of Open Access Journals (Sweden)

    Xiaona Ji

    2014-07-01

    Full Text Available p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy.

  2. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  3. Quantitative flux analysis reveals folate-dependent NADPH production

    Science.gov (United States)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  4. Protein phosphorylation during coconut zygotic embryo development

    International Nuclear Information System (INIS)

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

  5. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M

    2008-01-01

    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  6. Analysis of Hydrolysis Reaction of N-Phosphoryl Phenylalanine by HPLC-ESI-MS/MS

    Institute of Scientific and Technical Information of China (English)

    Shu Xia CAO; Jian Chen ZHANG; Jun XU; Xin Cheng LIAO; Yu Fen ZHAO

    2004-01-01

    Hydrolysis procedure of N-phosphoryl phenylalanine (DIPP-Phe) was studied by HPLC-ESI-MS/MS. The results showed that (HO)(i-PrO)P(O)Phe was the main intermediate and the hydrolysis of DIPP-Phe also occurred through a penta-coordinate transition state.

  7. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra;

    2015-01-01

    of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association...

  8. Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency.

    NARCIS (Netherlands)

    Hinttala, R.; Smeets, R.; Moilanen, J.S.; Ugalde, C.; Uusimaa, J.; Smeitink, J.A.M.; Majamaa, K.

    2006-01-01

    BACKGROUND: Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. OBJECTIVE: To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying g

  9. Reconstruction and analysis of nutrient-induced phosphorylation networks in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Guangyou eDuan

    2013-12-01

    Full Text Available Elucidating the dynamics of molecular processes in living organisms in response to external perturbations is a central goal in modern systems biology. We investigated the dynamics of protein phosphorylation events in Arabidopsis thaliana exposed to changing nutrient conditions. Phosphopeptide expression levels were detected at five consecutive time points over a time interval of 30 minutes after nutrient resupply following prior starvation. The three tested inorganic, ionic nutrients NH4+, NO3-, PO43- elicited similar phosphosignaling responses that were distinguishable from those invoked by the sugars mannitol, sucrose. When embedded in the protein-protein interaction network of Arabidopsis thaliana, phosphoproteins were found to exhibit a higher degree compared to average proteins. Based on the time-series data, we reconstructed a network of regulatory interactions mediated by phosphorylation. The performance of different network inference methods was evaluated by the observed likelihood of physical interactions within and across different subcellular compartments and based on gene ontology semantic similarity. The dynamic phosphorylation network was then reconstructed using a Pearson correlation method with added directionality based on partial variance differences. The topology of the inferred integrated network corresponds to an information dissemination architecture, in which the phosphorylation signal is passed on to an increasing number of phosphoproteins stratified into an initiation, processing, and effector layer. Specific phosphorylation peptide motifs associated with the distinct layers were identified indicating the action of layer-specific kinases. Despite the limited temporal resolution, combined with information on subcellular location, the available time-series data proved useful for reconstructing the dynamics of the molecular signaling cascade in response to nutrient stress conditions in the plant Arabidopsis thaliana.

  10. Localisation of the sites of action of cadmium on oxidative phosphorylation in potato tuber mitochondria using top-down elasticity analysis.

    Science.gov (United States)

    Kesseler, A; Brand, M D

    1994-11-01

    The aim of this study was to identify the significant sites of action of cadmium on oxidative phosphorylation in potato tuber mitocondria. We simplified the system to three convenient subsystems linked via the production or consumption of a common intermediate, namely protonmotive force. The three subsystems were substrate oxidation, which produces protonmotive force, and the proton leak reactions and the phosphorylation reactions, which consume protonmotive force. By measuring the effect of cadmium on the kinetic response of each subsystem to protonmotive force (top-down elasticity analysis), we found that cadmium stimulated proton leak reactions and strongly inhibited substrate oxidation, but had no measurable effect on the phosphorylation reactions. Cadmium therefore decreases the amount of ATP produced/oxygen consumed (the effective P/O ratio) not by inhibiting the phosphorylation reactions directly, but by inhibiting the production of protonmotive force and by diverting proton flux from phosphorylation reactions to the proton leak reactions. PMID:7957227

  11. Two-tier analysis of histone H2AX phosphorylation allows the identification of Ataxia Telangiectasia heterozygotes

    International Nuclear Information System (INIS)

    Background and purpose: Ataxia Telangiectasia (A-T) heterozygotes constitute 0.36-1% of the general population. They have a higher risk of developing several types of cancer and may be more likely to suffer side-effects following radiotherapy than the general population. Their identification is both labor- and time-consuming and the sensitivity and specificity of the methods employed has not been evaluated. This paper describes a new approach to the identification of A-T heterozygotes based on a two-tier analysis of histone H2AX phosphorylation. Materials and methods: We compared the T-cell phenotype after exposure to 2 Gy in nine obligate A-T heterozygotes and 17 normal donors. Examined end points were histone H2AX phosphorylation by flow cytometry 1 h after irradiation (kinase proficiency) and the residual γ-H2AX foci by confocal microscopy 72 h after irradiation (DSB repair proficiency). Results: The sequential use of these two methods results in 100% positive predictive value (PPV), 67% negative predictive value (NPV), 78% sensitivity, and 100% specificity. The overall hit rate, i.e. the ratio between the true positives plus the true negatives and the total number of observations was 85%. Conclusions: A-T heterozygotes can be identified by analysing irradiated T-cell H2AX phosphorylation level and residual γ-H2AX foci.

  12. Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Ino, Yoko; Arakawa, Noriaki; Ishiguro, Hitoshi; Uemura, Hiroji; Kubota, Yoshinobu; Hirano, Hisashi; Toda, Tosifusa

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

  13. Phosphorylation site analysis of the anti-inflammatory and mRNA destabilizing protein tristetraprolin

    OpenAIRE

    Cao, Heping; Deterding, Leesa J.; Blackshear, Perry J.

    2007-01-01

    Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been use...

  14. High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens.

    Science.gov (United States)

    Poquet, Y; Constant, P; Peyrat, M A; Poupot, R; Halary, F; Bonneville, M; Fournié, J J

    1996-12-01

    A rapid and sensitive high-pH anion-exchange chromatography (HPAEC) method for the separation and quantification of phosphorylated antigens in mycobacterial extracts has been developed. This method provides the separation of mono-, di-, or triphosphonucleotides and of various other phosphorylated molecules. Dual detection by conductimetry and UV absorption downstream of a chemical suppressor constitute nondegradative and highly sensitive tools for the physical detection and the quantification of phosphorylated compounds in biological samples. The lower limit of accurate quantification is around 1 nmol per sample. This method was used for the separation of several phosphorylated antigens activating human gamma delta T lymphocytes from semipurified mycobacterial fractions. Their quantification revealed that the minimal concentration activating a gamma delta T cell clone is between 1 and 5 nM. This approach can be used for more general preparative purposes with samples where minute amounts of biologically active phosphoanions are analyzed.

  15. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-01

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  16. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  17. In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

    Science.gov (United States)

    Agrawal, H C; Sprinkle, T J; Agrawal, D

    1994-06-01

    2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site. PMID:8065530

  18. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-04-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (/sup 32/P) PO/sub 4//sup 3 -/ and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone.

  19. Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato.

    Science.gov (United States)

    Bultema, Jelle B; Braun, Hans-Peter; Boekema, Egbert J; Kouril, Roman

    2009-01-01

    The individual protein complexes of the oxidative phosphorylation system (OXPHOS complexes I to V) specifically interact and form defined supramolecular structures, the so-called "respiratory supercomplexes". Some supercomplexes appear to associate into larger structures, or megacomplexes, such as a string of dimeric ATP synthase (complex V(2)). A row-like organization of OXPHOS complexes I, III and IV into respiratory strings has also been proposed. These transient strings cannot be purified after detergent solubilization. Hence the shape and composition of the respiratory string was approached by an extensive structural characterization of all its possible building blocks, which are the supercomplexes. About 400,000 molecular projections of supercomplexes from potato mitochondria were processed by single particle electron microscopy. We obtained two-dimensional projection maps of at least five different supercomplexes, including the supercomplex I+III(2), III(2)+IV(1), V(2), I+III(2)+IV(1) and I(2)+III(2) in different types of position. From these maps the relative position of the individual complexes in the largest unit, the I(2)+III(2)+IV(2) supercomplex, could be determined in a coherent way. The maps also show that the I+III(2)+IV(1) supercomplex, or respirasome, differs from its counterpart in bovine mitochondria. The new structural features allow us to propose a consistent model of the respiratory string, composed of repeating I(2)+III(2)+IV(2) units, which is in agreement with dimensions observed in former freeze-fracture electron microscopy data.

  20. Preservation Analysis of Macrophage Gene Coexpression Between Human and Mouse Identifies PARK2 as a Genetically Controlled Master Regulator of Oxidative Phosphorylation in Humans

    Science.gov (United States)

    Codoni, Veronica; Blum, Yuna; Civelek, Mete; Proust, Carole; Franzén, Oscar; Björkegren, Johan L. M.; Le Goff, Wilfried; Cambien, Francois; Lusis, Aldons J.; Trégouët, David-Alexandre

    2016-01-01

    Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules). Six modules were found preserved (P Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10−8) the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans. PMID:27558669

  1. Phosphorylation of mouse serine racemase regulates D-serine synthesis

    DEFF Research Database (Denmark)

    Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena;

    2010-01-01

    Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...

  2. Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter pylori strains.

    Directory of Open Access Journals (Sweden)

    Judith Lind

    Full Text Available The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors--in particular various SH2-domain containing human proteins--thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non

  3. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    Science.gov (United States)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  4. Transcriptome-wide Analysis Of Vernalization Reveals Conserved and Species-specific Mechanisms in Brachypodium

    Institute of Scientific and Technical Information of China (English)

    Qing Huan; Zhiwei Mao; Jingyu Zhang; Yunyuan Xu; Kang Chong

    2013-01-01

    Several temperate cereals need vernalization to promote flowering.Little,however,is known about the vernalization-memory-related genes,and almost no comparative analysis has been performed.Here,RNA-Seq was used for transcriptome analysis in non-vernalized,vernalized and post-vernalized Brachypodium distachyon (L.) Beauv.seedlings.In total,the expression of 1,665 genes showed significant changes (fold change ≥4) in response to vernalization.Among them,674 putative vernalization-memory-related genes with a constant response to vernalization were significantly enriched in transcriptional regulation and monooxygenase-mediated biological processes.Comparative analysis of vernalization-memory-related genes with barley demonstrated that the oxidative-stress response was the most conserved pathway between these two plant species.Moreover,Brachypodium preferred to regulate transcription and protein phosphorylation processes,while vernalization-memory-related genes,whose products are cytoplasmic membrane-bound-vesicle-located proteins,were preferred to be regulated in barley.Correlation analysis of the vernalization-related genes with barley revealed that the vernalization mechanism was conserved between these two plant species.In summary,vernalization,including its memory mechanism,is conserved between Brachypodium and barley,although several species-specific features also exist.The data reported here will provide primary resources for subsequent functional research in vernalization.

  5. NPM phosphorylation stimulates Cdk1, overrides G2/M checkpoint and increases leukemic blasts in mice.

    Science.gov (United States)

    Du, Wei; Zhou, Yun; Pike, Suzette; Pang, Qishen

    2010-02-01

    An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CDK) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Simultaneous inactivation of two CDK phosphorylation sites at Ser10 and Ser70 (NPM-AA) induced G(2)/M cell cycle arrest, phosphorylation of Cdk1 at Tyr15 (Cdc2(Tyr15)) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1(Tyr15) and Cdc25C sequestration was suppressed by expression of a phosphomimetic NPM mutant created on the same CDK sites (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 was required for proper interaction between Cdk1 and Cdc25C. Moreover, NPM-EE directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G(2)/M arrest and increased leukemia blasts in a NOD/SCID xenograft model. Thus, these findings reveal a novel function of NPM on regulation of cell cycle progression, in which phosphorylation of NPM controls cell cycle progression at G(2)/M transition through modulation of Cdk1 and Cdc25C activities.

  6. Informatics tools for the analysis and assignment of phosphorylation status in proteomics

    OpenAIRE

    Lee, Dave

    2015-01-01

    Presently, progress in the field of phosphoproteomics has been accelerated by mass spectrometry. This is not a surprise owing to not only the accuracy, precision and high-throughput capabilities of MS but also due to the support it receives from informaticians whom allow the automated analysis; making the task of going from a complex sample to a statistically satisfactory set of phosphopeptides and corresponding site positions with relative ease. However, the process of identifying and subseq...

  7. Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines.

    Science.gov (United States)

    Matsuda, Shun; Furuya, Kanji; Ikura, Masae; Matsuda, Tomonari; Ikura, Tsuyoshi

    2015-11-01

    Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.

  8. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J;

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  9. Phosphorylation of the androgen receptor by PIM1 in hormone refractory prostate cancer.

    Science.gov (United States)

    Ha, S; Iqbal, N J; Mita, P; Ruoff, R; Gerald, W L; Lepor, H; Taneja, S S; Lee, P; Melamed, J; Garabedian, M J; Logan, S K

    2013-08-22

    Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.

  10. Global gene expression analysis of fission yeast mutants impaired in Ser-2 phosphorylation of the RNA pol II carboxy terminal domain.

    Directory of Open Access Journals (Sweden)

    Reza Saberianfar

    Full Text Available In Schizosaccharomyces pombe the nuclear-localized Lsk1p-Lsc1p cyclin dependent kinase complex promotes Ser-2 phosphorylation of the heptad repeats found within the RNA pol II carboxy terminal domain (CTD. Here, we first provide evidence supporting the existence of a third previously uncharacterized Ser-2 CTD kinase subunit, Lsg1p. As expected for a component of the complex, Lsg1p localizes to the nucleus, promotes Ser-2 phosphorylation of the CTD, and physically interacts with both Lsk1p and Lsc1p in vivo. Interestingly, we also demonstrate that lsg1Δ mutants--just like lsk1Δ and lsc1Δ strains--are compromised in their ability to faithfully and reliably complete cytokinesis. Next, to address whether kinase mediated alterations in CTD phosphorylation might selectively alter the expression of genes with roles in cytokinesis and/or the cytoskeleton, global gene expression profiles were analyzed. Mutants impaired in Ser-2 phosphorylation display little change with respect to the level of transcription of most genes. However, genes affecting cytokinesis--including the actin interacting protein gene, aip1--as well as genes with roles in meiosis, are included in a small subset that are differentially regulated. Significantly, genetic analysis of lsk1Δ aip1Δ double mutants is consistent with Lsk1p and Aip1p acting in a linear pathway with respect to the regulation of cytokinesis.

  11. Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry

    NARCIS (Netherlands)

    Miliotis, Tasso; Olof Ericsson, Per; Marko-Varga, Gyorgy; Svensson, Robert; Nilsson, Johan; Laurell, Thomas; Bischoff, Rainer

    2001-01-01

    A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention t

  12. NetPhosBac - A predictor for Ser/Thr phosphorylation sites in bacterial proteins

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Soufi, Boumediene; Jers, Carsten;

    2009-01-01

    sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic-type protein kinases, which explains the poor performance of eukaryotic data-trained phosphorylation....... Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site-specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa-specific predictors and we hope it will provide a useful asset to the microbiological community....

  13. Properties of phosphorylated thymidylate synthase.

    Science.gov (United States)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Paweł; Rogowska-Wrzesinska, Adelina; Cieśla, Joanna; Zieliński, Zbigniew; Nizioł, Joanna; Jarmuła, Adam; Maj, Piotr; Gołos, Barbara; Wińska, Patrycja; Ostafil, Sylwia; Wałajtys-Rode, Elżbieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  14. Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

    KAUST Repository

    Rayapuram, Naganand

    2014-04-04

    Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

  15. Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3ζ and ZAP-70 tyrosine phosphorylation.

    Directory of Open Access Journals (Sweden)

    Mogjiborahman Salek

    Full Text Available Signaling through the T cell receptor (TCR initiates adaptive immunity and its perturbation may results in autoimmunity. The plasma membrane scaffolding protein LAT acts as a central organizer of the TCR signaling machinery to activate many functional pathways. LAT-deficient mice develop an autoimmune syndrome but the mechanism of this pathology is unknown. In this work we have compared global dynamics of TCR signaling by MS-based quantitative phosphoproteomics in LAT-sufficient and LAT-defective Jurkat T cells. Surprisingly, we found that many TCR-induced phosphorylation events persist in the absence of LAT, despite ERK and PLCγ1 phosphorylation being repressed. Most importantly, the absence of LAT resulted in augmented and persistent tyrosine phosphorylation of CD3ζ and ZAP70. This indicates that LAT signaling hub is also implicated in negative feedback signals to modulate upstream phosphorylation events. Phosphorylation kinetics data resulting from this investigation is documented in a database (phosphoTCR accessible online. The MS data have been deposited to the ProteomeXchange with identifier PXD000341.

  16. Linear stability analysis reveals exclusion zone for sliding bed transport

    Directory of Open Access Journals (Sweden)

    Talmon Arnold M.

    2015-06-01

    Full Text Available A bend or any another pipe component disturbs solids transport in pipes. Longitudinal pressure profiles downstream of such a component may show a stationary transient harmonic wave, as revealed by a recent settling slurry laboratory experiment. Therefore the fundamental transient response of the two-layer model for fully stratified flow is investigated as a first approach. A linear stability analysis of the sliding bed configuration is conducted. No stationary transient harmonic waves are found in this analysis, but adaptation lengths for exponential recovery are quantified. An example calculation is given for a 0.1 m diameter pipeline.

  17. Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

    Science.gov (United States)

    Narushima, Yuta; Kozuka-Hata, Hiroko; Koyama-Nasu, Ryo; Tsumoto, Kouhei; Inoue, Jun-ichiro; Akiyama, Tetsu; Oyama, Masaaki

    2016-03-01

    Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-β receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-β receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-β receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation. PMID:26670566

  18. Systematic toxicological analysis revealing a rare case of captan ingestion.

    Science.gov (United States)

    Gottzein, Anne K; Musshoff, Frank; Madea, Burkhard

    2013-07-01

    This article presents a case of suicide by intoxication with various pharmaceuticals, particularly anticonvulsants, combined with the fungicide captan. A cause of death could not be ascertained at autopsy. However, systematic toxicological analysis (STA) including a screening via solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) for (semi) volatile organic compounds revealed results suggesting a possible cause of death. The effects of captan on the human organism, its metabolism, and distribution will be discussed. Macroscopically, the cause of death was unascertained. STA revealed clonazepam, citalopram, and its metabolites, lamotrigine, levetiracetam, lacosamide, clonazepam, captan, and its metabolite tetrahydrophthalimide (THPI). For the first time, it was detected in human viscera. A quantification of THPI was performed to obtain distribution in the organs. The significance of a complete STA must be emphasized. The presence of THPI would have been missed without previous detection of captan. Consequently, this fatality would not have been investigated satisfactorily.

  19. Large-scale analysis of in Vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N;

    2003-01-01

    Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poo...

  20. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  1. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  2. Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms.

    Science.gov (United States)

    Gnad, Florian; Doll, Sophia; Song, Kyung; Stokes, Matthew P; Moffat, John; Liu, Bonnie; Arnott, David; Wallin, Jeffrey; Friedman, Lori S; Hatzivassiliou, Georgia; Belvin, Marcia

    2016-07-01

    The RAS-RAF-MEK-ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large-scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 (http://proteomecentral.proteomexchange.org/dataset/PXD003908). PMID:27273156

  3. Geometric morphometric analysis reveals sexual dimorphism in the distal femur.

    Science.gov (United States)

    Cavaignac, Etienne; Savall, Frederic; Faruch, Marie; Reina, Nicolas; Chiron, Philippe; Telmon, Norbert

    2016-02-01

    An individual's sex can be determined by the shape of their distal femur. The goal of this study was to show that differences in distal femur shape related to sexual dimorphism could be identified, visualized, and quantified using 3D geometric morphometric analysis. Geometric morphometric analysis was carried out on CT scans of the distal femur of 256 subjects living in the south of France. Ten landmarks were defined on 3D reconstructions of the distal femur. Both traditional metric and geometric morphometric analyses were carried out on these bone reconstructions; these analyses identified trends in bone shape in sex-based subgroups. Sex-related differences in shape were statistically significant. The subject's sex was correctly assigned in 77.3% of cases using geometric morphometric analysis. This study has shown that geometric morphometric analysis of the distal femur is feasible and has revealed sexual dimorphism differences in this bone segment. This reliable, accurate method could be used for virtual autopsy and be used to perform diachronic and interethnic comparisons. Moreover, this study provides updated morphometric data for a modern population in the south of France. PMID:26743712

  4. Morphological Analysis of CDC2 and Glycogen Synthase Kinase 3β Phosphorylation as Markers of G2 → M Transition in Glioma

    Directory of Open Access Journals (Sweden)

    José Javier Otero

    2011-01-01

    Full Text Available G2 → M transition is a strategic target for glioma chemotherapy. Key players in G2 → M transition include CDC2 and glycogen synthase kinase 3β (GSK3β, which are highly regulated by posttranslational phosphorylation. This report is a morphological analysis of CDC2 and GSK3β phosphorylation using immunohistochemistry in gliomas with different biological properties. GBM showed a 2.8-fold and 5.6-fold increase in number of cells positive for pThr161CDC2 and a 4.2- and 6.9-fold increase in number of cells positive for pTyr15CDC2 relative to oligodendroglioma and ependymoma, respectively. Elevated labeling for inhibited phospho-CDC2 (pTyr15CDC correlates with elevated levels of phosphorylated glycogen synthase kinase 3β (GSK3β. 71% of the GBM cases showed intermediate to high intensity staining for pSer9SGK3β 53% of oligodendroglioma, and 73% of ependymoma showed low intensity staining. CDC2 gene amplification correlates with increased survival in glioblastoma multiforme (GBM and astrocytoma WHO grades II-III, but not in oligodendroglioma WHO grades II-III.

  5. Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

    Science.gov (United States)

    Larsen, Sara C; Sylvestersen, Kathrine B; Mund, Andreas; Lyon, David; Mullari, Meeli; Madsen, Maria V; Daniel, Jeremy A; Jensen, Lars J; Nielsen, Michael L

    2016-01-01

    The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation. PMID:27577262

  6. Kinase-specific prediction of protein phosphorylation sites

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Blom, Nikolaj

    2009-01-01

    As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase...

  7. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    Science.gov (United States)

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  8. Single cell transcriptional analysis reveals novel innate immune cell types

    Directory of Open Access Journals (Sweden)

    Linda E. Kippner

    2014-06-01

    Full Text Available Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription

  9. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.

    Directory of Open Access Journals (Sweden)

    Zhen Dou

    Full Text Available BACKGROUND: Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC, a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1 is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. METHODOLOGY/PRINCIPAL FINDINGS: Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. CONCLUSIONS/SIGNIFICANCE: hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.

  10. Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro.

    Science.gov (United States)

    Cytryńska, M; Frajnt, M; Jakubowicz, T

    2001-09-25

    Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved. PMID:11583852

  11. Bypassing AMPK Phosphorylation

    OpenAIRE

    Viollet, Benoit; Foretz, Marc; Schlattner, Uwe

    2014-01-01

    AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event, and acts synergistically with AMP on naive AMPK.

  12. Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia.

    Directory of Open Access Journals (Sweden)

    Luca Fagnocchi

    Full Text Available Most bacterial small RNAs (sRNAs are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. Neisseria meningitides, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of N. meningitides in human blood ex vivo revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of N. meningitides to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation. and we identified putative sRNAs that were differentially expressed in vitro. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of N. meningitides infection in the in vivo infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.

  13. Sequential myosin phosphorylation activates tarantula thick filament via a disorder-order transition.

    Science.gov (United States)

    Espinoza-Fonseca, L Michel; Alamo, Lorenzo; Pinto, Antonio; Thomas, David D; Padrón, Raúl

    2015-08-01

    Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscles and a secondary (modulatory) role in striated muscles, which is regulated by Ca(2+)via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca(2+)4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectory analysis revealed that an inter-domain salt bridge network (R39/E58,E61) facilitates the formation of a stable helix-coil-helix (HCH) motif formed by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of the blocked head on Ser45 does not induce any substantial structural changes. However, phosphorylation of the free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH folding, phosphorylation of Ser35 unlocks a compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by the subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition. PMID

  14. Isotope analysis reveals foraging area dichotomy for atlantic leatherback turtles.

    Directory of Open Access Journals (Sweden)

    Stéphane Caut

    Full Text Available BACKGROUND: The leatherback turtle (Dermochelys coriacea has undergone a dramatic decline over the last 25 years, and this is believed to be primarily the result of mortality associated with fisheries bycatch followed by egg and nesting female harvest. Atlantic leatherback turtles undertake long migrations across ocean basins from subtropical and tropical nesting beaches to productive frontal areas. Migration between two nesting seasons can last 2 or 3 years, a time period termed the remigration interval (RI. Recent satellite transmitter data revealed that Atlantic leatherbacks follow two major dispersion patterns after nesting season, through the North Gulf Stream area or more eastward across the North Equatorial Current. However, information on the whole RI is lacking, precluding the accurate identification of feeding areas where conservation measures may need to be applied. METHODOLOGY/PRINCIPAL FINDINGS: Using stable isotopes as dietary tracers we determined the characteristics of feeding grounds of leatherback females nesting in French Guiana. During migration, 3-year RI females differed from 2-year RI females in their isotope values, implying differences in their choice of feeding habitats (offshore vs. more coastal and foraging latitude (North Atlantic vs. West African coasts, respectively. Egg-yolk and blood isotope values are correlated in nesting females, indicating that egg analysis is a useful tool for assessing isotope values in these turtles, including adults when not available. CONCLUSIONS/SIGNIFICANCE: Our results complement previous data on turtle movements during the first year following the nesting season, integrating the diet consumed during the year before nesting. We suggest that the French Guiana leatherback population segregates into two distinct isotopic groupings, and highlight the urgent need to determine the feeding habitats of the turtle in the Atlantic in order to protect this species from incidental take by

  15. Changes in Phosphorylation of Connexin43 in Rats during Acute Myocardial Hypoxia and Effects of Antiarrhythmic Peptide on the Phosphorylation

    Institute of Scientific and Technical Information of China (English)

    WANG Rong; ZHANG Cuntai; RUAN Yanfei; LIU Nian; WANG Lin

    2007-01-01

    In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43),isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribution of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis revealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia,while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercelluar communication by enhancing phosphorylation of Cx43.

  16. Proteomic analysis reveals key proteins and phosphoproteins upon seed germination of wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Kun eDong

    2015-11-01

    Full Text Available Wheat (Triticum aestivum L. is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE-based proteomic approach. A total of 166 differentially expressed protein (DEP spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/ transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

  17. Power analysis attacks revealing the secrets of smart cards

    CERN Document Server

    Mangard, Stefan; Popp, Thomas

    2008-01-01

    A comprehensive treatment of power analysis attacks and countermeasures. Based on the principle that the only way to defend against power analysis attacks is to understand them, this book explains how power analysis attacks work. It discusses simple and differential power analysis as well as advanced techniques like template attacks.

  18. Oxidative phosphorylation revisited

    DEFF Research Database (Denmark)

    Nath, Sunil; Villadsen, John

    2015-01-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...... are proton‐dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes...

  19. Integrated Experimental and Model-based Analysis Reveals the Spatial Aspects of EGFR Activation Dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Harish; Zhang, Yi; Chrisler, William B.; Ewald, Jonathan A.; Wiley, H. S.; Resat, Haluk

    2012-10-02

    The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases, and controls a diverse set of cellular responses relevant to development and tumorigenesis. ErbB activation is a complex process involving receptor-ligand binding, receptor dimerization, phosphorylation, and trafficking (internalization, recycling and degradation), which together dictate the spatio-temporal distribution of active receptors within the cell. The ability to predict this distribution, and elucidation of the factors regulating it, would help to establish a mechanistic link between ErbB expression levels and the cellular response. Towards this end, we constructed mathematical models for deconvolving the contributions of receptor dimerization and phosphorylation to EGFR activation, and to examine the dependence of these processes on sub-cellular location. We collected experimental datasets for EGFR activation dynamics in human mammary epithelial cells, with the specific goal of model parameterization, and used the data to estimate parameters for several alternate models. Model-based analysis indicated that: 1) signal termination via receptor dephosphorylation in late endosomes, prior to degradation, is an important component of the response, 2) less than 40% of the receptors in the cell are phosphorylated at any given time, even at saturating ligand doses, and 3) receptor dephosphorylation rates at the cell surface and early endosomes are comparable. We validated the last finding by measuring EGFR dephosphorylation rates at various times following ligand addition both in whole cells, and in endosomes using ELISAs and fluorescent imaging. Overall, our results provide important information on how EGFR phosphorylation levels are regulated within cells. Further, the mathematical model described here can be extended to determine receptor dimer abundances in cells co-expressing various levels of ErbB receptors. This study demonstrates that an iterative cycle of

  20. Directional and quantitative phosphorylation networks

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Linding, Rune

    2008-01-01

    for unravelling phosphorylation-mediated cellular interaction networks. In particular, we will discuss how the combination of new quantitative mass-spectrometric technologies and computational algorithms together are enhancing mapping of these largely uncharted dynamic networks. By combining quantitative......Directionality in protein signalling networks is due to modulated protein-protein interactions and is fundamental for proper signal progression and response to external and internal cues. This property is in part enabled by linear motifs embedding post-translational modification sites. These serve...... as recognition sites, guiding phosphorylation by kinases and subsequent binding of modular domains (e.g. SH2 and BRCT). Characterization of such modification-modulated interactions on a proteome-wide scale requires extensive computational and experimental analysis. Here, we review the latest advances in methods...

  1. Identification and characterization of a leucine-rich repeat kinase 2 (LRRK2 consensus phosphorylation motif.

    Directory of Open Access Journals (Sweden)

    Pooja P Pungaliya

    Full Text Available Mutations in LRRK2 (leucine-rich repeat kinase 2 have been identified as major genetic determinants of Parkinson's disease (PD. The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410 within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.

  2. Epidermal growth factor (EGF) sensitive phosphorylation of calmodulin (CAM) in A431 cell membrane

    International Nuclear Information System (INIS)

    A431, a transformed cell line, is known to contain a high concentration of EGF receptors (EGFR). Exogenous CAM, when combined with purified membrane from A431 is strongly phosphorylated in the presence of EGF. The EGF-dependent phosphorylation of CAM did not alter the normal profile of the A431 EGFR autophosphorylation, as demonstrated by SDS-PAGE and autoradiography. In addition to its EGF dependency, the presence of divalent cations is also critical for CAM phosphorylation. The presence of a divalent cation chelator, such as EGTA, caused a complete inhibition of CAM phosphorylation, which can be reversed with cations in the following order of effectiveness: Mg++ > Mn++ > Ca++. Divalent cations also break up CAM into four co-migrating bands as indicated by Coomassie Blue stained gels and the corresponding autoradiograms. Double antibody precipitation followed by phospho-amino acid analysis revealed that the EGF-sensitive CAM phosphorylation occurs exclusively on the serine residue. Using radioimmunoassay, purified A431 membrane was shown to contain a significant amount of endogenous CAM. The implications of the EGF-sensitive CAM phosphorylation are currently under investigation

  3. Feedback phosphorylation of an RGS protein by MAP kinase in yeast.

    Science.gov (United States)

    Garrison, T R; Zhang, Y; Pausch, M; Apanovitch, D; Aebersold, R; Dohlman, H G

    1999-12-17

    Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling. PMID:10593933

  4. Flux control analysis of mitochondrial oxidative phosphorylation in rat skeletal muscle: pyruvate and palmitoyl-carnitine as substrates give different control patterns

    DEFF Research Database (Denmark)

    Fritzen, Anette J; Grunnet, Niels; Quistorff, Bjørn

    2007-01-01

    Flux control analysis of eight reactions involved in oxidative phosphorylation of mitochondria from rat quadriceps muscle was performed under circumstances resembling in vivo conditions of carbohydrate or fatty acid oxidation. The major flux control at a respiration rate of 55% of state 3...... with the fatty acid oxidation or transport. Flux control of ADP generation was specifically tested at three different respiration rates, 30, 55 and 75% of state 3. At all respiration rates control was higher with pyruvate and pyruvate + palmitoyl-carnitine compared with palmitoyl-carnitine as substrate. Also...... the control was lower at 75% compared to 30% of the state 3 respiration both with pyruvate and pyruvate + palmitoyl-carnitine as substrate, suggesting that muscle respiration moves from "demand control" to "supply control" as respiration increases....

  5. Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria.

    Science.gov (United States)

    Kavanagh, N I; Ainscow, E K; Brand, M D

    2000-02-24

    Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.

  6. Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation

    Science.gov (United States)

    Rigatti, Marc; Le, Andrew V.; Gerber, Claire; Moraru, Ion I.; Dodge-Kafka, Kimberly L.

    2016-01-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca2+ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca2+ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100–200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100–200 fold lower concentrations. PMID:26027516

  7. Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation.

    Science.gov (United States)

    Chidiac, Rony; Zhang, Ying; Tessier, Sylvain; Faubert, Denis; Delisle, Chantal; Gratton, Jean-Philippe

    2016-05-01

    VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis. PMID:26846344

  8. BAHAMAS: new SNIa analysis reveals inconsistencies with standard cosmology

    CERN Document Server

    Shariff, H; Trotta, R; van Dyk, D A

    2015-01-01

    We present results obtained by applying our BAyesian HierArchical Modeling for the Analysis of Supernova cosmology (BAHAMAS) software package to the 740 spectroscopically confirmed supernovae type Ia (SNIa) from the "Joint Light-curve Analysis" (JLA) dataset. We simultaneously determine cosmological parameters and standardization parameters, including host galaxy mass corrections, residual scatter and object-by-object intrinsic magnitudes. Combining JLA and Planck Cosmic Microwave Background data, we find significant discrepancies in cosmological parameter constraints with respect to the standard analysis: we find Omega_M = 0.399+/-0.027, 2.8\\sigma\\ higher than previously reported and w = -0.910+/-0.045, 1.6\\sigma\\ higher than the standard analysis. We determine the residual scatter to be sigma_res = 0.104+/-0.005. We confirm (at the 95% probability level) the existence of two sub-populations segregated by host galaxy mass, separated at log_{10}(M/M_solar) = 10, differing in mean intrinsic magnitude by 0.055+...

  9. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P;

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK...

  10. Penicillium simile sp. nov. revealed by morphological and phylogenetic analysis.

    Science.gov (United States)

    Davolos, Domenico; Pietrangeli, Biancamaria; Persiani, Anna Maria; Maggi, Oriana

    2012-02-01

    The morphology of three phenetically identical Penicillium isolates, collected from the bioaerosol in a restoration laboratory in Italy, displayed macro- and microscopic characteristics that were similar though not completely ascribable to Penicillium raistrickii. For this reason, a phylogenetic approach based on DNA sequencing analysis was performed to establish both the taxonomic status and the evolutionary relationships of these three peculiar isolates in relation to previously described species of the genus Penicillium. We used four nuclear loci (both rRNA and protein coding genes) that have previously proved useful for the molecular investigation of taxa belonging to the genus Penicillium at various evolutionary levels. The internal transcribed spacer region (ITS1-5.8S-ITS2), domains D1 and D2 of the 28S rDNA, a region of the tubulin beta chain gene (benA) and part of the calmodulin gene (cmd) were amplified by PCR and sequenced. Analysis of the rRNA genes and of the benA and cmd sequence data indicates the presence of three isogenic isolates belonging to a genetically distinct species of the genus Penicillium, here described and named Penicillium simile sp. nov. (ATCC MYA-4591(T)  = CBS 129191(T)). This novel species is phylogenetically different from P. raistrickii and other related species of the genus Penicillium (e.g. Penicillium scabrosum), from which it can be distinguished on the basis of morphological trait analysis.

  11. Fibronectin phosphorylation by ecto-protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru (Meiji Institute of Health Science, Odawara (Japan))

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  12. Subfield profitability analysis reveals an economic case for cropland diversification

    Science.gov (United States)

    Brandes, E.; McNunn, G. S.; Schulte, L. A.; Bonner, I. J.; Muth, D. J.; Babcock, B. A.; Sharma, B.; Heaton, E. A.

    2016-01-01

    Public agencies and private enterprises increasingly desire to achieve ecosystem service outcomes in agricultural systems, but are limited by perceived conflicts between economic and ecosystem service goals and a lack of tools enabling effective operational management. Here we use Iowa—an agriculturally homogeneous state representative of the Maize Belt—to demonstrate an economic rationale for cropland diversification at the subfield scale. We used a novel computational framework that integrates disparate but publicly available data to map ˜3.3 million unique potential management polygons (9.3 Mha) and reveal subfield opportunities to increase overall field profitability. We analyzed subfield profitability for maize/soybean fields during 2010-2013—four of the most profitable years in recent history—and projected results for 2015. While cropland operating at a loss of US 250 ha-1 or more was negligible between 2010 and 2013 at 18 000-190 000 ha (profitable areas, incorporating conservation management that breaks even (e.g., planting low-input perennials), into low-yielding portions of fields could increase overall cropland profitability by 80%. This approach is applicable to the broader region and differs substantially from the status quo of ‘top-down’ land management for conservation by harnessing private interest to align profitability with the production of ecosystem services.

  13. Leukemia surfaceome analysis reveals new disease-associated features.

    Science.gov (United States)

    Mirkowska, Paulina; Hofmann, Andreas; Sedek, Lukasz; Slamova, Lucie; Mejstrikova, Ester; Szczepanski, Tomasz; Schmitz, Maike; Cario, Gunnar; Stanulla, Martin; Schrappe, Martin; van der Velden, Vincent H J; Bornhauser, Beat C; Wollscheid, Bernd; Bourquin, Jean-Pierre

    2013-06-20

    A better description of the leukemia cell surface proteome (surfaceome) is a prerequisite for the development of diagnostic and therapeutic tools. Insights into the complexity of the surfaceome have been limited by the lack of suitable methodologies. We combined a leukemia xenograft model with the discovery-driven chemoproteomic Cell Surface Capture technology to explore the B-cell precursor acute lymphoblastic leukemia (BCP-ALL) surfaceome; 713 cell surface proteins, including 181 CD proteins, were detected through combined analysis of 19 BCP-ALL cases. Diagnostic immunophenotypes were recapitulated in each case, and subtype specific markers were detected. To identify new leukemia-associated markers, we filtered the surfaceome data set against gene expression information from sorted, normal hematopoietic cells. Nine candidate markers (CD18, CD63, CD31, CD97, CD102, CD157, CD217, CD305, and CD317) were validated by flow cytometry in patient samples at diagnosis and during chemotherapy. CD97, CD157, CD63, and CD305 accounted for the most informative differences between normal and malignant cells. The ALL surfaceome constitutes a valuable resource to assist the functional exploration of surface markers in normal and malignant lymphopoiesis. This unbiased approach will also contribute to the development of strategies that rely on complex information for multidimensional flow cytometry data analysis to improve its diagnostic applications. PMID:23649467

  14. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  15. Modes of embayed beach dynamics: analysis reveals emergent timescales

    Science.gov (United States)

    Murray, K. T.; Murray, A.; Limber, P. W.; Ells, K. D.

    2013-12-01

    Embayed beaches, or beaches positioned between rocky headlands, exhibit morphologic changes over many length and time scales. Beach sediment is transported as a result of the day-to-day wave forcing, causing patterns of erosion and accretion. We use the Rocky Coastline Evolution Model (RCEM) to investigate how patterns of shoreline change depend on wave climate (the distribution of wave-approach angles) and beach characteristics. Measuring changes in beach width through time allows us to track the evolution of the shape of the beach and the movement of sand within it. By using Principle Component Analysis (PCA), these changes can be categorized into modes, where the first few modes explain the majority of the variation in the time series. We analyze these modes and how they vary as a function of wave climate and headland/bay aspect ratio. In the purposefully simple RCEM, sediment transport is wave-driven and affected by wave shadowing behind the headlands. The rock elements in our model experiments (including the headlands) are fixed and unerodable so that this analysis can focus purely on sand dynamics between the headlands, without a sand contribution from the headlands or cliffs behind the beach. The wave climate is characterized by dictating the percentage of offshore waves arriving from the left and the percentage of waves arriving from high angles (very oblique to the coastline orientation). A high-angle dominated wave climate tends to amplify coastline perturbations, whereas a lower-angle wave climate is diffusive. By changing the headland/bay aspect ratio and wave climate, we can perform PCA analysis of generalized embayed beaches with differing anatomy and wave climate forcings. Previous work using PCA analysis of embayed beaches focused on specific locations and shorter timescales (beach dynamics over longer timescales. The first two PCA modes, which explain a majority of the beach width time series variation (typically >70%), are a 'breathing' mode and a

  16. Genome analysis of the platypus reveals unique signatures of evolution

    Science.gov (United States)

    Warren, Wesley C.; Hillier, LaDeana W.; Marshall Graves, Jennifer A.; Birney, Ewan; Ponting, Chris P.; Grützner, Frank; Belov, Katherine; Miller, Webb; Clarke, Laura; Chinwalla, Asif T.; Yang, Shiaw-Pyng; Heger, Andreas; Locke, Devin P.; Miethke, Pat; Waters, Paul D.; Veyrunes, Frédéric; Fulton, Lucinda; Fulton, Bob; Graves, Tina; Wallis, John; Puente, Xose S.; López-Otín, Carlos; Ordóñez, Gonzalo R.; Eichler, Evan E.; Chen, Lin; Cheng, Ze; Deakin, Janine E.; Alsop, Amber; Thompson, Katherine; Kirby, Patrick; Papenfuss, Anthony T.; Wakefield, Matthew J.; Olender, Tsviya; Lancet, Doron; Huttley, Gavin A.; Smit, Arian F. A.; Pask, Andrew; Temple-Smith, Peter; Batzer, Mark A.; Walker, Jerilyn A.; Konkel, Miriam K.; Harris, Robert S.; Whittington, Camilla M.; Wong, Emily S. W.; Gemmell, Neil J.; Buschiazzo, Emmanuel; Vargas Jentzsch, Iris M.; Merkel, Angelika; Schmitz, Juergen; Zemann, Anja; Churakov, Gennady; Kriegs, Jan Ole; Brosius, Juergen; Murchison, Elizabeth P.; Sachidanandam, Ravi; Smith, Carly; Hannon, Gregory J.; Tsend-Ayush, Enkhjargal; McMillan, Daniel; Attenborough, Rosalind; Rens, Willem; Ferguson-Smith, Malcolm; Lefèvre, Christophe M.; Sharp, Julie A.; Nicholas, Kevin R.; Ray, David A.; Kube, Michael; Reinhardt, Richard; Pringle, Thomas H.; Taylor, James; Jones, Russell C.; Nixon, Brett; Dacheux, Jean-Louis; Niwa, Hitoshi; Sekita, Yoko; Huang, Xiaoqiu; Stark, Alexander; Kheradpour, Pouya; Kellis, Manolis; Flicek, Paul; Chen, Yuan; Webber, Caleb; Hardison, Ross; Nelson, Joanne; Hallsworth-Pepin, Kym; Delehaunty, Kim; Markovic, Chris; Minx, Pat; Feng, Yucheng; Kremitzki, Colin; Mitreva, Makedonka; Glasscock, Jarret; Wylie, Todd; Wohldmann, Patricia; Thiru, Prathapan; Nhan, Michael N.; Pohl, Craig S.; Smith, Scott M.; Hou, Shunfeng; Renfree, Marilyn B.; Mardis, Elaine R.; Wilson, Richard K.

    2009-01-01

    We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation. PMID:18464734

  17. Hereditary hemochromatosis: HFE mutation analysis in Greeks reveals genetic heterogeneity.

    Science.gov (United States)

    Papanikolaou, G; Politou, M; Terpos, E; Fourlemadis, S; Sakellaropoulos, N; Loukopoulos, D

    2000-04-01

    Hereditary hemochromatosis (HH) is common among Caucasians; reported disease frequencies vary from 0.3 to 0.8%. Identification of a candidate HFE gene in 1996 was soon followed by the description of two ancestral mutations, i.e., c.845G-->A (C282Y) and c.187C-->G (H63D). To these was recently added the mutation S65C, which may represent a simple polymorphism. The incidence of HH in Greece is unknown but clinical cases are rare. Also unknown is the carrier frequency of the two mutant alleles. A first estimate of the latter is given in the present report. It is based on data from the genetic analysis of 10 unrelated patients of Greek origin who were referred to our center for genotyping and 158 unselected male blood donors. The allele frequencies for the C282Y and H63D mutations were 0.003 and 0.145, respectively. The C282Y allele was detected in 50% of HH patients. This is considerably lower than the frequencies reported for HH patients in the U.S.A. (82%) and France (91 %) and closer to that reported in Italy (64%). Five patients did not carry any known HFE mutation; three may represent cases of juvenile hemochromatosis, given their early onset with iron overload, hypogonadism, and heart disease. We suggest that genetic heterogeneity is more prominent in Southern Europe. It is also possible that the penetrance of the responsible genes is different across the Mediterranean.

  18. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners

    Science.gov (United States)

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W. Thomas; Bush, Nicole R.; Adler, Nancy; Levine, Joel D.

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of “reciprocal” interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  19. Network analysis reveals distinct clinical syndromes underlying acute mountain sickness.

    Directory of Open Access Journals (Sweden)

    David P Hall

    Full Text Available Acute mountain sickness (AMS is a common problem among visitors at high altitude, and may progress to life-threatening pulmonary and cerebral oedema in a minority of cases. International consensus defines AMS as a constellation of subjective, non-specific symptoms. Specifically, headache, sleep disturbance, fatigue and dizziness are given equal diagnostic weighting. Different pathophysiological mechanisms are now thought to underlie headache and sleep disturbance during acute exposure to high altitude. Hence, these symptoms may not belong together as a single syndrome. Using a novel visual analogue scale (VAS, we sought to undertake a systematic exploration of the symptomatology of AMS using an unbiased, data-driven approach originally designed for analysis of gene expression. Symptom scores were collected from 292 subjects during 1110 subject-days at altitudes between 3650 m and 5200 m on Apex expeditions to Bolivia and Kilimanjaro. Three distinct patterns of symptoms were consistently identified. Although fatigue is a ubiquitous finding, sleep disturbance and headache are each commonly reported without the other. The commonest pattern of symptoms was sleep disturbance and fatigue, with little or no headache. In subjects reporting severe headache, 40% did not report sleep disturbance. Sleep disturbance correlates poorly with other symptoms of AMS (Mean Spearman correlation 0.25. These results challenge the accepted paradigm that AMS is a single disease process and describe at least two distinct syndromes following acute ascent to high altitude. This approach to analysing symptom patterns has potential utility in other clinical syndromes.

  20. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners.

    Science.gov (United States)

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W Thomas; Bush, Nicole R; Adler, Nancy; Levine, Joel D

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of "reciprocal" interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  1. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners

    Directory of Open Access Journals (Sweden)

    Mireille eGolemiec

    2016-03-01

    Full Text Available The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioural interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of ‘reciprocal’ interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored.

  2. Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production.

    Science.gov (United States)

    Li, Jian-ying; Ye, Lu-peng; Che, Jia-qian; Song, Jia; You, Zheng-ying; Yun, Ki-chan; Wang, Shao-hua; Zhong, Bo-xiong

    2015-08-01

    The silkworm middle silk gland (MSG) is the sericin synthesis and secretion unique sub-organ. The molecular mechanisms of regulating MSG protein synthesis are largely unknown. Here, we performed shotgun proteomic analysis on the three MSG subsections: the anterior (MSG-A), middle (MSG-M), and posterior (MSG-P) regions. The results showed that more strongly expressed proteins in the MSG-A were involved in multiple processes, such as silk gland development and silk protein protection. The proteins that were highly expressed in the MSG-M were enriched in the ribosome pathway. MSG-P proteins with stronger expression were mainly involved in the oxidative phosphorylation and citrate cycle pathways. These results suggest that the MSG-M is the most active region in the sericin synthesis. Furthermore, comparing the proteome of the MSG with the posterior silk gland (PSG) revealed that the specific and highly expressed proteins in the MSG were primarily involved in the ribosome and aminoacyl-tRNA biosynthesis pathways. These results indicate that silk protein synthesis is much more active as a result of the enhancement of translation-related pathways in the MSG. These results also suggest that enhancing ribosome biogenesis is important to the efficient synthesis of silk proteins. PMID:26051239

  3. Constitutive phosphorylation of Shc proteins in human tumors

    DEFF Research Database (Denmark)

    Pelicci, G; Lanfrancone, L; Salcini, A E;

    1995-01-01

    cells. In tumor cells with known TK gene alterations Shc proteins were constitutively phosphorylated and complexed with the activated TK. No constitutive Shc phosphorylation was found in primary cell cultures and normal tissues. In 14 of 27 tumor cell lines with no reported TK alterations, Shc proteins...... activated TKs and that the analysis of Shc phosphorylation allow the identification of tumors with constitutive TK activation....

  4. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-04-27

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  5. miR-106b inhibits tau phosphorylation at Tyr18 by targeting Fyn in a model of Alzheimer's disease.

    Science.gov (United States)

    Liu, Wei; Zhao, Jingya; Lu, Guangxiu

    2016-09-16

    Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by β-amyloid deposits and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Increasing evidence has revealed that microRNAs (miRNAs) are implicated in the pathogenesis of AD. However, the effect of miRNAs on abnormal tau phosphorylation remains largely unclear so far. In this study, we investigated the role of miR-106b in tau phosphorylation and identified a new molecular mechanism of the hyperphosphorylation of tau. The results of qRT-PCR showed that the expression level of miR-106b was decreased, but Fyn was increased in the temporal cortex of AD patients. Overexpression of miR-106b inhibited Aβ1-42-induced tau phosphorylation at Tyr18 in SH-SY5Y cells stably expressing tau (SH-SY5Y/tau), whereas no changes were observed in tau phosphorylation at Ser396/404. Dual-luciferase reporter gene assay validated that Fyn was a direct target gene of miR-106b. In addition, western blot analysis revealed that Fyn protein expression was suppressed when SH-SY5Y cells were transfected with miR-106b mimics. Endogenous Fyn expression was knockdown by transfection with a small interfering RNA specific for Fyn (si-Fyn). The phosphorylation level of tau at Tyr 18 was decreased in the si-Fyn group compared with the negative control group, but the inhibitory effect of si-Fyn on tau phosphorylation was attenuated when miR-106b expression was inhibited. Taken together, these data suggest that miR-106b inhibits Aβ1-42-induced tau phosphorylation at Tyr18 by targeting Fyn. Our findings extend the knowledge about the regulation of tau phosphorylation and the regulatory mechanism of Fyn gene expression. PMID:27520374

  6. Prognostic significance of STAT3 and phosphorylated STAT3 in human soft tissue tumors - a clinicopathological analysis

    Directory of Open Access Journals (Sweden)

    Nair Asha S

    2011-05-01

    Full Text Available Abstract Background Signal transducer and activator of transcription 3 (STAT3 is a key signaling molecule and a central cytoplasmic transcription factor, implicated in the regulation of growth. Its aberrant activation has been demonstrated to correlate with many types of human malignancy. However, whether constitutive STAT3 signaling plays a key role in the survival and growth of soft-tissue tumors is still unclear and hence needs to be elucidated further. In our study we examined the expression levels of STAT3 and pSTAT3 in different grades of soft tissue tumors and correlated with its clinicopathological characteristics. Methods Expression levels of STAT3 and pSTAT3 in soft tissue tumors were studied using Immunohistochemistry, Western blotting and Reverse transcriptase- PCR and correlated with its clinicopathological characteristics using Chi squared or Fisher's exact test and by logistic regression analysis. Statistical analysis was done using Intercooled Stata software (Intercooled Stata 8.2 version. Results Of the 82 soft tissue tumor samples, fifty four (65.8% showed immunoreactivity for STAT3 and twenty eight (34.1% for pSTAT3. Expression of STAT3 and pSTAT3 was significantly associated with tumor grade (P Conclusion These findings suggest that constitutive activation of STAT3 is an important factor related to carcinogenesis of human soft tissue tumors and is significantly associated with its clinicopathological parameters which may possibly have potential diagnostic implications.

  7. Site-specific IGFBP-1 hyper-phosphorylation in fetal growth restriction: clinical and functional relevance.

    Science.gov (United States)

    Abu Shehab, Majida; Khosravi, Javad; Han, Victor K M; Shilton, Brian H; Gupta, Madhulika B

    2010-04-01

    Phosphorylation enhances IGFBP-1 binding to IGF-I, thereby limiting the bioavailability of IGF-I that may be important in fetal growth. Our goal in this study was to determine whether changes in site-specific IGFBP-1 phosphorylation were unique to fetal growth restriction. To establish a link, we compared IGFBP-1 phosphorylation (sites and degree) in amniotic fluid from FGR (N = 10) and controls (N = 12). The concentration of serine phosphorylated IGFBP-1 showed a negative correlation with birth weight in FGR (P = 0.049). LC-MS/MS analysis revealed all four previously identified phosphorylation sites (Ser98, Ser101, Ser119, and Ser169) to be common to FGR and control groups. Relative phosphopeptide intensities (LC-MS) between FGR and controls demonstrated 4-fold higher intensity for Ser101 (P = 0.026), 7-fold for Ser98/Ser101 (P = 0.02), and 23-fold for Ser169 (P = 0.002) in the FGR group. Preliminary BIAcore data revealed 4-fold higher association and 1.7-fold lower dissociation constants for IGFBP-1/IGF-I in FGR. A structural model of IGFBP-1 bound to IGF-I indicates that all the phosphorylation sites are on relatively mobile regions of the IGFBP-1 sequence. Residues Ser98, Ser101, and Ser169 are close to structured regions that are involved in IGF-I binding and, therefore, could potentially make direct contact with IGF-I. On the other hand, residue Ser119 is in the middle of the unstructured linker that connects the N- and C-terminal domains of IGFBP-1. The model is consistent with the assumption that residues Ser98, Ser101, and Ser169 could directly interact with IGF-I, and therefore phosphorylation at these sites could change IGF-I interactions. We suggest that site-specific increase in IGFBP-1 phosphorylation limits IGF-I bioavailability, which directly contributes to the development of FGR. This study delineates the potential role of higher phosphorylation of IGFBP-1 in FGR and provides the basis to substantiate these findings with larger sample size. PMID

  8. The structure of a dual-specificity tyrosine phosphorylation-regulated kinase 1A-PKC412 complex reveals disulfide-bridge formation with the anomalous catalytic loop HRD(HCD) cysteine.

    Science.gov (United States)

    Alexeeva, Marina; Åberg, Espen; Engh, Richard A; Rothweiler, Ulli

    2015-05-01

    Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery. PMID:25945585

  9. Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Helen L Hellmich

    Full Text Available Developing new pharmacotherapies for traumatic brain injury (TBI requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT and carbenoxolone (CB, which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11β hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks. We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1, cytochrome c oxidase (Complex IV and ATP synthase (Complex V. This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that

  10. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B;

    2013-01-01

    used quantitative in vivo phosphoproteomics to identify 670 site-specific phosphorylation changes in murine hearts in response to acute treatment with specific βAR agonists. The residues adjacent to the regulated phosphorylation sites exhibited a sequence-specific preference (R-X-X-pS/T), and...

  11. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

    Science.gov (United States)

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans

    2007-03-01

    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  12. Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

    Science.gov (United States)

    Llorens, M Candelaria; Lorenzatti, Guadalupe; Cavallo, Natalia L; Vaglienti, Maria V; Perrone, Ana P; Carenbauer, Anne L; Darling, Douglas S; Cabanillas, Ana M

    2016-10-01

    ZEB1 transcription factor is important in both development and disease, including many TGFβ-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc. PMID:26868487

  13. Comparative phosphoproteomic analysis of the mouse testis reveals changes in phosphopeptide abundance in response to Ppp1cc deletion

    Directory of Open Access Journals (Sweden)

    Graham MacLeod

    2014-03-01

    Full Text Available Male Ppp1cc knockout mice are infertile due to failure of spermatogenesis, thought to arise from a deficiency of the predominant testis isoform PPP1CC2. We hypothesize that substrates of the PPP1CC2 will be hyperphosphorylated in Ppp1cc mutant testes. Quantitative RT-PCR and histological data suggest a role for PPP1CC2 by 3 weeks of age in the testis. Comparative phosphoproteomic analysis identified 828 proteins phosphorylated in the 3 week mouse testis, and confidently assigned 1026 unique phosphorylation sites. Thirty-two peptides corresponding to 30 proteins were found to be more abundant in Ppp1cc mutant samples than in wild-type, representing candidate substrates of PPP1CC2.

  14. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr;

    2015-01-01

    , Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous m...

  15. Phosphorylation of chicken growth hormone

    Energy Technology Data Exchange (ETDEWEB)

    Aramburo, C.; Montiel, J.L. (Universidad Nacional Autonoma de Mexico (Mexico)); Donoghue, D.; Scanes, C.G. (Rutgers Univ., New Brunswick, NJ (USA)); Berghman, L.R. (Laboratory for Neuroendocrinology and Immunological Biotechnology, Louvain (Belgium))

    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 {gamma}-{sup 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 {sup 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 {sup 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.

  16. Identification of extracellularly phosphorylated membrane proteins.

    Science.gov (United States)

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

  17. Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process.

    Directory of Open Access Journals (Sweden)

    Moritz Treeck

    2009-03-01

    Full Text Available A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1, a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

  18. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  19. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

    Science.gov (United States)

    McDonough, Patrick M; Ingermanson, Randall S; Loy, Patricia A; Koon, Erick D; Whittaker, Ross; Laris, Casey A; Hilton, Jeffrey M; Nicoll, James B; Buehrer, Benjamin M; Price, Jeffrey H

    2011-06-01

    Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

  20. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

    Institute of Scientific and Technical Information of China (English)

    Yonglong Yu; Dong Zhu; Chaoying Ma; Hui Cao; Yaping Wang; Yanhao Xu; Wenying Zhang; Yueming Yan

    2016-01-01

    Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20) during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA) was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further informa-tion about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  1. Transcriptome analysis reveals key differentially expressed genes involved in wheat grain development

    Directory of Open Access Journals (Sweden)

    Yonglong Yu

    2016-04-01

    Full Text Available Wheat seed development is an important physiological process of seed maturation and directly affects wheat yield and quality. In this study, we performed dynamic transcriptome microarray analysis of an elite Chinese bread wheat cultivar (Jimai 20 during grain development using the GeneChip Wheat Genome Array. Grain morphology and scanning electron microscope observations showed that the period of 11–15 days post-anthesis (DPA was a key stage for the synthesis and accumulation of seed starch. Genome-wide transcriptional profiling and significance analysis of microarrays revealed that the period from 11 to 15 DPA was more important than the 15–20 DPA stage for the synthesis and accumulation of nutritive reserves. Series test of cluster analysis of differential genes revealed five statistically significant gene expression profiles. Gene ontology annotation and enrichment analysis gave further information about differentially expressed genes, and MapMan analysis revealed expression changes within functional groups during seed development. Metabolic pathway network analysis showed that major and minor metabolic pathways regulate one another to ensure regular seed development and nutritive reserve accumulation. We performed gene co-expression network analysis to identify genes that play vital roles in seed development and identified several key genes involved in important metabolic pathways. The transcriptional expression of eight key genes involved in starch and protein synthesis and stress defense was further validated by qRT-PCR. Our results provide new insight into the molecular mechanisms of wheat seed development and the determinants of yield and quality.

  2. Using team cognitive work analysis to reveal healthcare team interactions in a birthing unit

    OpenAIRE

    Ashoori, Maryam; Burns, Catherine M.; d'Entremont, Barbara; Momtahan, Kathryn

    2014-01-01

    Cognitive work analysis (CWA) as an analytical approach for examining complex sociotechnical systems has shown success in modelling the work of single operators. The CWA approach incorporates social and team interactions, but a more explicit analysis of team aspects can reveal more information for systems design. In this paper, Team CWA is explored to understand teamwork within a birthing unit at a hospital. Team CWA models are derived from theories and models of teamworkand leverage the exis...

  3. Bioinformatic Analysis Reveals Genome Size Reduction and the Emergence of Tyrosine Phosphorylation Site in the Movement Protein of New World Bipartite Begomoviruses

    OpenAIRE

    Ho, Eric S; Kuchie, Joan; Duffy, Siobain

    2014-01-01

    Begomovirus (genus Begomovirus, family Geminiviridae) infection is devastating to a wide variety of agricultural crops including tomato, squash, and cassava. Thus, understanding the replication and adaptation of begomoviruses has important translational value in alleviating substantial economic loss, particularly in developing countries. The bipartite genome of begomoviruses prevalent in the New World and their counterparts in the Old World share a high degree of genome homology except for a ...

  4. Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

  5. Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

    Science.gov (United States)

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

  6. Phosphorylation and activation of the plasma membrane Na+/H+ exchanger (NHE1 during osmotic cell shrinkage.

    Directory of Open Access Journals (Sweden)

    Robert R Rigor

    Full Text Available The Na(+/H(+Exchanger isoform 1 (NHE1 is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na(+/H(+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS. We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC. Na(+/H(+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1 that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA increase Na(+ transport capacity without affecting transport affinity (K(m=44 mM in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ(32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na(+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events.

  7. Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

    Science.gov (United States)

    Crow, T; Xue-Bian, J J; Siddiqi, V; Kang, Y; Neary, J T

    1998-05-01

    The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

  8. PD trafficking of potato leaf roll virus movement protein in Arabidopsis depends on site-specific protein phosphorylation

    Directory of Open Access Journals (Sweden)

    Katrin eLink

    2011-06-01

    Full Text Available Many plant viruses encode for specialised movement proteins (MP to facilitate passage of viral material to and through plasmodesmata (PD. To analyse intracellular trafficking of potato leaf roll virus (PLRV movement protein (MP17 we performed GFP fusion experiments with distinct deletion variants of MP17. These studies revealed that the C-terminus of MP17 is essential but not sufficient for PD targeting. Interestingly, fusion of GFP to three C-terminal MP17 deletion variants resulted in the accumulation of GFP in chloroplasts. This indicates that MP17 harbours hidden plastid targeting sequences. Previous studies showed that posttranslational protein phosphorylation influences PD targeting of MP and virus spread. Analysis of MP17-derived phospho-peptides by mass spectrometry revealed four phosphorylated serine residues (S71, S79, S137 and S140. Site-directed mutagenesis of S71/S79 and S137/S140 showed that the C-terminal serine residues S137/S140 are dispensable for PD targeting. However, exchange of S71/S79 to A71/A79 abolished PD targeting of the mutated MP17 protein. To mimic phosphorylation of S71/S79 both amino acids were substituted by aspartic acid. The resulting D71/D79 variant of MP17 was efficiently targeted to PD. Further deletion analysis showed that PD targeting of MP17 is dependent on the C-terminus, phosphorylation of S71 and/or S79 and a N-terminal domain.

  9. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

    Directory of Open Access Journals (Sweden)

    Danming Tang

    2012-09-01

    GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

  10. An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas

    DEFF Research Database (Denmark)

    Cirilo, Priscila Daniele Ramos; Marchi, Fábio Albuquerque; Barros Filho, Mateus de Camargo;

    2013-01-01

    integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell...... transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs....

  11. Regulation of peroxisome dynamics by phosphorylation.

    Science.gov (United States)

    Oeljeklaus, Silke; Schummer, Andreas; Mastalski, Thomas; Platta, Harald W; Warscheid, Bettina

    2016-05-01

    Peroxisomes are highly dynamic organelles that can rapidly change in size, abundance, and protein content in response to alterations in nutritional and other environmental conditions. These dynamic changes in peroxisome features, referred to as peroxisome dynamics, rely on the coordinated action of several processes of peroxisome biogenesis. Revealing the regulatory mechanisms of peroxisome dynamics is an emerging theme in cell biology. These mechanisms are inevitably linked to and synchronized with the biogenesis and degradation of peroxisomes. To date, the key players and basic principles of virtually all steps in the peroxisomal life cycle are known, but regulatory mechanisms remained largely elusive. A number of recent studies put the spotlight on reversible protein phosphorylation for the control of peroxisome dynamics and highlighted peroxisomes as hubs for cellular signal integration and regulation. Here, we will present and discuss the results of several studies performed using yeast and mammalian cells that convey a sense of the impact protein phosphorylation may have on the modulation of peroxisome dynamics by regulating peroxisomal matrix and membrane protein import, proliferation, inheritance, and degradation. We further put forward the idea to make use of current data on phosphorylation sites of peroxisomal and peroxisome-associated proteins reported in advanced large-scale phosphoproteomic studies. PMID:26775584

  12. Analysis of self-overlap reveals trade-offs in plankton swimming trajectories

    DEFF Research Database (Denmark)

    Mariani, Patrizio; Visser, Andre W.; Mazzocchi, Maria Grazia;

    2014-01-01

    these contrasting processes. This trade-off can be hypothesized as being evident in the behaviour of plankton, which inhabit a dilute three-dimensional environment with few refuges or orienting landmarks. We present an analysis of the swimming path geometries based on a volumetric Monte Carlo sampling approach......, which is particularly adept at revealing such trade-offs by measuring the self-overlap of the trajectories. Application of this method to experimentally measured trajectories reveals that swimming patterns in copepods are shaped to efficiently explore volumes at small scales, while achieving a large...

  13. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

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    Denis Avey

    2015-07-01

    Full Text Available Kaposi's Sarcoma-Associated Herpesvirus (KSHV is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK mitogen-activated protein kinase (MAPK pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45

  14. MtDNA analysis reveals enriched pathogenic mutations in Tibetan highlanders

    Science.gov (United States)

    Kang, Longli; Zheng, Hong-Xiang; Zhang, Menghan; Yan, Shi; Li, Lei; Liu, Lijun; Liu, Kai; Hu, Kang; Chen, Feng; Ma, Lifeng; Qin, Zhendong; Wang, Yi; Wang, Xiaofeng; Jin, Li

    2016-01-01

    Tibetan highlanders, including Tibetans, Monpas, Lhobas, Dengs and Sherpas, are considered highly adaptive to severe hypoxic environments. Mitochondrial DNA (mtDNA) might be important in hypoxia adaptation given its role in coding core subunits of oxidative phosphorylation. In this study, we employed 549 complete highlander mtDNA sequences (including 432 random samples) to obtain a comprehensive view of highlander mtDNA profile. In the phylogeny of a total of 36,914 sequences, we identified 21 major haplogroups representing founding events of highlanders, most of which were coalesced in 10 kya. Through founder analysis, we proposed a three-phase model of colonizing the plateau, i.e., pre-LGM Time (30 kya, 4.68%), post-LGM Paleolithic Time (16.8 kya, 29.31%) and Neolithic Time (after 8 kya, 66.01% in total). We observed that pathogenic mutations occurred far more frequently in 22 highlander-specific lineages (five lineages carrying two pathogenic mutations and six carrying one) than in the 6,857 haplogroups of all the 36,914 sequences (P = 4.87 × 10−8). Furthermore, the number of possible pathogenic mutations carried by highlanders (in average 3.18 ± 1.27) were significantly higher than that in controls (2.82 ± 1.40) (P = 1.89 × 10−4). Considering that function-altering and pathogenic mutations are enriched in highlanders, we therefore hypothesize that they may have played a role in hypoxia adaptation. PMID:27498855

  15. An analysis of the effects of Mn2+ on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

    International Nuclear Information System (INIS)

    Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn2+ inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn2+ inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn2+ inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F1F0 ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn2+ inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

  16. Cytochrome P450 genes in coronary artery diseases: Codon usage analysis reveals genomic GC adaptation.

    Science.gov (United States)

    Malakar, Arup Kumar; Halder, Binata; Paul, Prosenjit; Chakraborty, Supriyo

    2016-09-15

    Establishing codon usage biases are imperative for understanding the etiology of coronary artery diseases (CAD) as well as the genetic factors associated with these diseases. The aim of this study was to evaluate the contribution of 18 responsible cytochrome P450 (CYP) genes for the risk of CAD. Effective number of codon (Nc) showed a negative correlation with both GC3 and synonymous codon usage order (SCUO) suggesting an antagonistic relationship between codon usage and Nc of genes. The dinucleotide analysis revealed that CG and TA dinucleotides have the lowest odds ratio in these genes. Principal component analysis showed that GC composition has a profound effect in separating the genes along the first major axis. Our findings revealed that mutational pressure and natural selection could possibly be the major factors responsible for codon bias in these genes. The study not only offers an insight into the mechanisms of genomic GC adaptation, but also illustrates the complexity of CYP genes in CAD. PMID:27275533

  17. Chemistry of Phosphorylated Formaldehyde Derivatives. Part I

    OpenAIRE

    Vasily P. Morgalyuk

    2014-01-01

    The underinvestigated derivatives of unstable phosphorylated formaldehyde acetals and some of the structurally related compounds, such as thioacetals, aminonitriles, aminomethylphosphinoyl compounds, are considered. Separately considered are halogen aminals of phosphorylated formaldehyde, acetals of phosphorylated formaldehyde of H-phosphinate-type and a phosphorylated gem-diol of formaldehyde. Synthetic methods, chemical properties and examples of practical applications are given.

  18. Revealing spatio-spectral electroencephalographic dynamics of musical mode and tempo perception by independent component analysis

    OpenAIRE

    Lin, Yuan-Pin; Duann, Jeng-Ren; Feng, Wenfeng; Chen, Jyh-Horng; Jung, Tzyy-Ping

    2014-01-01

    Background Music conveys emotion by manipulating musical structures, particularly musical mode- and tempo-impact. The neural correlates of musical mode and tempo perception revealed by electroencephalography (EEG) have not been adequately addressed in the literature. Method This study used independent component analysis (ICA) to systematically assess spatio-spectral EEG dynamics associated with the changes of musical mode and tempo. Results Empirical results showed that music with major mode ...

  19. Reticulate evolutionary history and extensive introgression in mosquito species revealed by phylogenetic network analysis.

    Science.gov (United States)

    Wen, Dingqiao; Yu, Yun; Hahn, Matthew W; Nakhleh, Luay

    2016-06-01

    The role of hybridization and subsequent introgression has been demonstrated in an increasing number of species. Recently, Fontaine et al. (Science, 347, 2015, 1258524) conducted a phylogenomic analysis of six members of the Anopheles gambiae species complex. Their analysis revealed a reticulate evolutionary history and pointed to extensive introgression on all four autosomal arms. The study further highlighted the complex evolutionary signals that the co-occurrence of incomplete lineage sorting (ILS) and introgression can give rise to in phylogenomic analyses. While tree-based methodologies were used in the study, phylogenetic networks provide a more natural model to capture reticulate evolutionary histories. In this work, we reanalyse the Anopheles data using a recently devised framework that combines the multispecies coalescent with phylogenetic networks. This framework allows us to capture ILS and introgression simultaneously, and forms the basis for statistical methods for inferring reticulate evolutionary histories. The new analysis reveals a phylogenetic network with multiple hybridization events, some of which differ from those reported in the original study. To elucidate the extent and patterns of introgression across the genome, we devise a new method that quantifies the use of reticulation branches in the phylogenetic network by each genomic region. Applying the method to the mosquito data set reveals the evolutionary history of all the chromosomes. This study highlights the utility of 'network thinking' and the new insights it can uncover, in particular in phylogenomic analyses of large data sets with extensive gene tree incongruence. PMID:26808290

  20. Protein phosphorylation in chloroplasts - a survey of phosphorylation targets.

    Science.gov (United States)

    Baginsky, Sacha

    2016-06-01

    The development of new software tools, improved mass spectrometry equipment, a suite of optimized scan types, and better-quality phosphopeptide affinity capture have paved the way for an explosion of mass spectrometry data on phosphopeptides. Because phosphoproteomics achieves good sensitivity, most studies use complete cell extracts for phosphopeptide enrichment and identification without prior enrichment of proteins or subcellular compartments. As a consequence, the phosphoproteome of cell organelles often comes as a by-product from large-scale studies and is commonly assembled from these in meta-analyses. This review aims at providing some guidance on the limitations of meta-analyses that combine data from analyses with different scopes, reports on the current status of knowledge on chloroplast phosphorylation targets, provides initial insights into phosphorylation site conservation in different plant species, and highlights emerging information on the integration of gene expression with metabolism and photosynthesis by means of protein phosphorylation. PMID:26969742

  1. Mixed mechanisms of multi-site phosphorylation.

    Science.gov (United States)

    Suwanmajo, Thapanar; Krishnan, J

    2015-06-01

    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose-response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction. PMID:25972433

  2. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    Science.gov (United States)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  3. Recent adaptive events in human brain revealed by meta-analysis of positively selected genes.

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    Yue Huang

    Full Text Available BACKGROUND AND OBJECTIVES: Analysis of positively-selected genes can help us understand how human evolved, especially the evolution of highly developed cognitive functions. However, previous works have reached conflicting conclusions regarding whether human neuronal genes are over-represented among genes under positive selection. METHODS AND RESULTS: We divided positively-selected genes into four groups according to the identification approaches, compiling a comprehensive list from 27 previous studies. We showed that genes that are highly expressed in the central nervous system are enriched in recent positive selection events in human history identified by intra-species genomic scan, especially in brain regions related to cognitive functions. This pattern holds when different datasets, parameters and analysis pipelines were used. Functional category enrichment analysis supported these findings, showing that synapse-related functions are enriched in genes under recent positive selection. In contrast, immune-related functions, for instance, are enriched in genes under ancient positive selection revealed by inter-species coding region comparison. We further demonstrated that most of these patterns still hold even after controlling for genomic characteristics that might bias genome-wide identification of positively-selected genes including gene length, gene density, GC composition, and intensity of negative selection. CONCLUSION: Our rigorous analysis resolved previous conflicting conclusions and revealed recent adaptation of human brain functions.

  4. Phosphorylation of ATPase subunits of the 26S proteasome.

    Science.gov (United States)

    Mason, G G; Murray, R Z; Pappin, D; Rivett, A J

    1998-07-01

    The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

  5. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  6. Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death.

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    Marie-Noëlle Delyfer

    Full Text Available BACKGROUND: Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. METHODOLOGY/PRINCIPAL FINDINGS: Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. CONCLUSIONS/SIGNIFICANCE: This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery.

  7. Network analysis of oyster transcriptome revealed a cascade of cellular responses during recovery after heat shock.

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    Lingling Zhang

    Full Text Available Oysters, as a major group of marine bivalves, can tolerate a wide range of natural and anthropogenic stressors including heat stress. Recent studies have shown that oysters pretreated with heat shock can result in induced heat tolerance. A systematic study of cellular recovery from heat shock may provide insights into the mechanism of acquired thermal tolerance. In this study, we performed the first network analysis of oyster transcriptome by reanalyzing microarray data from a previous study. Network analysis revealed a cascade of cellular responses during oyster recovery after heat shock and identified responsive gene modules and key genes. Our study demonstrates the power of network analysis in a non-model organism with poor gene annotations, which can lead to new discoveries that go beyond the focus on individual genes.

  8. Local coexistence of VO2 phases revealed by deep data analysis

    Science.gov (United States)

    Strelcov, Evgheni; Ievlev, Anton; Belianinov, Alex; Tselev, Alexander; Kolmakov, Andrei; Kalinin, Sergei V.

    2016-07-01

    We report a synergistic approach of micro-Raman spectroscopic mapping and deep data analysis to study the distribution of crystallographic phases and ferroelastic domains in a defected Al-doped VO2 microcrystal. Bayesian linear unmixing revealed an uneven distribution of the T phase, which is stabilized by the surface defects and uneven local doping that went undetectable by other classical analysis techniques such as PCA and SIMPLISMA. This work demonstrates the impact of information recovery via statistical analysis and full mapping in spectroscopic studies of vanadium dioxide systems, which is commonly substituted by averaging or single point-probing approaches, both of which suffer from information misinterpretation due to low resolving power.

  9. The Revealed Competitiveness of Major Ports in the East Asian Region: An Additive Market Share Analysis

    Directory of Open Access Journals (Sweden)

    Tae Seung Kim

    2015-12-01

    Full Text Available In the single cargo market, the ordinary market share analysis method has been the representative tool for revealed competitiveness analysis. This paper develops and employs an applied market share index called the additive market share (AMS. Data are collected from 15 major container ports for the 1998-2013 period. In comparison to the results of an ordinary market share analysis, the highest AMS is observed for the Bohai Rim port cluster from 2008, not for the Yangtze River cluster or the Pearl River cluster. There are substitutable relationships between Yangtze River and non-Chinese ports and between Pearl River and Bohai Rim ports from 2001. Finally, there is an internal competition at Pearl River and Yangtze River ports, whereas Bohai Rim and non-Chinese ports show internally complementary relationships.

  10. A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles.

    Science.gov (United States)

    Grenfell, Andrew W; Strzelecka, Magdalena; Crowder, Marina E; Helmke, Kara J; Schlaitz, Anne-Lore; Heald, Rebecca

    2016-04-11

    Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset ofXenopusextract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems. PMID:27044897

  11. The phosphorylation specificity of B-RAF(WT), B-RAF(D594V), B-RAF(V600E) and B-RAF(K601E) kinases: An in silico study

    DEFF Research Database (Denmark)

    Fratev, Filip Filipov; Jonsdottir, Svava Osk

    2010-01-01

    dynamics (MD) simulations and GRID molecular interaction field analysis. According to our analysis, Thr599 and Ser602 were the only residues in the activation segment in B-RAF(WT) that were well exposed to ATP binding, which is in agreement with the experimental results, and provide a molecular basis...... of the observed phosphorylation. The phosphorylation specificity was altered significantly for each of the three different mutants studied due to the large conformational changes and subsequent alterations in the electrostatic forces between several residues for each of these mutants. Thus the analysis revealed...

  12. Proteomic and genomic analysis reveals novel Campylobacter jejuni outer membrane proteins and potential heterogeneity

    Directory of Open Access Journals (Sweden)

    Eleanor Watson

    2014-09-01

    Full Text Available Gram-negative bacterial outer membrane proteins play important roles in the interaction of bacteria with their environment including nutrient acquisition, adhesion and invasion, and antibiotic resistance. In this study we identified 47 proteins within the Sarkosyl-insoluble fraction of Campylobacter jejuni 81-176, using LC–ESI-MS/MS. Comparative analysis of outer membrane protein sequences was visualised to reveal protein distribution within a panel of Campylobacter spp., identifying several C. jejuni-specific proteins. Smith–Waterman analyses of C. jejuni homologues revealed high sequence conservation amongst a number of hypothetical proteins, sequence heterogeneity of other proteins and several proteins which are absent in a proportion of strains.

  13. DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.

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    Andreas Lamkowski

    Full Text Available Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs. The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI with 49 Gy (± 6% Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available

  14. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

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    Sergio Alvarez-Pérez

    Full Text Available The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA of four core housekeeping genes (rrs, gyrB, rpoB and rpoD. A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1; P. fluorescens, P. lutea and P. syringae (NG 2; and P. rhizosphaerae (NG 3. Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria.

  15. Phylogenomic Analysis Reveals Extensive Phylogenetic Mosaicism in the Human GPCR Superfamily

    Directory of Open Access Journals (Sweden)

    Mathew Woodwark

    2007-01-01

    Full Text Available A novel high throughput phylogenomic analysis (HTP was applied to the rhodopsin G-protein coupled receptor (GPCR family. Instances of phylogenetic mosaicism between receptors were found to be frequent, often as instances of correlated mosaicism and repeated mosaicism. A null data set was constructed with the same phylogenetic topology as the rhodopsin GPCRs. Comparison of the two data sets revealed that mosaicism was found in GPCRs in a higher frequency than would be expected by homoplasy or the effects of topology alone. Various evolutionary models of differential conservation, recombination and homoplasy are explored which could result in the patterns observed in this analysis. We find that the results are most consistent with frequent recombination events. A complex evolutionary history is illustrated in which it is likely frequent recombination has endowed GPCRs with new functions. The pattern of mosaicism is shown to be informative for functional prediction for orphan receptors. HTP analysis is complementary to conventional phylogenomic analyses revealing mosaicism that would not otherwise have been detectable through conventional phylogenetics.

  16. Transmural heterogeneity of myofilament function and sarcomeric protein phosphorylation in remodelled myocardium of pigs with a recent myocardial infarction

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    Jolanda evan der Velden

    2011-11-01

    Full Text Available Transmural differences in sarcomeric protein composition and function across the left ventricular (LV wall have been reported. We studied sarcomeric function and protein phosphorylation in subepicardial (EPI and subendocardial (ENDO layers of remote LV pig myocardium after infarction (MI, induced by left circumflex coronary artery ligation. EPI and ENDO samples were taken 3 weeks after sham surgery (n=12 or induction of MI (n=12 at baseline and after β-adrenergic receptor (βAR stimulation with dobutamine. Isometric force was measured in single cardiomyocytes at various [Ca2+] and 2.2 μm sarcomere length. In sham hearts, no significant transmural differences were observed in myofilament function or protein phosphorylation. Myofilament Ca2+-sensitivity was significantly higher in both EPI and ENDO of MI compared to sham hearts. Maximal force was significantly reduced in MI compared to sham, but solely in ENDO cells. A higher passive force was observed in MI hearts, but only in EPI cells. The proportion of stiff N2B isoform was higher in EPI than in ENDO in both sham and MI hearts, and a trend towards increased N2B-proportion appeared in MI EPI, but not MI Endo. Analysis of myofilament protein phosphorylation did not reveal significant transmural differences in phosphorylation of myosin binding protein C, desmin, troponin T, troponin I (cTnI and myosin light chain 2 (MLC-2 both at baseline and after βAR stimulation with dobutamine infusion. A significant increase in MLC-2 phosphorylation was observed during dobutamine only in sham. In addition, the increase in cTnI phosphorylation upon dobutamine was 2-fold lower in MI than in sham.Myofilament dysfunction is present in both EPI and ENDO in post-MI remodelled myocardium, but shows a high degree of qualitative heterogeneity across the LV wall. These heterogeneous transmural changes in sarcomeric properties likely contribute differently to systolic versus diastolic global LV dysfunction after MI.

  17. Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

    International Nuclear Information System (INIS)

    The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor β subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4β-phorbol 12β-myristate 13α-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled β subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor β subunit. This threonine, which resides in a receptor domain also containing tyrosine phosphorylation sites, is located eight amino acids from the carboxyl terminus of the β subunit and may play a role in the protein kinase C induced inhibition of insulin receptor tyrosine kinase activity

  18. Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, R.E.; Cao, Lin; Perregaux, D.; Czech, M.P. (Univ. of Massachusetts Medical Center, Worcester (USA))

    1990-02-20

    The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor {beta} subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with ({sup 32}P)phosphate and treated with or without 100 nM 4{beta}-phorbol 12{beta}-myristate 13{alpha}-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the {sup 32}P-labeled {beta} subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor {beta} subunit. This threonine, which resides in a receptor domain also containing tyrosine phosphorylation sites, is located eight amino acids from the carboxyl terminus of the {beta} subunit and may play a role in the protein kinase C induced inhibition of insulin receptor tyrosine kinase activity.

  19. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    Science.gov (United States)

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  20. Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

    Science.gov (United States)

    Pérez-Lluch, Sílvia; Blanco, Enrique; Carbonell, Albert; Raha, Debasish; Snyder, Michael; Serras, Florenci; Corominas, Montserrat

    2011-06-01

    An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

  1. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    Energy Technology Data Exchange (ETDEWEB)

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  2. VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia

    Directory of Open Access Journals (Sweden)

    Ayling Roger D

    2008-11-01

    Full Text Available Abstract Background Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of M. agalactiae have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation. Results We have developed variable number tandem repeat (VNTR analysis using the sequenced genome of the M. agalactiae type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320. We have investigated the genetic diversity of 88 M. agalactiae isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE and random amplified polymorphic DNA (RAPD analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within M. agalactiae with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates. Conclusion VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of M. agalactiae for outbreak tracing and control.

  3. Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

    Science.gov (United States)

    Kolinko, Sebastian; Jogler, Christian; Katzmann, Emanuel; Wanner, Gerhard; Peplies, Jörg; Schüler, Dirk

    2012-07-01

    Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum. PMID:22003954

  4. Genetic Manipulation of Neurofilament Protein Phosphorylation.

    Science.gov (United States)

    Jones, Maria R; Villalón, Eric; Garcia, Michael L

    2016-01-01

    Neurofilament biology is important to understanding structural properties of axons, such as establishment of axonal diameter by radial growth. In order to study the function of neurofilaments, a series of genetically modified mice have been generated. Here, we describe a brief history of genetic modifications used to study neurofilaments, as well as an overview of the steps required to generate a gene-targeted mouse. In addition, we describe steps utilized to analyze neurofilament phosphorylation status using immunoblotting. Taken together, these provide comprehensive analysis of neurofilament function in vivo, which can be applied to many systems.

  5. Standardization of Assay Procedures for Analysis of the CSF Biomarkers Amyloid β(1-42, Tau, and Phosphorylated Tau in Alzheimer's Disease: Report of an International Workshop

    Directory of Open Access Journals (Sweden)

    Charlotte E. Teunissen

    2010-01-01

    Full Text Available Large variation in assay performance and outcomes of CSF Aβ1-42, total Tau (Tau, and phosphorylated Tau (pTau (at amino acid 181 levels is observed between laboratories. The aim of this study was to assess the differences in assay procedures between several experienced international laboratories, as potential sources of error. 14 groups performed the Aβ42, Tau, and pTau assays according to the guidelines of the manufacturer. Differences in analytical procedures between the laboratories were monitored. At least 23 items in assay procedures were identified that varied between the laboratories, including procedures for washing, pipetting, incubation, finishing, and sample handing. In general, the inter- and intra-assay variation between the groups was generally below 10% for all three assays. We concluded that 17 international centers that use the same assays for Aβ42, Tau and pTau on a regular basis do not uniformly adhere to the procedures recommended by the manufacturer. For harmonization of intercenter results of these biomarkers standardization of protocols is highly needed.

  6. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

    Science.gov (United States)

    Kaminski, Naftali; Allard, John D.; Pittet, Jean F.; Zuo, Fengrong; Griffiths, Mark J. D.; Morris, David; Huang, Xiaozhu; Sheppard, Dean; Heller, Renu A.

    2000-02-01

    The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin 6 subunit (6/-), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

  7. The phosphorylation pattern of bovine heart complex I subunits

    DEFF Research Database (Denmark)

    Palmisano, Giuseppe; Sardanelli, Anna Maria; Signorile, Anna;

    2007-01-01

    The phosphoproteome of bovine heart complex I of the respiratory chain has been analysed with a procedure based on nondenaturing gel electrophoretic separation of complex I from small quantities of mitochondria samples, in-gel digestion, in combination with phosphopeptide enrichment by titanium...... dioxide and MS. The results, complemented by analyses of purified samples of complex I, showed phosphorylation of five subunits of the complex, 42 kDa (human gene NDUFA10), ESSS, B14.5a (human gene NDUFA7), B14.5b (human gene NDUFC2) and B16.6 (GRIM-19). MS also revealed the presence of phosphorylated...

  8. Phenotypic Analysis Reveals that the 2010 Haiti Cholera Epidemic Is Linked to a Hypervirulent Strain.

    Science.gov (United States)

    Satchell, Karla J F; Jones, Christopher J; Wong, Jennifer; Queen, Jessica; Agarwal, Shivani; Yildiz, Fitnat H

    2016-09-01

    Vibrio cholerae O1 El Tor strains have been responsible for pandemic cholera since 1961. These strains have evolved over time, spreading globally in three separate waves. Wave 3 is caused by altered El Tor (AET) variant strains, which include the strain with the signature ctxB7 allele that was introduced in 2010 into Haiti, where it caused a devastating epidemic. In this study, we used phenotypic analysis to compare an early isolate from the Haiti epidemic to wave 1 El Tor isolates commonly used for research. It is demonstrated that the Haiti isolate has increased production of cholera toxin (CT) and hemolysin, increased motility, and a reduced ability to form biofilms. This strain also outcompetes common wave 1 El Tor isolates for colonization of infant mice, indicating that it has increased virulence. Monitoring of CT production and motility in additional wave 3 isolates revealed that this phenotypic variation likely evolved over time rather than in a single genetic event. Analysis of available whole-genome sequences and phylogenetic analyses suggested that increased virulence arose from positive selection for mutations found in known and putative regulatory genes, including hns and vieA, diguanylate cyclase genes, and genes belonging to the lysR and gntR regulatory families. Overall, the studies presented here revealed that V. cholerae virulence potential can evolve and that the currently prevalent wave 3 AET strains are both phenotypically distinct from and more virulent than many El Tor isolates. PMID:27297393

  9. A beamformer analysis of MEG data reveals frontal generators of the musically elicited mismatch negativity.

    Directory of Open Access Journals (Sweden)

    Claudia Lappe

    Full Text Available To localize the neural generators of the musically elicited mismatch negativity with high temporal resolution we conducted a beamformer analysis (Synthetic Aperture Magnetometry, SAM on magnetoencephalography (MEG data from a previous musical mismatch study. The stimuli consisted of a six-tone melodic sequence comprising broken chords in C- and G-major. The musical sequence was presented within an oddball paradigm in which the last tone was lowered occasionally (20% by a minor third. The beamforming analysis revealed significant right hemispheric neural activation in the superior temporal (STC, inferior frontal (IFC, superior frontal (SFC and orbitofrontal (OFC cortices within a time window of 100-200 ms after the occurrence of a deviant tone. IFC and SFC activation was also observed in the left hemisphere. The pronounced early right inferior frontal activation of the auditory mismatch negativity has not been shown in MEG studies so far. The activation in STC and IFC is consistent with earlier electroencephalography (EEG, optical imaging and functional magnetic resonance imaging (fMRI studies that reveal the auditory and inferior frontal cortices as main generators of the auditory MMN. The observed right hemispheric IFC is also in line with some previous music studies showing similar activation patterns after harmonic syntactic violations. The results demonstrate that a deviant tone within a musical sequence recruits immediately a distributed neural network in frontal and prefrontal areas suggesting that top-down processes are involved when expectation violation occurs within well-known stimuli.

  10. CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock

    Science.gov (United States)

    Tamaru, Teruya; Hattori, Mitsuru; Honda, Kousuke; Nakahata, Yasukazu; Sassone-Corsi, Paolo; van der Horst, Gijsbertus T. J.; Ozawa, Takeaki; Takamatsu, Ken

    2015-01-01

    Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK)-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P) in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein–protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1–CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1–P-BMAL1 loop is an integral part of the core clock oscillator. PMID:26562092

  11. CRY Drives Cyclic CK2-Mediated BMAL1 Phosphorylation to Control the Mammalian Circadian Clock.

    Directory of Open Access Journals (Sweden)

    Teruya Tamaru

    Full Text Available Intracellular circadian clocks, composed of clock genes that act in transcription-translation feedback loops, drive global rhythmic expression of the mammalian transcriptome and allow an organism to anticipate to the momentum of the day. Using a novel clock-perturbing peptide, we established a pivotal role for casein kinase (CK-2-mediated circadian BMAL1-Ser90 phosphorylation (BMAL1-P in regulating central and peripheral core clocks. Subsequent analysis of the underlying mechanism showed a novel role of CRY as a repressor for protein kinase. Co-immunoprecipitation experiments and real-time monitoring of protein-protein interactions revealed that CRY-mediated periodic binding of CK2β to BMAL1 inhibits BMAL1-Ser90 phosphorylation by CK2α. The FAD binding domain of CRY1, two C-terminal BMAL1 domains, and particularly BMAL1-Lys537 acetylation/deacetylation by CLOCK/SIRT1, were shown to be critical for CRY-mediated BMAL1-CK2β binding. Reciprocally, BMAL1-Ser90 phosphorylation is prerequisite for BMAL1-Lys537 acetylation. We propose a dual negative-feedback model in which a CRY-dependent CK2-driven posttranslational BMAL1-P-BMAL1 loop is an integral part of the core clock oscillator.

  12. Network Analysis of a Comprehensive Knowledge Repository Reveals a Dual Role for Ceramide in Alzheimer's Disease.

    Science.gov (United States)

    Mizuno, Satoshi; Ogishima, Soichi; Kitatani, Kazuyuki; Kikuchi, Masataka; Tanaka, Hiroshi; Yaegashi, Nobuo; Nakaya, Jun

    2016-01-01

    Alzheimer's disease (AD) is the most common cause of senile dementia. Many inflammatory factors such as amyloid-β and pro-inflammatory cytokines are known to contribute to the inflammatory response in the AD brain. Sphingolipids are widely known to have roles in the pathogenesis of inflammatory diseases, where the precise roles for sphingolipids in inflammation-associated pathogenesis of AD are not well understood. Here we performed a network analysis to clarify the importance of sphingolipids and to model relationships among inflammatory factors and sphingolipids in AD. In this study, we have updated sphingolipid signaling and metabolic cascades in a map of AD signaling networks that we named "AlzPathway," a comprehensive knowledge repository of signaling pathways in AD. Our network analysis of the updated AlzPathway indicates that the pathways related to ceramide are one of the primary pathways and that ceramide is one of the important players in the pathogenesis of AD. The results of our analysis suggest the following two prospects about inflammation in AD: (1) ceramide could play important roles in both inflammatory and anti-inflammatory pathways of AD, and (2) several factors such as Sphingomyelinase and Siglec-11 may be associated with ceramide related inflammation and anti-inflammation pathways in AD. In this study, network analysis of comprehensive knowledge repository reveals a dual role for ceramide in AD. This result provides a clue to clarify sphingolipids related inflammatory and anti-inflammatory pathways in AD. PMID:26849355

  13. Network Analysis of a Comprehensive Knowledge Repository Reveals a Dual Role for Ceramide in Alzheimer's Disease.

    Directory of Open Access Journals (Sweden)

    Satoshi Mizuno

    Full Text Available Alzheimer's disease (AD is the most common cause of senile dementia. Many inflammatory factors such as amyloid-β and pro-inflammatory cytokines are known to contribute to the inflammatory response in the AD brain. Sphingolipids are widely known to have roles in the pathogenesis of inflammatory diseases, where the precise roles for sphingolipids in inflammation-associated pathogenesis of AD are not well understood. Here we performed a network analysis to clarify the importance of sphingolipids and to model relationships among inflammatory factors and sphingolipids in AD. In this study, we have updated sphingolipid signaling and metabolic cascades in a map of AD signaling networks that we named "AlzPathway," a comprehensive knowledge repository of signaling pathways in AD. Our network analysis of the updated AlzPathway indicates that the pathways related to ceramide are one of the primary pathways and that ceramide is one of the important players in the pathogenesis of AD. The results of our analysis suggest the following two prospects about inflammation in AD: (1 ceramide could play important roles in both inflammatory and anti-inflammatory pathways of AD, and (2 several factors such as Sphingomyelinase and Siglec-11 may be associated with ceramide related inflammation and anti-inflammation pathways in AD. In this study, network analysis of comprehensive knowledge repository reveals a dual role for ceramide in AD. This result provides a clue to clarify sphingolipids related inflammatory and anti-inflammatory pathways in AD.

  14. Importance of glycolysis and oxidative phosphorylation in advanced melanoma

    Directory of Open Access Journals (Sweden)

    Ho Jonhan

    2012-10-01

    Full Text Available Abstract Serum lactate dehydrogenase (LDH is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS, we subsequently determined using tissue microarray (TMA analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

  15. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Staehr, Peter; Hansen, Bo Falck;

    2003-01-01

    In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites, but the mecha......In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH(2)- and COOH-terminal sites......, but the mechanism involved in human muscle and the defect in type 2 diabetes remain unclear. We studied the effect of insulin at physiological concentrations on glucose metabolism, insulin signaling and phosphorylation of GS in skeletal muscle from type 2 diabetic and well-matched control subjects during euglycemic......-hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact...

  16. Diversity of eukaryotic DNA replication origins revealed by genome-wide analysis of chromatin structure.

    Directory of Open Access Journals (Sweden)

    Nicolas M Berbenetz

    2010-09-01

    Full Text Available Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers positions nucleosomes adjacent to the origin to promote replication origin function.

  17. A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.

    Science.gov (United States)

    Gee, Christine L; Papavinasasundaram, Kadamba G; Blair, Sloane R; Baer, Christina E; Falick, Arnold M; King, David S; Griffin, Jennifer E; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K; Crick, Dean C; Rubin, Eric J; Sassetti, Christopher M; Alber, Tom

    2012-01-24

    Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase-FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

  18. Dynamic functional connectivity analysis reveals transient states of dysconnectivity in schizophrenia

    Directory of Open Access Journals (Sweden)

    E. Damaraju

    2014-01-01

    Full Text Available Schizophrenia is a psychotic disorder characterized by functional dysconnectivity or abnormal integration between distant brain regions. Recent functional imaging studies have implicated large-scale thalamo-cortical connectivity as being disrupted in patients. However, observed connectivity differences in schizophrenia have been inconsistent between studies, with reports of hyperconnectivity and hypoconnectivity between the same brain regions. Using resting state eyes-closed functional imaging and independent component analysis on a multi-site data that included 151 schizophrenia patients and 163 age- and gender matched healthy controls, we decomposed the functional brain data into 100 components and identified 47 as functionally relevant intrinsic connectivity networks. We subsequently evaluated group differences in functional network connectivity, both in a static sense, computed as the pairwise Pearson correlations between the full network time courses (5.4 minutes in length, and a dynamic sense, computed using sliding windows (44 s in length and k-means clustering to characterize five discrete functional connectivity states. Static connectivity analysis revealed that compared to healthy controls, patients show significantly stronger connectivity, i.e., hyperconnectivity, between the thalamus and sensory networks (auditory, motor and visual, as well as reduced connectivity (hypoconnectivity between sensory networks from all modalities. Dynamic analysis suggests that (1, on average, schizophrenia patients spend much less time than healthy controls in states typified by strong, large-scale connectivity, and (2, that abnormal connectivity patterns are more pronounced during these connectivity states. In particular, states exhibiting cortical–subcortical antagonism (anti-correlations and strong positive connectivity between sensory networks are those that show the group differences of thalamic hyperconnectivity and sensory hypoconnectivity

  19. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  20. Molecular analysis of endothelial progenitor cell (EPC subtypes reveals two distinct cell populations with different identities

    Directory of Open Access Journals (Sweden)

    Simpson David A

    2010-05-01

    Full Text Available Abstract Background The term endothelial progenitor cells (EPCs is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs and outgrowth endothelial cells (OECs. Methods Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN with links to immunity and inflammation (TLRs, CD14, HLAs, whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

  1. Cellular regulation by protein phosphorylation.

    Science.gov (United States)

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

  2. Phototaxis of Haloarcula marismortui revealed through a novel microbial motion analysis algorithm.

    Science.gov (United States)

    Lin, Yu-Cheng; Fu, Hsu-Yuan; Yang, Chii-Shen

    2010-01-01

    Haloarcula marismortui has been described to be nonmotile prior to the recent identification of flagellar filaments, suggesting the motile nature of H. marismortui. Here we observed the locomotion of freshly cultured H. marismortui cells and tracked the swimming trajectories via ImageJ. Trajectories of H. marismortui are intrinsically noisy, posing difficulties in motion analysis with previously established algorithms. By introducing the concept of "window vector," a Microsoft Excel-VBA-implemented microbial motion analysis algorithm reported here was able to (1) discriminate nonswimming objects from swimming cells without empirical customization by applying a power-law relationship and (2) reduce the noise caused by Brownian motion, thus enhancing the accuracy of swim reversal identification. Based on this motion analysis algorithm, two recently identified sensory rhodopsins, HmSRI and HmSRII, were shown to mediate photoattractant and photorepellent responses, respectively, revealing the phototactic activity of H. marismortui, the only archaeon showing such phenomenon other than Halobacterium salinarum. PMID:20553410

  3. Comparative Analysis of 35 Basidiomycete Genomes Reveals Diversity and Uniqueness of the Phylum

    Energy Technology Data Exchange (ETDEWEB)

    Riley, Robert; Salamov, Asaf; Otillar, Robert; Fagnan, Kirsten; Boussau, Bastien; Brown, Daren; Henrissat, Bernard; Levasseur, Anthony; Held, Benjamin; Nagy, Laszlo; Floudas, Dimitris; Morin, Emmanuelle; Manning, Gerard; Baker, Scott; Martin, Francis; Blanchette, Robert; Hibbett, David; Grigoriev, Igor V.

    2013-03-11

    Fungi of the phylum Basidiomycota (basidiomycetes), make up some 37percent of the described fungi, and are important in forestry, agriculture, medicine, and bioenergy. This diverse phylum includes symbionts, pathogens, and saprobes including wood decaying fungi. To better understand the diversity of this phylum we compared the genomes of 35 basidiomycete fungi including 6 newly sequenced genomes. The genomes of basidiomycetes span extremes of genome size, gene number, and repeat content. A phylogenetic tree of Basidiomycota was generated using the Phyldog software, which uses all available protein sequence data to simultaneously infer gene and species trees. Analysis of core genes reveals that some 48percent of basidiomycete proteins are unique to the phylum with nearly half of those (22percent) comprising proteins found in only one organism. Phylogenetic patterns of plant biomass-degrading genes suggest a continuum rather than a sharp dichotomy between the white rot and brown rot modes of wood decay among the members of Agaricomycotina subphylum. There is a correlation of the profile of certain gene families to nutritional mode in Agaricomycotina. Based on phylogenetically-informed PCA analysis of such profiles, we predict that that Botryobasidium botryosum and Jaapia argillacea have properties similar to white rot species, although neither has liginolytic class II fungal peroxidases. Furthermore, we find that both fungi exhibit wood decay with white rot-like characteristics in growth assays. Analysis of the rate of discovery of proteins with no or few homologs suggests the high value of continued sequencing of basidiomycete fungi.

  4. Potential microRNA-mediated oncogenic intercellular communication revealed by pan-cancer analysis

    Science.gov (United States)

    Li, Yue; Zhang, Zhaolei

    2014-11-01

    Carcinogenesis consists of oncogenesis and metastasis, and intriguingly microRNAs (miRNAs) are involved in both processes. Although aberrant miRNA activities are prevalent in diverse tumor types, the exact mechanisms for how they regulate cancerous processes are not always clear. To this end, we performed a large-scale pan-cancer analysis via a novel probabilistic approach to infer recurrent miRNA-target interactions implicated in 12 cancer types using data from The Cancer Genome Atlas. We discovered ~20,000 recurrent miRNA regulations, which are enriched for cancer-related miRNAs/genes. Notably, miRNA 200 family (miR-200/141/429) is among the most prominent miRNA regulators, which is known to be involved in metastasis. Importantly, the recurrent miRNA regulatory network is not only enriched for cancer pathways but also for extracellular matrix (ECM) organization and ECM-receptor interactions. The results suggest an intriguing cancer mechanism involving miRNA-mediated cell-to-cell communication, which possibly involves delivery of tumorigenic miRNA messengers to adjacent cells via exosomes. Finally, survival analysis revealed 414 recurrent-prognostic associations, where both gene and miRNA involved in each interaction conferred significant prognostic power in one or more cancer types. Together, our comprehensive pan-cancer analysis provided not only biological insights into metastasis but also brought to bear the clinical relevance of the proposed recurrent miRNA-gene associations.

  5. Rapid genome evolution in Pms1 region of rice revealed by comparative sequence analysis

    Institute of Scientific and Technical Information of China (English)

    YU JinSheng; FAN YouRong; LIU Nan; SHAN Yan; LI XiangHua; ZHANG QiFa

    2007-01-01

    Pms1, a locus for photoperiod sensitive genic male sterility in rice, was identified and mapped to chromosome 7 in previous studies. Here we report an effort to identify the candidate genes for Pms1 by comparative sequencing of BAC clones from two cultivars Minghui 63 and Nongken 58, the parents for the initial mapping population. Annotation and comparison of the sequences of the two clones resulted in a total of five potential candidates which should be functionally tested. We also conducted comparative analysis of sequences of these two cultivars with two other cultivars, Nipponbare and 93-11,for which sequence data were available in public databases. The analysis revealed large differences in sequence composition among the four genotypes in the Pms1 region primarily due to retroelement activity leading to rapid recent growth and divergence of the genomes. High levels of polymorphism in the forms of indels and SNPs were found both in intra- and inter-subspecific comparisons. Dating analysis using LTRs of the retroelements in this region showed that the substitution rate of LTRs was much higher than reported in the literature. The results provided strong evidence for rapid genomic evolution of this region as a consequence of natural and artificial selection.

  6. Meta-Analysis of EMT Datasets Reveals Different Types of EMT

    Science.gov (United States)

    Zhang, Wei; Zhang, Mengdan; Yang, Xiao; Kuang, Rui; Zheng, Hui

    2016-01-01

    As a critical process during embryonic development, cancer progression and cell fate conversions, epithelial-mesenchymal transition (EMT) has been extensively studied over the last several decades. To further understand the nature of EMT, we performed meta-analysis of multiple microarray datasets to identify the related generic signature. In this study, 24 human and 17 mouse microarray datasets were integrated to identify conserved gene expression changes in different types of EMT. Our integrative analysis revealed that there is low agreement among the list of the identified signature genes and three other lists in previous studies. Since removing the datasets with weakly-induced EMT from the analysis did not significantly improve the overlapping in the signature-gene lists, we hypothesized the existence of different types of EMT. This hypothesis was further supported by the grouping of 74 human EMT-induction samples into five distinct clusters, and the identification of distinct pathways in these different clusters of EMT samples. The five clusters of EMT-induction samples also improves the understanding of the characteristics of different EMT types. Therefore, we concluded the existence of different types of EMT was the possible reason for its complex role in multiple biological processes. PMID:27258544

  7. Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

    Science.gov (United States)

    Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

    2015-11-01

    Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

  8. Dependency Network Analysis (DEPNA) Reveals Context Related Influence of Brain Network Nodes

    Science.gov (United States)

    Jacob, Yael; Winetraub, Yonatan; Raz, Gal; Ben-Simon, Eti; Okon-Singer, Hadas; Rosenberg-Katz, Keren; Hendler, Talma; Ben-Jacob, Eshel

    2016-01-01

    Communication between and within brain regions is essential for information processing within functional networks. The current methods to determine the influence of one region on another are either based on temporal resolution, or require a predefined model for the connectivity direction. However these requirements are not always achieved, especially in fMRI studies, which have poor temporal resolution. We thus propose a new graph theory approach that focuses on the correlation influence between selected brain regions, entitled Dependency Network Analysis (DEPNA). Partial correlations are used to quantify the level of influence of each node during task performance. As a proof of concept, we conducted the DEPNA on simulated datasets and on two empirical motor and working memory fMRI tasks. The simulations revealed that the DEPNA correctly captures the network’s hierarchy of influence. Applying DEPNA to the functional tasks reveals the dynamics between specific nodes as would be expected from prior knowledge. To conclude, we demonstrate that DEPNA can capture the most influencing nodes in the network, as they emerge during specific cognitive processes. This ability opens a new horizon for example in delineating critical nodes for specific clinical interventions. PMID:27271458

  9. Genetic analysis reveals candidate species in the Scinax catharinae clade (Amphibia: Anura) from Central Brazil.

    Science.gov (United States)

    Nogueira, Lídia; Solé, Mirco; Siqueira, Sérgio; Affonso, Paulo Roberto Antunes de Mello; Strüssmann, Christine; Sampaio, Iracilda

    2016-03-01

    Scinax (Anura: Hylidae) is a species-rich genus of amphibians (113 spp.), divided into five species groups by morphological features. Cladistic analyses however revealed only two monophyletic clades in these groups: Scinax catharinae and Scinax ruber. Most species from the S. catharinae clade are found in Atlantic rainforest, except for Scinax canastrensis,S. centralis, S. luizotavioi, S. machadoi,S. pombali and S. skaios. In the present work, specimens of Scinax collected in Chapada dos Guimarães, central Brazil, were morphologically compatible with species from theS. catharinae group. On the other hand, genetic analysis based on mitochondrial (16S and 12S) and nuclear (rhodopsin) sequences revealed a nucleotide divergence of 6 to 20% between Scinax sp. and other congeners from the Brazilian savannah (Cerrado). Accordingly, Bayesian inference placed Scinax sp. in the S. catharinae clade with high support values. Hence, these findings strongly indicate the presence of a new species in the S. catharinae clade from the southwestern portion of the Brazilian savannah. To be properly validated as a novel species, detailed comparative morphological and bioacustic studies with other taxa from Brazil such asS. canastrensis, S. centralis, S. luizotavioi, S. machadoi, S. pombali and S. skaios are required. PMID:27007898

  10. Stable isotope analysis of vertebrae reveals ontogenetic changes in habitat in an endothermic pelagic shark.

    Science.gov (United States)

    Carlisle, Aaron B; Goldman, Kenneth J; Litvin, Steven Y; Madigan, Daniel J; Bigman, Jennifer S; Swithenbank, Alan M; Kline, Thomas C; Block, Barbara A

    2015-01-22

    Ontogenetic changes in habitat are driven by shifting life-history requirements and play an important role in population dynamics. However, large portions of the life history of many pelagic species are still poorly understood or unknown. We used a novel combination of stable isotope analysis of vertebral annuli, Bayesian mixing models, isoscapes and electronic tag data to reconstruct ontogenetic patterns of habitat and resource use in a pelagic apex predator, the salmon shark (Lamna ditropis). Results identified the North Pacific Transition Zone as the major nursery area for salmon sharks and revealed an ontogenetic shift around the age of maturity from oceanic to increased use of neritic habitats. The nursery habitat may reflect trade-offs between prey availability, predation pressure and thermal constraints on juvenile endothermic sharks. The ontogenetic shift in habitat coincided with a reduction of isotopic niche, possibly reflecting specialization upon particular prey or habitats. Using tagging data to inform Bayesian isotopic mixing models revealed that adult sharks primarily use neritic habitats of Alaska yet receive a trophic subsidy from oceanic habitats. Integrating the multiple methods used here provides a powerful approach to retrospectively study the ecology and life history of migratory species throughout their ontogeny. PMID:25621332

  11. Precursors of stall and surge processes in gas turbines revealed by wavelet analysis

    Energy Technology Data Exchange (ETDEWEB)

    Dremin, I.M.; Ivanov, O.V.; Nechitailo, V.A. [P.N. Lebedev Physical Institute, Moscow (Russian Federation); Furletov, V.I. [Central Institute for Aviation Motors, Moscow (Russian Federation); Terziev, V.G. [TEKO, Moscow (Russian Federation)

    2002-06-01

    Multiresolution wavelet analysis of pressure variations in a gas turbine compressor reveals the existence of precursors of stall and surge processes. Signals from eight pressure sensors positioned at various places within the compressor were recorded and digitized in three different operating modes in stationary conditions with a recording interval of 1 ms during 5-6 s. It has been discovered that there exists a scale of 32 intervals over which the dispersion (variance) of the wavelet coefficients shows a remarkable drop of about 40% for more than 1 s prior to the development of the malfunction. A shuffled sample of the same values of the pressure does not show such a drop demonstrating the dynamical origin of this effect. Higher order correlation moments reveal different slopes in these two regions differing by the variance values. The log-log dependence of the moments does not show clear fractal behavior because the scales of 16 and 32 intervals are not on the straight line of monofractals. This is a clear indication of the nonlinear response of the system at this scale. These results provide a means for automatic regulation of an engine, preventing possible failures. (author)

  12. Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut.

    Directory of Open Access Journals (Sweden)

    Francesca Bottacini

    Full Text Available Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration.

  13. Lateral spreading in the Svecofennian mid-crust revealed by seismic attribute analysis

    DEFF Research Database (Denmark)

    Torvela, Taija; Moreau, Julien; Butler, Robert;

    orogen. Details of this spreading are, however, still not clear. The Svecofennian crust is imaged by the Finnish Reflection Experiment (FIRE) that was designed to image the major crustal structures in Finland. Here, we show an example of how seismic attributes, combined with the seismic facies...... interpretation technique, can be used to enhance the FIRE data. We focussed our study to the West Uusimaa complex WUC, where the middle crust of the Svecofennian is exposed. We compare the interpreted, enhanced seismic data with field observations. The attribute analysis of the seismic data reveals......In orogenic belts, crustal thickening precedes rheological weakening of the middle and lower orogenic crust leading to lateral spreading and, ultimately, to collapse of an orogen. Recent studies have implied similar development in the middle crust during the Palaeoproterozoic composite Svecofennian...

  14. Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways.

    Science.gov (United States)

    Müller, Sebastian; Balaz, Miroslav; Stefanicka, Patrik; Varga, Lukas; Amri, Ez-Zoubir; Ukropec, Jozef; Wollscheid, Bernd; Wolfrum, Christian

    2016-07-15

    Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology.

  15. Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways.

    Science.gov (United States)

    Müller, Sebastian; Balaz, Miroslav; Stefanicka, Patrik; Varga, Lukas; Amri, Ez-Zoubir; Ukropec, Jozef; Wollscheid, Bernd; Wolfrum, Christian

    2016-01-01

    Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology. PMID:27418403

  16. Anomalous dispersion of Lagrangian particles in local regions of turbulent flows revealed by convex hull analysis

    CERN Document Server

    Pratt, J; Mueller, W -C; Chapman, S C; Watkins, N W

    2014-01-01

    Local regions of anomalous particle dispersion, and intermittent events that occur in turbulent flows can greatly influence the global statistical description of the flow. These local behaviors can be identified and analyzed by comparing the growth of neighboring convex hulls of Lagrangian tracer particles. Although in our simulations of homogeneous turbulence the convex hulls generally grow in size, after the Lagrangian particles that define the convex hulls begin to disperse, our analysis reveals short periods when the convex hulls of the Lagrangian particles shrink, evidence that particles are not dispersing simply. Shrinkage can be associated with anisotropic flows, since it occurs most frequently in the presence of a mean magnetic field or thermal convection. We compare dispersion between a wide range of statistically homogeneous and stationary turbulent flows ranging from homogeneous isotropic Navier-Stokes turbulence over different configurations of magnetohydrodynamic turbulence and Boussinesq convect...

  17. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    Science.gov (United States)

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  18. Proteomic Analysis of Human Brown Adipose Tissue Reveals Utilization of Coupled and Uncoupled Energy Expenditure Pathways

    Science.gov (United States)

    Müller, Sebastian; Balaz, Miroslav; Stefanicka, Patrik; Varga, Lukas; Amri, Ez-Zoubir; Ukropec, Jozef; Wollscheid, Bernd; Wolfrum, Christian

    2016-01-01

    Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology. PMID:27418403

  19. Metabolomic analysis reveals metabolic disturbance in the cortex and hippocampus of subchronic MK-801 treated rats.

    Directory of Open Access Journals (Sweden)

    Liya Sun

    Full Text Available BACKGROUND: Although a number of proteins and genes relevant to schizophrenia have been identified in recent years, few are known about the exact metabolic pathway involved in this disease. Our previous proteomic study has revealed the energy metabolism abnormality in subchronic MK-801 treated rat, a well-established animal model for schizophrenia. This prompted us to further investigate metabolite levels in the same rat model to better delineate the metabolism dysfunctions and provide insights into the pathology of schizophrenia. METHODS: Metabolomics, a high-throughput investigatory strategy developed in recent years, can offer comprehensive metabolite-level insights that complement protein and genetic findings. In this study, we employed a nondestructive metabolomic approach (1H-MAS-NMR to investigate the metabolic traits in cortex and hippocampus of MK-801 treated rats. Multivariate statistics and ingenuity pathways analyses (IPA were applied in data processing. The result was further integrated with our previous proteomic findings by IPA analysis to obtain a systematic view on our observations. RESULTS: Clear distinctions between the MK-801 treated group and the control group in both cortex and hippocampus were found by OPLS-DA models (with R(2X = 0.441, Q(2Y = 0.413 and R(2X = 0.698, Q(2Y = 0.677, respectively. The change of a series of metabolites accounted for the separation, such as glutamate, glutamine, citrate and succinate. Most of these metabolites fell in a pathway characterized by down-regulated glutamate synthesis and disturbed Krebs cycle. IPA analysis further confirmed the involvement of energy metabolism abnormality induced by MK-801 treatment. CONCLUSIONS: Our metabolomics findings reveal systematic changes in pathways of glutamate metabolism and Krebs cycle in the MK-801 treated rats' cortex and hippocampus, which confirmed and improved our previous proteomic observation and served as a valuable reference to

  20. Partial sequencing of the bottle gourd genome reveals markers useful for phylogenetic analysis and breeding

    Directory of Open Access Journals (Sweden)

    Wang Sha

    2011-09-01

    Full Text Available Abstract Background Bottle gourd [Lagenaria siceraria (Mol. Standl.] is an important cucurbit crop worldwide. Archaeological research indicates that bottle gourd was domesticated more than 10,000 years ago, making it one of the earliest plants cultivated by man. In spite of its widespread importance and long history of cultivation almost nothing has been known about the genome of this species thus far. Results We report here the partial sequencing of bottle gourd genome using the 454 GS-FLX Titanium sequencing platform. A total of 150,253 sequence reads, which were assembled into 3,994 contigs and 82,522 singletons were generated. The total length of the non-redundant singletons/assemblies is 32 Mb, theoretically covering ~ 10% of the bottle gourd genome. Functional annotation of the sequences revealed a broad range of functional types, covering all the three top-level ontologies. Comparison of the gene sequences between bottle gourd and the model cucurbit cucumber (Cucumis sativus revealed a 90% sequence similarity on average. Using the sequence information, 4395 microsatellite-containing sequences were identified and 400 SSR markers were developed, of which 94% amplified bands of anticipated sizes. Transferability of these markers to four other cucurbit species showed obvious decline with increasing phylogenetic distance. From analyzing polymorphisms of a subset of 14 SSR markers assayed on 44 representative China bottle gourd varieties/landraces, a principal coordinates (PCo analysis output and a UPGMA-based dendrogram were constructed. Bottle gourd accessions tended to group by fruit shape rather than geographic origin, although in certain subclades the lines from the same or close origin did tend to cluster. Conclusions This work provides an initial basis for genome characterization, gene isolation and comparative genomics analysis in bottle gourd. The SSR markers developed would facilitate marker assisted breeding schemes for efficient

  1. Ovarian hormones and prolactin increase renal NaCl cotransporter phosphorylation.

    Science.gov (United States)

    Rojas-Vega, Lorena; Reyes-Castro, Luis A; Ramírez, Victoria; Bautista-Pérez, Rocío; Rafael, Chloe; Castañeda-Bueno, María; Meade, Patricia; de Los Heros, Paola; Arroyo-Garza, Isidora; Bernard, Valérie; Binart, Nadine; Bobadilla, Norma A; Hadchouel, Juliette; Zambrano, Elena; Gamba, Gerardo

    2015-04-15

    Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

  2. Comparative transcriptomic analysis reveals similarities and dissimilarities in Saccharomyces cerevisiae wine strains response to nitrogen availability.

    Directory of Open Access Journals (Sweden)

    Catarina Barbosa

    Full Text Available Nitrogen levels in grape-juices are of major importance in winemaking ensuring adequate yeast growth and fermentation performance. Here we used a comparative transcriptome analysis to uncover wine yeasts responses to nitrogen availability during fermentation. Gene expression was assessed in three genetically and phenotypically divergent commercial wine strains (CEG, VL1 and QA23, under low (67 mg/L and high nitrogen (670 mg/L regimes, at three time points during fermentation (12 h, 24 h and 96 h. Two-way ANOVA analysis of each fermentation condition led to the identification of genes whose expression was dependent on strain, fermentation stage and on the interaction of both factors. The high fermenter yeast strain QA23 was more clearly distinct from the other two strains, by differential expression of genes involved in flocculation, mitochondrial functions, energy generation and protein folding and stabilization. For all strains, higher transcriptional variability due to fermentation stage was seen in the high nitrogen fermentations. A positive correlation between maximum fermentation rate and the expression of genes involved in stress response was observed. The finding of common genes correlated with both fermentation activity and nitrogen up-take underlies the role of nitrogen on yeast fermentative fitness. The comparative analysis of genes differentially expressed between both fermentation conditions at 12 h, where the main difference was the level of nitrogen available, showed the highest variability amongst strains revealing strain-specific responses. Nevertheless, we were able to identify a small set of genes whose expression profiles can quantitatively assess the common response of the yeast strains to varying nitrogen conditions. The use of three contrasting yeast strains in gene expression analysis prompts the identification of more reliable, accurate and reproducible biomarkers that will facilitate the diagnosis of deficiency of this

  3. Comparative Transcriptome Analysis Reveals Substantial Tissue Specificity in Human Aortic Valve

    Science.gov (United States)

    Wang, Jun; Wang, Ying; Gu, Weidong; Ni, Buqing; Sun, Haoliang; Yu, Tong; Gu, Wanjun; Chen, Liang; Shao, Yongfeng

    2016-01-01

    RNA sequencing (RNA-seq) has revolutionary roles in transcriptome identification and quantification of different types of tissues and cells in many organisms. Although numerous RNA-seq data derived from many types of human tissues and cell lines, little is known on the transcriptome repertoire of human aortic valve. In this study, we sequenced the total RNA prepared from two calcified human aortic valves and reported the whole transcriptome of human aortic valve. Integrating RNA-seq data of 13 human tissues from Human Body Map 2 Project, we constructed a transcriptome repertoire of human tissues, including 19,505 protein-coding genes and 4,948 long intergenic noncoding RNAs (lincRNAs). Among them, 263 lincRNAs were identified as novel noncoding transcripts in our data. By comparing transcriptome data among different human tissues, we observed substantial tissue specificity of RNA transcripts, both protein-coding genes and lincRNAs, in human aortic valve. Further analysis revealed that aortic valve-specific lincRNAs were more likely to be recently derived from repetitive elements in the primate lineage, but were less likely to be conserved at the nucleotide level. Expression profiling analysis showed significant lower expression levels of aortic valve-specific protein-coding genes and lincRNA genes, when compared with genes that were universally expressed in various tissues. Isoform-level expression analysis also showed that a majority of mRNA genes had a major isoform expressed in the human aortic valve. To our knowledge, this is the first comparative transcriptome analysis between human aortic valve and other human tissues. Our results are helpful to understand the transcriptome diversity of human tissues and the underlying mechanisms that drive tissue specificity of protein-coding genes and lincRNAs in human aortic valve. PMID:27493474

  4. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  5. Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis.

    Science.gov (United States)

    Kohorn, Bruce D; Hoon, Divya; Minkoff, Benjamin B; Sussman, Michael R; Kohorn, Susan L

    2016-04-01

    The wall-associated kinases (WAKs)(1)are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGsin planta Using N(14/)N(15)isotopicin vivometabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominantWAKallele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase

  6. Effects of phosphorylation on function of the Rad GTPase.

    Science.gov (United States)

    Moyers, J S; Zhu, J; Kahn, C R

    1998-08-01

    Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases. PMID:9677319

  7. Metagenomic analysis reveals symbiotic relationship among bacteria in Microcystis-dominated community

    Directory of Open Access Journals (Sweden)

    Meili eXie

    2016-02-01

    Full Text Available Microcystis bloom, a cyanobacterial mass occurrence often found in eutrophicated water bodies, is one of the most serious threats to freshwater ecosystems worldwide. In nature, Microcystis forms aggregates or colonies that contain heterotrophic bacteria. The Microcystis-bacteria colonies were persistent even when they were maintained in lab culture for a long period. The relationship between Microcystis and the associated bacteria was investigated by a metagenomic approach in this study. We developed a visualization-guided method of binning for genome assembly after total colony DNA sequencing. We found that the method was effective in grouping sequences and it did not require reference genome sequence. Individual genomes of the colony bacteria were obtained and they provided valuable insights into microbial community structures. Analysis of metabolic pathways based on these genomes revealed that while all heterotrophic bacteria were dependent upon Microcystis for carbon and energy, Vitamin B12 biosynthesis, which is required for growth by Microcystis, was accomplished in a cooperative fashion among the bacteria. Our analysis also suggests that individual bacteria in the colony community contributed a complete pathway for degradation of benzoate, which is inhibitory to the cyanobacterial growth, and its ecological implication for Microcystis bloom is discussed.

  8. Bach Is the Father of Harmony: Revealed by a 1/f Fluctuation Analysis across Musical Genres.

    Science.gov (United States)

    Wu, Dan; Kendrick, Keith M; Levitin, Daniel J; Li, Chaoyi; Yao, Dezhong

    2015-01-01

    Harmony is a fundamental attribute of music. Close connections exist between music and mathematics since both pursue harmony and unity. In music, the consonance of notes played simultaneously partly determines our perception of harmony; associates with aesthetic responses; and influences the emotion expression. The consonance could be considered as a window to understand and analyze harmony. Here for the first time we used a 1/f fluctuation analysis to investigate whether the consonance fluctuation structure in music with a wide range of composers and genres followed the scale free pattern that has been found for pitch, melody, rhythm, human body movements, brain activity, natural images and geographical features. We then used a network graph approach to investigate which composers were the most influential both within and across genres. Our results showed that patterns of consonance in music did follow scale-free characteristics, suggesting that this feature is a universally evolved one in both music and the living world. Furthermore, our network analysis revealed that Bach's harmony patterns were having the most influence on those used by other composers, followed closely by Mozart. PMID:26545104

  9. Expression analysis of five zebrafish RXFP3 homologues reveals evolutionary conservation of gene expression pattern.

    Science.gov (United States)

    Donizetti, Aldo; Fiengo, Marcella; Iazzetti, Giovanni; del Gaudio, Rosanna; Di Giaimo, Rossella; Pariante, Paolo; Minucci, Sergio; Aniello, Francesco

    2015-01-01

    Relaxin peptides exert different functions in reproduction and neuroendocrine processes via interaction with two evolutionarily unrelated groups of receptors: RXFP1 and RXFP2 on one hand, RXFP3 and RXFP4 on the other hand. Evolution of receptor genes after splitting of tetrapods and teleost lineage led to a different retention rate between mammals and fish, with the latter having more gene copies compared to the former. In order to improve our knowledge on the evolution of the relaxin ligands/receptors system and have insights on their function in early stages of life, in the present paper we analyzed the expression pattern of five zebrafish RXFP3 homologue genes during embryonic development. In our analysis, we show that only two of the five genes are expressed during embryogenesis and that their transcripts are present in all the developmental stages. Spatial localization analysis of these transcripts revealed that the gene expression is restricted in specific territories starting from early pharyngula stage. Both genes are expressed in the brain but in different cell clusters and in extra-neural territories, one gene in the interrenal gland and the other in the pancreas. These two genes share expression territories with the homologue mammalian counterpart, highlighting a general conservation of gene expression regulatory processes and their putative function during evolution that are established early in vertebrate embryogenesis. PMID:25384467

  10. Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes.

    Science.gov (United States)

    Subirats, Jéssica; Sànchez-Melsió, Alexandre; Borrego, Carles M; Balcázar, José Luis; Simonet, Pascal

    2016-08-01

    A metagenomics approach was applied to explore the presence of antibiotic resistance genes (ARGs) in bacteriophages from hospital wastewater. Metagenomic analysis showed that most phage sequences affiliated to the order Caudovirales, comprising the tailed phage families Podoviridae, Siphoviridae and Myoviridae. Moreover, the relative abundance of ARGs in the phage DNA fraction (0.26%) was higher than in the bacterial DNA fraction (0.18%). These differences were particularly evident for genes encoding ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND) proteins, phosphotransferases, β-lactamases and plasmid-mediated quinolone resistance. Analysis of assembled contigs also revealed that blaOXA-10, blaOXA-58 and blaOXA-24 genes belonging to class D β-lactamases as well as a novel blaTEM (98.9% sequence similarity to the blaTEM-1 gene) belonging to class A β-lactamases were detected in a higher proportion in phage DNA. Although preliminary, these findings corroborate the role of bacteriophages as reservoirs of resistance genes and thus highlight the necessity to include them in future studies on the emergence and spread of antibiotic resistance in the environment.

  11. Transcriptome analysis reveals dynamic changes in the gene expression of tobacco seedlings under low potassium stress

    Indian Academy of Sciences (India)

    Liming Lu; Yong Chen; Lin Lu; Yifei Lu; Liqin Li

    2015-09-01

    Potassium plays a key role in plant development and reproduction. In agricultural practice, potassium deficiency is common worldwide, and leads to crop growth inhibition and output reduction. In this study, we analysed the transcriptome of tobacco seedlings under low potassium stress. Tobacco seedlings with or without decreased potassium treatment were harvested after 0 (control), 6, 12, or 24 h and were submitted for microarray analysis. The results showed that up to 3790 genes were upregulated or downregulated more than 2-fold as a result of the decreased potassium treatment. Gene ontology analysis revealed significantly differentially expressed genes that were categorized as cation binding, transcription regulation, metabolic processes, transporter activity and enzyme regulation. Some potassium, nitrogen and phosphorus transporters; transcription factors; and plant signal molecules, such as CPKs were also significantly differentially expressed under potassium deficiency. Our results indicate that the expression profiles of a large number of genes involved in various plant physiological processes are significantly altered in response to potassium deficiency, which can result in physiological and morphological changes in tobacco plants.

  12. Meta-analysis of Dense Genecentric Association Studies Reveals Common and Uncommon Variants Associated with Height

    Science.gov (United States)

    Lanktree, Matthew B.; Guo, Yiran; Murtaza, Muhammed; Glessner, Joseph T.; Bailey, Swneke D.; Onland-Moret, N. Charlotte; Lettre, Guillaume; Ongen, Halit; Rajagopalan, Ramakrishnan; Johnson, Toby; Shen, Haiqing; Nelson, Christopher P.; Klopp, Norman; Baumert, Jens; Padmanabhan, Sandosh; Pankratz, Nathan; Pankow, James S.; Shah, Sonia; Taylor, Kira; Barnard, John; Peters, Bas J.; M. Maloney, Cliona; Lobmeyer, Maximilian T.; Stanton, Alice; Zafarmand, M. Hadi; Romaine, Simon P.R.; Mehta, Amar; van Iperen, Erik P.A.; Gong, Yan; Price, Tom S.; Smith, Erin N.; Kim, Cecilia E.; Li, Yun R.; Asselbergs, Folkert W.; Atwood, Larry D.; Bailey, Kristian M.; Bhatt, Deepak; Bauer, Florianne; Behr, Elijah R.; Bhangale, Tushar; Boer, Jolanda M.A.; Boehm, Bernhard O.; Bradfield, Jonathan P.; Brown, Morris; Braund, Peter S.; Burton, Paul R.; Carty, Cara; Chandrupatla, Hareesh R.; Chen, Wei; Connell, John; Dalgeorgou, Chrysoula; Boer, Anthonius de; Drenos, Fotios; Elbers, Clara C.; Fang, James C.; Fox, Caroline S.; Frackelton, Edward C.; Fuchs, Barry; Furlong, Clement E.; Gibson, Quince; Gieger, Christian; Goel, Anuj; Grobbee, Diederik E.; Hastie, Claire; Howard, Philip J.; Huang, Guan-Hua; Johnson, W. Craig; Li, Qing; Kleber, Marcus E.; Klein, Barbara E.K.; Klein, Ronald; Kooperberg, Charles; Ky, Bonnie; LaCroix, Andrea; Lanken, Paul; Lathrop, Mark; Li, Mingyao; Marshall, Vanessa; Melander, Olle; Mentch, Frank D.; J. Meyer, Nuala; Monda, Keri L.; Montpetit, Alexandre; Murugesan, Gurunathan; Nakayama, Karen; Nondahl, Dave; Onipinla, Abiodun; Rafelt, Suzanne; Newhouse, Stephen J.; Otieno, F. George; Patel, Sanjey R.; Putt, Mary E.; Rodriguez, Santiago; Safa, Radwan N.; Sawyer, Douglas B.; Schreiner, Pamela J.; Simpson, Claire; Sivapalaratnam, Suthesh; Srinivasan, Sathanur R.; Suver, Christine; Swergold, Gary; Sweitzer, Nancy K.; Thomas, Kelly A.; Thorand, Barbara; Timpson, Nicholas J.; Tischfield, Sam; Tobin, Martin; Tomaszweski, Maciej; Verschuren, W.M. Monique; Wallace, Chris; Winkelmann, Bernhard; Zhang, Haitao; Zheng, Dongling; Zhang, Li; Zmuda, Joseph M.; Clarke, Robert; Balmforth, Anthony J.; Danesh, John; Day, Ian N.; Schork, Nicholas J.; de Bakker, Paul I.W.; Delles, Christian; Duggan, David; Hingorani, Aroon D.; Hirschhorn, Joel N.; Hofker, Marten H.; Humphries, Steve E.; Kivimaki, Mika; Lawlor, Debbie A.; Kottke-Marchant, Kandice; Mega, Jessica L.; Mitchell, Braxton D.; Morrow, David A.; Palmen, Jutta; Redline, Susan; Shields, Denis C.; Shuldiner, Alan R.; Sleiman, Patrick M.; Smith, George Davey; Farrall, Martin; Jamshidi, Yalda; Christiani, David C.; Casas, Juan P.; Hall, Alistair S.; Doevendans, Pieter A.; D. Christie, Jason; Berenson, Gerald S.; Murray, Sarah S.; Illig, Thomas; Dorn, Gerald W.; Cappola, Thomas P.; Boerwinkle, Eric; Sever, Peter; Rader, Daniel J.; Reilly, Muredach P.; Caulfield, Mark; Talmud, Philippa J.; Topol, Eric; Engert, James C.; Wang, Kai; Dominiczak, Anna; Hamsten, Anders; Curtis, Sean P.; Silverstein, Roy L.; Lange, Leslie A.; Sabatine, Marc S.; Trip, Mieke; Saleheen, Danish; Peden, John F.; Cruickshanks, Karen J.; März, Winfried; O'Connell, Jeffrey R.; Klungel, Olaf H.; Wijmenga, Cisca; Maitland-van der Zee, Anke Hilse; Schadt, Eric E.; Johnson, Julie A.; Jarvik, Gail P.; Papanicolaou, George J.; Grant, Struan F.A.; Munroe, Patricia B.; North, Kari E.; Samani, Nilesh J.; Koenig, Wolfgang; Gaunt, Tom R.; Anand, Sonia S.; van der Schouw, Yvonne T.; Soranzo, Nicole; FitzGerald, Garret A.; Reiner, Alex; Hegele, Robert A.; Hakonarson, Hakon; Keating, Brendan J.

    2011-01-01

    Height is a classic complex trait with common variants in a growing list of genes known to contribute to the phenotype. Using a genecentric genotyping array targeted toward cardiovascular-related loci, comprising 49,320 SNPs across approximately 2000 loci, we evaluated the association of common and uncommon SNPs with adult height in 114,223 individuals from 47 studies and six ethnicities. A total of 64 loci contained a SNP associated with height at array-wide significance (p < 2.4 × 10−6), with 42 loci surpassing the conventional genome-wide significance threshold (p < 5 × 10−8). Common variants with minor allele frequencies greater than 5% were observed to be associated with height in 37 previously reported loci. In individuals of European ancestry, uncommon SNPs in IL11 and SMAD3, which would not be genotyped with the use of standard genome-wide genotyping arrays, were strongly associated with height (p < 3 × 10−11). Conditional analysis within associated regions revealed five additional variants associated with height independent of lead SNPs within the locus, suggesting allelic heterogeneity. Although underpowered to replicate findings from individuals of European ancestry, the direction of effect of associated variants was largely consistent in African American, South Asian, and Hispanic populations. Overall, we show that dense coverage of genes for uncommon SNPs, coupled with large-scale meta-analysis, can successfully identify additional variants associated with a common complex trait. PMID:21194676

  13. Comparative Metagenomic Analysis Reveals Mechanisms for Stress Response in Hypoliths from Extreme Hyperarid Deserts.

    Science.gov (United States)

    Le, Phuong Thi; Makhalanyane, Thulani P; Guerrero, Leandro D; Vikram, Surendra; Van de Peer, Yves; Cowan, Don A

    2016-01-01

    Understanding microbial adaptation to environmental stressors is crucial for interpreting broader ecological patterns. In the most extreme hot and cold deserts, cryptic niche communities are thought to play key roles in ecosystem processes and represent excellent model systems for investigating microbial responses to environmental stressors. However, relatively little is known about the genetic diversity underlying such functional processes in climatically extreme desert systems. This study presents the first comparative metagenome analysis of cyanobacteria-dominated hypolithic communities in hot (Namib Desert, Namibia) and cold (Miers Valley, Antarctica) hyperarid deserts. The most abundant phyla in both hypolith metagenomes were Actinobacteria, Proteobacteria, Cyanobacteria and Bacteroidetes with Cyanobacteria dominating in Antarctic hypoliths. However, no significant differences between the two metagenomes were identified. The Antarctic hypolithic metagenome displayed a high number of sequences assigned to sigma factors, replication, recombination and repair, translation, ribosomal structure, and biogenesis. In contrast, the Namib Desert metagenome showed a high abundance of sequences assigned to carbohydrate transport and metabolism. Metagenome data analysis also revealed significant divergence in the genetic determinants of amino acid and nucleotide metabolism between these two metagenomes and those of soil from other polar deserts, hot deserts, and non-desert soils. Our results suggest extensive niche differentiation in hypolithic microbial communities from these two extreme environments and a high genetic capacity for survival under environmental extremes. PMID:27503299

  14. Multifractal analysis of Barkhausen noise reveals the dynamic nature of criticality at hysteresis loop

    Science.gov (United States)

    Tadić, Bosiljka

    2016-06-01

    The field-driven magnetisation reversal processes in disordered systems exhibit a collective behaviour that is manifested in the scale-invariance of avalanches, closely related to underlying dynamical mechanisms. Using the multifractal time series analysis, we study the structure of fluctuations at different scales in the accompanying Barkhausen noise. The stochastic signal represents the magnetisation discontinuities along the hysteresis loop of a three-dimensional random field Ising model simulated for varied disorder strength and driving rates. The analysis of the spectrum of the generalised Hurst exponents reveals that the dominant segments of the signal with large fluctuations represent two distinct classes of stochastic processes in weak and strong pinning regimes. Furthermore, in the weak pinning regime, the part of the signal originating from the beginning of the hysteresis loop has a different multifractal spectrum than the signal near the coercive field. The enhanced fluctuations (primarily in the central part of the hysteresis loop) for increased driving rate and larger system size, lead to a further broadening of the spectrum. The analysed Barkhausen signals are also shown to exhibit temporal correlations and power-law distributions of the magnetisation discontinuity and avalanche sizes, in agreement with previous studies. The multifractal properties of Barkhausen noise describe the dynamical state of domains and precisely discriminate the weak pinning, permitting the motion of individual walls, from the mechanisms occurring in strongly disordered systems.

  15. DNA polymorphisms in banana and sugar cane varieties revealed by RAPD analysis

    International Nuclear Information System (INIS)

    Sugar cane is the fourth most important cash crop of Pakistan and is grown on 1 million hectares of land, with a total production of 37 million tonnes. It does not flower under existing environmental conditions. Sugar cane is vegetatively propagated and the national breeding programmes is restricted to the adaptation and multiplication of exotic varieties. Random amplified polymorphic DNA (RAPD) markers were used to establish polymorphisms among various local sugar cane varieties. DNA from the varieties L-118, L-116, BL-4, BF-162, Col-44, Col-54, Triton and Puri was isolated and amplified by polymerase chain reaction using ten nucleotide primers. The amplification profiles of all the sugar cane varieties were compared and the polymorphisms detected. DNA was isolated from the embryogenic calli of sugar cane subjected to gamma irradiation at different doses (0, 0.5, 2.0, 4.0 and 6.0 krad) and salt stresses (NaCl: 0, 50, 100, 150 and 200 mM), and was amplified with random primers to detect the polymorphisms introduced by stress. The banana is another important vegetatively propagated crop in Pakistan. DNA isolation from micropropagated banana was optimized and RAPD analysis performed on several clones of the banana variety Williams. The level of genetic variability revealed from calli and vegetatively propagated sugar cane and banana by RAPD analysis is discussed. (author). 10 refs, 5 figs, 2 tabs

  16. Automated image analysis reveals the dynamic 3-dimensional organization of multi-ciliary arrays

    Directory of Open Access Journals (Sweden)

    Domenico F. Galati

    2016-01-01

    Full Text Available Multi-ciliated cells (MCCs use polarized fields of undulating cilia (ciliary array to produce fluid flow that is essential for many biological processes. Cilia are positioned by microtubule scaffolds called basal bodies (BBs that are arranged within a spatially complex 3-dimensional geometry (3D. Here, we develop a robust and automated computational image analysis routine to quantify 3D BB organization in the ciliate, Tetrahymena thermophila. Using this routine, we generate the first morphologically constrained 3D reconstructions of Tetrahymena cells and elucidate rules that govern the kinetics of MCC organization. We demonstrate the interplay between BB duplication and cell size expansion through the cell cycle. In mutant cells, we identify a potential BB surveillance mechanism that balances large gaps in BB spacing by increasing the frequency of closely spaced BBs in other regions of the cell. Finally, by taking advantage of a mutant predisposed to BB disorganization, we locate the spatial domains that are most prone to disorganization by environmental stimuli. Collectively, our analyses reveal the importance of quantitative image analysis to understand the principles that guide the 3D organization of MCCs.

  17. Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes.

    Science.gov (United States)

    Subirats, Jéssica; Sànchez-Melsió, Alexandre; Borrego, Carles M; Balcázar, José Luis; Simonet, Pascal

    2016-08-01

    A metagenomics approach was applied to explore the presence of antibiotic resistance genes (ARGs) in bacteriophages from hospital wastewater. Metagenomic analysis showed that most phage sequences affiliated to the order Caudovirales, comprising the tailed phage families Podoviridae, Siphoviridae and Myoviridae. Moreover, the relative abundance of ARGs in the phage DNA fraction (0.26%) was higher than in the bacterial DNA fraction (0.18%). These differences were particularly evident for genes encoding ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND) proteins, phosphotransferases, β-lactamases and plasmid-mediated quinolone resistance. Analysis of assembled contigs also revealed that blaOXA-10, blaOXA-58 and blaOXA-24 genes belonging to class D β-lactamases as well as a novel blaTEM (98.9% sequence similarity to the blaTEM-1 gene) belonging to class A β-lactamases were detected in a higher proportion in phage DNA. Although preliminary, these findings corroborate the role of bacteriophages as reservoirs of resistance genes and thus highlight the necessity to include them in future studies on the emergence and spread of antibiotic resistance in the environment. PMID:27312355

  18. Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions

    Science.gov (United States)

    Law, Yingyu; Kirkegaard, Rasmus Hansen; Cokro, Angel Anisa; Liu, Xianghui; Arumugam, Krithika; Xie, Chao; Stokholm-Bjerregaard, Mikkel; Drautz-Moses, Daniela I.; Nielsen, Per Halkjær; Wuertz, Stefan; Williams, Rohan B. H.

    2016-05-01

    Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

  19. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    Science.gov (United States)

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  20. Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications.

    Science.gov (United States)

    Halim, Adnan; Carlsson, Michael C; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L; Wandall, Hans H

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

  1. Bach Is the Father of Harmony: Revealed by a 1/f Fluctuation Analysis across Musical Genres.

    Directory of Open Access Journals (Sweden)

    Dan Wu

    Full Text Available Harmony is a fundamental attribute of music. Close connections exist between music and mathematics since both pursue harmony and unity. In music, the consonance of notes played simultaneously partly determines our perception of harmony; associates with aesthetic responses; and influences the emotion expression. The consonance could be considered as a window to understand and analyze harmony. Here for the first time we used a 1/f fluctuation analysis to investigate whether the consonance fluctuation structure in music with a wide range of composers and genres followed the scale free pattern that has been found for pitch, melody, rhythm, human body movements, brain activity, natural images and geographical features. We then used a network graph approach to investigate which composers were the most influential both within and across genres. Our results showed that patterns of consonance in music did follow scale-free characteristics, suggesting that this feature is a universally evolved one in both music and the living world. Furthermore, our network analysis revealed that Bach's harmony patterns were having the most influence on those used by other composers, followed closely by Mozart.

  2. Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions

    Science.gov (United States)

    Law, Yingyu; Kirkegaard, Rasmus Hansen; Cokro, Angel Anisa; Liu, Xianghui; Arumugam, Krithika; Xie, Chao; Stokholm-Bjerregaard, Mikkel; Drautz-Moses, Daniela I.; Nielsen, Per Halkjær; Wuertz, Stefan; Williams, Rohan B. H.

    2016-01-01

    Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions. PMID:27193869

  3. Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action

    Directory of Open Access Journals (Sweden)

    Faure Claudine

    2007-10-01

    Full Text Available Abstract Background: The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAα proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R. v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor. However, v-ErbA target genes involved in its transforming activity still remain to be identified. Results: By using Serial Analysis of Gene Expression (SAGE, we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription and unexpected (protein metabolism functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA. Conclusion: This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

  4. Time-series analysis reveals genetic responses to intensive management of razorback sucker (Xyrauchen texanus).

    Science.gov (United States)

    Dowling, Thomas E; Turner, Thomas F; Carson, Evan W; Saltzgiver, Melody J; Adams, Deborah; Kesner, Brian; Marsh, Paul C

    2014-03-01

    Time-series analysis is used widely in ecology to study complex phenomena and may have considerable potential to clarify relationships of genetic and demographic processes in natural and exploited populations. We explored the utility of this approach to evaluate population responses to management in razorback sucker, a long-lived and fecund, but declining freshwater fish species. A core population in Lake Mohave (Arizona-Nevada, USA) has experienced no natural recruitment for decades and is maintained by harvesting naturally produced larvae from the lake, rearing them in protective custody, and repatriating them at sizes less vulnerable to predation. Analyses of mtDNA and 15 microsatellites characterized for sequential larval cohorts collected over a 15-year time series revealed no changes in geographic structuring but indicated significant increase in mtDNA diversity for the entire population over time. Likewise, ratios of annual effective breeders to annual census size (N b /N a) increased significantly despite sevenfold reduction of N a. These results indicated that conservation actions diminished near-term extinction risk due to genetic factors and should now focus on increasing numbers of fish in Lake Mohave to ameliorate longer-term risks. More generally, time-series analysis permitted robust testing of trends in genetic diversity, despite low precision of some metrics. PMID:24665337

  5. Principal Component Analysis reveals correlation of cavities evolution and functional motions in proteins.

    Science.gov (United States)

    Desdouits, Nathan; Nilges, Michael; Blondel, Arnaud

    2015-02-01

    Protein conformation has been recognized as the key feature determining biological function, as it determines the position of the essential groups specifically interacting with substrates. Hence, the shape of the cavities or grooves at the protein surface appears to drive those functions. However, only a few studies describe the geometrical evolution of protein cavities during molecular dynamics simulations (MD), usually with a crude representation. To unveil the dynamics of cavity geometry evolution, we developed an approach combining cavity detection and Principal Component Analysis (PCA). This approach was applied to four systems subjected to MD (lysozyme, sperm whale myoglobin, Dengue envelope protein and EF-CaM complex). PCA on cavities allows us to perform efficient analysis and classification of the geometry diversity explored by a cavity. Additionally, it reveals correlations between the evolutions of the cavities and structures, and can even suggest how to modify the protein conformation to induce a given cavity geometry. It also helps to perform fast and consensual clustering of conformations according to cavity geometry. Finally, using this approach, we show that both carbon monoxide (CO) location and transfer among the different xenon sites of myoglobin are correlated with few cavity evolution modes of high amplitude. This correlation illustrates the link between ligand diffusion and the dynamic network of internal cavities.

  6. Bioinformatic analysis of the neprilysin (M13 family of peptidases reveals complex evolutionary and functional relationships

    Directory of Open Access Journals (Sweden)

    Pinney John W

    2008-01-01

    Full Text Available Abstract Background The neprilysin (M13 family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2, which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. Results The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates

  7. Functional and Complementary Phosphorylation State Attributes of Human Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) Isoforms Resolved by Free Flow Electrophoresis

    Science.gov (United States)

    Nissum, Mikkel; Shehab, Majida Abu; Sukop, Ute; Khosravi, Javad M.; Wildgruber, Robert; Eckerskorn, Christoph; Han, Victor K. M.; Gupta, Madhulika B.

    2009-01-01

    Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR. In this study a novel strategy was designed to determine a link between IGF-I affinity and the concomitant phosphorylation state characteristics of IGFBP-1 phosphoisoforms. Using free flow electrophoresis (FFE), multiple IGFBP-1 phosphoisoforms in amniotic fluid were resolved within pH 4.43–5.09. The binding of IGFBP-1 for IGF-I in each FFE fraction was determined with BIAcore biosensor analysis. The IGF-I affinity (K) for different IGFBP-1 isoforms ranged between 1.12e−08 and 4.59e−07. LC-MS/MS characterization revealed four phosphorylation sites, Ser(P)98, Ser(P)101, Ser(P)119, and Ser(P)169, of which Ser(P)98 was new. Although the IGF-I binding affinity for IGFBP-1 phosphoisoforms across the FFE fractions did not correlate with phosphopeptide intensities for Ser(P)101, Ser(P)98, and Ser(P)169 sites, a clear association was recorded with Ser(P)119. Our data demonstrate that phosphorylation at Ser119 plays a significant role in modulating affinity of IGFBP-1 for IGF-I. In addition, an altered profile of IGFBP-1 phosphoisoforms was revealed between FGR and healthy pregnancies that may result from potential site-specific phosphorylation. This study provides a strong basis for use of this novel approach in establishing the linkage between phosphorylation of IGFBP-1 and FGR. This overall strategy will also be broadly applicable to other phosphoproteins with clinical and functional significance. PMID:19193607

  8. Time series analysis of satellite data reveals continuous deforestation of New England since the 1980s

    Science.gov (United States)

    Olofsson, Pontus; Holden, Christopher E.; Bullock, Eric L.; Woodcock, Curtis E.

    2016-06-01

    Land cover and land change were monitored continuously between 1985 and 2011 at 30 m resolution across New England in the Northeastern United States in support of modeling the terrestrial carbon budget. It was found that the forest area has been decreasing throughout the study period in each state of the region since the 1980s. A total of 386 657 ± 98 137 ha (95% confidence interval) of forest has been converted to other land covers since 1985. Mainly driven by low density residential development, the deforestation accelerated in the mid-1990s until 2007 when it plateaued as a result of declining new residential construction and in turn, the financial crisis of 2007–08. The area of forest harvest, estimated at 226 519 ± 66 682 ha, was mapped separately and excluded from the deforestation estimate, while the area of forest expansion on non-forested lands was found to not be significantly different from zero. New England is often held as a principal example of a forest transition with historical widespread deforestation followed by recovery of forestlands as farming activities diminished, but the results of this study support the notion of a reversal of the forest transition as the region again is experiencing widespread deforestation. All available Landsat imagery acquired after 1985 for the study area were collected and used in the analysis. Areas of land cover and land change were estimated from a random sample of reference observations stratified by a twelve-class land change map encompassing the entire study area and period. The statistical analysis revealed that the net change in forest area and the associated modeled impact on the terrestrial carbon balance would have been considerably different if the results of the map were used without inferring the area of forest change by analysis of a reference sample.

  9. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    Science.gov (United States)

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Time series analysis of satellite data reveals continuous deforestation of New England since the 1980s

    Science.gov (United States)

    Olofsson, Pontus; Holden, Christopher E.; Bullock, Eric L.; Woodcock, Curtis E.

    2016-06-01

    Land cover and land change were monitored continuously between 1985 and 2011 at 30 m resolution across New England in the Northeastern United States in support of modeling the terrestrial carbon budget. It was found that the forest area has been decreasing throughout the study period in each state of the region since the 1980s. A total of 386 657 ± 98 137 ha (95% confidence interval) of forest has been converted to other land covers since 1985. Mainly driven by low density residential development, the deforestation accelerated in the mid-1990s until 2007 when it plateaued as a result of declining new residential construction and in turn, the financial crisis of 2007-08. The area of forest harvest, estimated at 226 519 ± 66 682 ha, was mapped separately and excluded from the deforestation estimate, while the area of forest expansion on non-forested lands was found to not be significantly different from zero. New England is often held as a principal example of a forest transition with historical widespread deforestation followed by recovery of forestlands as farming activities diminished, but the results of this study support the notion of a reversal of the forest transition as the region again is experiencing widespread deforestation. All available Landsat imagery acquired after 1985 for the study area were collected and used in the analysis. Areas of land cover and land change were estimated from a random sample of reference observations stratified by a twelve-class land change map encompassing the entire study area and period. The statistical analysis revealed that the net change in forest area and the associated modeled impact on the terrestrial carbon balance would have been considerably different if the results of the map were used without inferring the area of forest change by analysis of a reference sample.

  12. Genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes.

    Directory of Open Access Journals (Sweden)

    Peter Hevezi

    Full Text Available Efforts to unravel the mechanisms underlying taste sensation (gustation have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM procured fungiform (FG and circumvallate (CV taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology.

  13. Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

    Science.gov (United States)

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

    2016-05-10

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  14. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    The uptake of exogenous 32Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[32P]ATP, and suggests a possible reinterpretation of those experiments

  15. Negative Role of RIG-I Serine 8 Phosphorylation in the Regulatin of Interferon-beta Production

    Energy Technology Data Exchange (ETDEWEB)

    E Nistal-Villan; M Gack; G Martinez-Delgado; N Maharaj; K Inn; H Yang; R Wang; A Aggarwal; J Jung; A Garcia-Sastre

    2011-12-31

    RIG-I (retinoic acid-inducible gene I) and TRIM25 (tripartite motif protein 25) have emerged as key regulatory factors to induce interferon (IFN)-mediated innate immune responses to limit viral replication. Upon recognition of viral RNA, TRIM25 E3 ligase binds the first caspase recruitment domain (CARD) of RIG-I and subsequently induces lysine 172 ubiquitination of the second CARD of RIG-I, which is essential for the interaction with downstream MAVS/IPS-1/CARDIF/VISA and, thereby, IFN-beta mRNA production. Although ubiquitination has emerged as a major factor involved in RIG-I activation, the potential contribution of other post-translational modifications, such as phosphorylation, to the regulation of RIG-I activity has not been addressed. Here, we report the identification of serine 8 phosphorylation at the first CARD of RIG-I as a negative regulatory mechanism of RIG-I-mediated IFN-beta production. Immunoblot analysis with a phosphospecific antibody showed that RIG-I serine 8 phosphorylation steady-state levels were decreased upon stimulation of cells with IFN-beta or virus infection. Substitution of serine 8 in the CARD RIG-I functional domain with phosphomimetic aspartate or glutamate results in decreased TRIM25 binding, RIG-I ubiquitination, MAVS binding, and downstream signaling. Finally, sequence comparison reveals that only primate species carry serine 8, whereas other animal species carry an asparagine, indicating that serine 8 phosphorylation may represent a primate-specific regulation of RIG-I activation. Collectively, these data suggest that the phosphorylation of RIG-I serine 8 operates as a negative switch of RIG-I activation by suppressing TRIM25 interaction, further underscoring the importance of RIG-I and TRIM25 connection in type I IFN signal transduction.

  16. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard;

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  17. Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

    Science.gov (United States)

    Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

    2011-11-01

    Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive

  18. Comorbid Analysis of Genes Associated with Autism Spectrum Disorders Reveals Differential Evolutionary Constraints

    Science.gov (United States)

    David, Maude M.; Enard, David; Ozturk, Alp; Daniels, Jena; Jung, Jae-Yoon; Diaz-Beltran, Leticia; Wall, Dennis. P.

    2016-01-01

    The burden of comorbidity in Autism Spectrum Disorder (ASD) is substantial. The symptoms of autism overlap with many other human conditions, reflecting common molecular pathologies suggesting that cross-disorder analysis will help prioritize autism gene candidates. Genes in the intersection between autism and related conditions may represent nonspecific indicators of dysregulation while genes unique to autism may play a more causal role. Thorough literature review allowed us to extract 125 ICD-9 codes comorbid to ASD that we mapped to 30 specific human disorders. In the present work, we performed an automated extraction of genes associated with ASD and its comorbid disorders, and found 1031 genes involved in ASD, among which 262 are involved in ASD only, with the remaining 779 involved in ASD and at least one comorbid disorder. A pathway analysis revealed 13 pathways not involved in any other comorbid disorders and therefore unique to ASD, all associated with basal cellular functions. These pathways differ from the pathways associated with both ASD and its comorbid conditions, with the latter being more specific to neural function. To determine whether the sequence of these genes have been subjected to differential evolutionary constraints, we studied long term constraints by looking into Genomic Evolutionary Rate Profiling, and showed that genes involved in several comorbid disorders seem to have undergone more purifying selection than the genes involved in ASD only. This result was corroborated by a higher dN/dS ratio for genes unique to ASD as compare to those that are shared between ASD and its comorbid disorders. Short-term evolutionary constraints showed the same trend as the pN/pS ratio indicates that genes unique to ASD were under significantly less evolutionary constraint than the genes associated with all other disorders. PMID:27414027

  19. Analysis of the full genome of human group C rotaviruses reveals lineage diversification and reassortment.

    Science.gov (United States)

    Medici, Maria Cristina; Tummolo, Fabio; Martella, Vito; Arcangeletti, Maria Cristina; De Conto, Flora; Chezzi, Carlo; Fehér, Enikő; Marton, Szilvia; Calderaro, Adriana; Bányai, Krisztián

    2016-08-01

    Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

  20. RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

    Directory of Open Access Journals (Sweden)

    Nawar Naseer

    Full Text Available The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving > 100 genes against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers.

  1. RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

    Science.gov (United States)

    Naseer, Nawar; Shapiro, Joshua A; Chander, Monica

    2014-01-01

    The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving > 100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers. PMID:25162599

  2. Proteomic analysis of Ketogulonicigenium vulgare under glutathione reveals high demand for thiamin transport and antioxidant protection.

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    Qian Ma

    Full Text Available Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4-6 folds elongated, and formed intracytoplasmic membranes (ICM. To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare.

  3. Analysis of gene expression during parabolic flights reveals distinct early gravity responses in Arabidopsis roots.

    Science.gov (United States)

    Aubry-Hivet, D; Nziengui, H; Rapp, K; Oliveira, O; Paponov, I A; Li, Y; Hauslage, J; Vagt, N; Braun, M; Ditengou, F A; Dovzhenko, A; Palme, K

    2014-01-01

    Plant roots are among most intensively studied biological systems in gravity research. Altered gravity induces asymmetric cell growth leading to root bending. Differential distribution of the phytohormone auxin underlies root responses to gravity, being coordinated by auxin efflux transporters from the PIN family. The objective of this study was to compare early transcriptomic changes in roots of Arabidopsis thaliana wild type, and pin2 and pin3 mutants under parabolic flight conditions and to correlate these changes to auxin distribution. Parabolic flights allow comparison of transient 1-g, hypergravity and microgravity effects in living organisms in parallel. We found common and mutation-related genes differentially expressed in response to transient microgravity phases. Gene ontology analysis of common genes revealed lipid metabolism, response to stress factors and light categories as primarily involved in response to transient microgravity phases, suggesting that fundamental reorganisation of metabolic pathways functions upstream of a further signal mediating hormonal network. Gene expression changes in roots lacking the columella-located PIN3 were stronger than in those deprived of the epidermis and cortex cell-specific PIN2. Moreover, repetitive exposure to microgravity/hypergravity and gravity/hypergravity flight phases induced an up-regulation of auxin responsive genes in wild type and pin2 roots, but not in pin3 roots, suggesting a critical function of PIN3 in mediating auxin fluxes in response to transient microgravity phases. Our study provides important insights towards understanding signal transduction processes in transient microgravity conditions by combining for the first time the parabolic flight platform with the transcriptome analysis of different genetic mutants in the model plant, Arabidopsis.

  4. Comparative Genomic Analysis of Clinical and Environmental Vibrio Vulnificus Isolates Revealed Biotype 3 Evolutionary Relationships

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    Yael eKotton

    2015-01-01

    Full Text Available In 1996 a common-source outbreak of severe soft tissue and bloodstream infections erupted among Israeli fish farmers and fish consumers due to changes in fish marketing policies. The causative pathogen was a new strain of Vibrio vulnificus, named biotype 3, which displayed a unique biochemical and genotypic profile. Initial observations suggested that the pathogen erupted as a result of genetic recombination between two distinct populations. We applied a whole genome shotgun sequencing approach using several V. vulnificus strains from Israel in order to study the pan genome of V. vulnificus and determine the phylogenetic relationship of biotype 3 with existing populations. The core genome of V. vulnificus based on 16 draft and complete genomes consisted of 3068 genes, representing between 59% and 78% of the whole genome of 16 strains. The accessory genome varied in size from 781 kbp to 2044 kbp. Phylogenetic analysis based on whole, core, and accessory genomes displayed similar clustering patterns with two main clusters, clinical (C and environmental (E, all biotype 3 strains formed a distinct group within the E cluster. Annotation of accessory genomic regions found in biotype 3 strains and absent from the core genome yielded 1732 genes, of which the vast majority encoded hypothetical proteins, phage-related proteins, and mobile element proteins. A total of 1916 proteins (including 713 hypothetical proteins were present in all human pathogenic strains (both biotype 3 and non-biotype 3 and absent from the environmental strains. Clustering analysis of the non-hypothetical proteins revealed 148 protein clusters shared by all human pathogenic strains; these included transcriptional regulators, arylsulfatases, methyl-accepting chemotaxis proteins, acetyltransferases, GGDEF family proteins, transposases, type IV secretory system (T4SS proteins, and integrases. Our study showed that V. vulnificus biotype 3 evolved from environmental populations and

  5. Interspecies insertion polymorphism analysis reveals recent activity of transposable elements in extant coelacanths.

    Science.gov (United States)

    Naville, Magali; Chalopin, Domitille; Volff, Jean-Nicolas

    2014-01-01

    Coelacanths are lobe-finned fish represented by two extant species, Latimeria chalumnae in South Africa and Comoros and L. menadoensis in Indonesia. Due to their intermediate phylogenetic position between ray-finned fish and tetrapods in the vertebrate lineage, they are of great interest from an evolutionary point of view. In addition, extant specimens look similar to 300 million-year-old fossils; because of their apparent slowly evolving morphology, coelacanths have been often described as « living fossils ». As an underlying cause of such a morphological stasis, several authors have proposed a slow evolution of the coelacanth genome. Accordingly, sequencing of the L. chalumnae genome has revealed a globally low substitution rate for protein-coding regions compared to other vertebrates. However, genome and gene evolution can also be influenced by transposable elements, which form a major and dynamic part of vertebrate genomes through their ability to move, duplicate and recombine. In this work, we have searched for evidence of transposition activity in coelacanth genomes through the comparative analysis of orthologous genomic regions from both Latimeria species. Comparison of 5.7 Mb (0.2%) of the L. chalumnae genome with orthologous Bacterial Artificial Chromosome clones from L. menadoensis allowed the identification of 27 species-specific transposable element insertions, with a strong relative contribution of CR1 non-LTR retrotransposons. Species-specific homologous recombination between the long terminal repeats of a new coelacanth endogenous retrovirus was also detected. Our analysis suggests that transposon activity is responsible for at least 0.6% of genome divergence between both Latimeria species. Taken together, this study demonstrates that coelacanth genomes are not evolutionary inert: they contain recently active transposable elements, which have significantly contributed to post-speciation genome divergence in Latimeria.

  6. Interspecies insertion polymorphism analysis reveals recent activity of transposable elements in extant coelacanths.

    Directory of Open Access Journals (Sweden)

    Magali Naville

    Full Text Available Coelacanths are lobe-finned fish represented by two extant species, Latimeria chalumnae in South Africa and Comoros and L. menadoensis in Indonesia. Due to their intermediate phylogenetic position between ray-finned fish and tetrapods in the vertebrate lineage, they are of great interest from an evolutionary point of view. In addition, extant specimens look similar to 300 million-year-old fossils; because of their apparent slowly evolving morphology, coelacanths have been often described as « living fossils ». As an underlying cause of such a morphological stasis, several authors have proposed a slow evolution of the coelacanth genome. Accordingly, sequencing of the L. chalumnae genome has revealed a globally low substitution rate for protein-coding regions compared to other vertebrates. However, genome and gene evolution can also be influenced by transposable elements, which form a major and dynamic part of vertebrate genomes through their ability to move, duplicate and recombine. In this work, we have searched for evidence of transposition activity in coelacanth genomes through the comparative analysis of orthologous genomic regions from both Latimeria species. Comparison of 5.7 Mb (0.2% of the L. chalumnae genome with orthologous Bacterial Artificial Chromosome clones from L. menadoensis allowed the identification of 27 species-specific transposable element insertions, with a strong relative contribution of CR1 non-LTR retrotransposons. Species-specific homologous recombination between the long terminal repeats of a new coelacanth endogenous retrovirus was also detected. Our analysis suggests that transposon activity is responsible for at least 0.6% of genome divergence between both Latimeria species. Taken together, this study demonstrates that coelacanth genomes are not evolutionary inert: they contain recently active transposable elements, which have significantly contributed to post-speciation genome divergence in Latimeria.

  7. Single cell analysis reveals gametic and tissue-specific instability of the SCA1 CAG repeat

    Energy Technology Data Exchange (ETDEWEB)

    Chong, S.S.; McCall, A.E.; Cota, J. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Spinocerebellar ataxia type 1 is an autosomal dominant neurodegenerative disease caused by expansion of a CAG trinucleotide repeat within the SCA1 gene on chromosome 6p22-23. We performed a comparative analysis of the SCA1 CAG repeat from blood and sperm of an affected male. Genomic amplification revealed a broader smear of the SCA1 allele product from sperm compared to that from peripheral blood leukocytes (PBL). To resolve this observed difference, we analyzed single sperm directly and demonstrate that the SCA1 allele in PBL is also heterogeneous, although the range of variability in allele sizes is much less than that observed in sperm. Limited genome analysis was also performed on PBL DNA from an unaffected individual with an upper normal allele of 36 repeats in parallel with an affected individual with an expanded allele of 40 repeats. The 36 repeat normal allele, which contains a CAT interruption, was completely stable compared to the uninterrupted repeat of the SCA1 allele, demonstrating a direct correlation between absence of a CAT interruption and somatic instability of the repeat. We also analyzed the size of the CAG repeat in tissues derived from various brain regions from a patient with juvenile-onset disease to determine if the size of the expansion correlated with the site of neuropathology. The results clearly show tissue-specific differences in mosaicism of repeat length. More importantly, the pattern of tissue-specific differences in repeat-length mosaicism in SCA1 within the brain parallels those seen in Huntington disease. In both disorders the expanded alleles are smaller in cerebellar tissue. These results suggest that the observed tissue-specific differences in instability of the SCA1 CAG repeat, either within the brain or between blood and sperm, are a function of the intracellular milieu or the intrinsic replicative potential of the various celltypes.

  8. Molecular systematic analysis reveals cryptic tertiary diversification of a widespread tropical rain forest tree.

    Science.gov (United States)

    Dick, Christopher W; Abdul-Salim, Kobinah; Bermingham, Eldredge

    2003-12-01

    The broad geographic range of many Neotropical rain forest tree species implies excellent dispersal abilities or range establishment that preceded the formation of current dispersal barriers. In order to initiate historical analyses of such widespread Neotropical trees, we sequenced the nuclear ribosomal spacer (ITS) region of Symphonia globulifera L. f. (Clusiaceae) from populations spanning the Neotropics and western Africa. This rain forest tree has left unmistakable Miocene fossils in Mesoamerica (15.5-18.2 Ma) and in South America ( approximately 15 Ma). Although marine dispersal of S. globulifera is considered improbable, our study establishes three marine dispersal events leading to the colonization of Mesoamerica, the Amazon basin, and the West Indies, thus supporting the paleontological data. Our phylogeographic analysis revealed the spatial extent of the three Neotropical S. globulifera clades, which represent trans-Andes (Mesoamerica+west Ecuador), cis-Andes (Amazonia+Guiana), and the West Indies. Strong phylogeographic structure found among trans-Andean populations of S. globulifera stands in contrast to an absence of ITS nucleotide variation across the Amazon basin and indicates profound regional differences in the demographic history of this rain forest tree. Drawing from these results, we provide a historical biogeographic hypothesis to account for differences in the patterns of beta diversity within Mesoamerican and Amazonian forests. PMID:14737707

  9. GC--MS analysis reveals production of 2--Phenylethanol from Aspergillus niger endophytic in rose.

    Science.gov (United States)

    Wani, Masood Ahmed; Sanjana, Kaul; Kumar, Dhar Manoj; Lal, Dhar Kanahya

    2010-02-01

    Endophytes include all organisms that during a variable period of their life, colonize the living internal tissues of their hosts without causing detectable symptoms. Several fungal endophytes have been isolated from a variety of plant species which have proved themselves as a rich source of secondary metabolites. The reported natural products from endophytes include antibiotics, immunosuppresants, anticancer compounds, antioxidant agents, etc. For the first time Rosa damacaena (rose) has been explored for its endophytes. The rose oil industry is the major identified deligence for its application in perfumery, flavouring, ointments, and pharmaceuticals including various herbal products. During the present investigation fungal endophytes were isolated from Rosa damacaena. A total of fifty four isolates were isolated out of which sixteen isolates were screened for the production of secondary metabolites. GCMS analysis reveals the production of 2-phenylethanol by one of the isolates JUBT 3M which was identified as Aspergillus niger. This is the first report of production of 2-phenylethanol from endophytic A. niger. 2-phenylethanol is an important constituent of rose oil constituting about 4.06% of rose oil. Presence of 2-phenylethanol indicates that the endophyte of rose may duplicate the biosynthesis of phenyl propanoids by rose plant. Besides this, the other commercial applications of phenylethanol include its use in antiseptics, disinfectants, anti-microbials and preservative in pharmaceuticals. PMID:20082377

  10. Mosaic Analysis with Double Markers Reveals Cell-Type-Specific Paternal Growth Dominance

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    Simon Hippenmeyer

    2013-03-01

    Full Text Available Genomic imprinting leads to preferred expression of either the maternal or paternal alleles of a subset of genes. Imprinting is essential for mammalian development, and its deregulation causes many diseases. However, the functional relevance of imprinting at the cellular level is poorly understood for most imprinted genes. We used mosaic analysis with double markers (MADM in mice to create uniparental disomies (UPDs and to visualize imprinting effects with single-cell resolution. Although chromosome 12 UPD did not produce detectable phenotypes, chromosome 7 UPD caused highly significant paternal growth dominance in the liver and lung, but not in the brain or heart. A single gene on chromosome 7, encoding the secreted insulin-like growth factor 2 (IGF2, accounts for most of the paternal dominance effect. Mosaic analyses implied additional imprinted loci on chromosome 7 acting cell autonomously to transmit the IGF2 signal. Our study reveals chromosome- and cell-type specificity of genomic imprinting effects.

  11. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    Science.gov (United States)

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  12. Gamma-Tocotrienol Modulated Gene Expression in Senescent Human Diploid Fibroblasts as Revealed by Microarray Analysis

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    Suzana Makpol

    2013-01-01

    Full Text Available The effect of γ-tocotrienol, a vitamin E isomer, in modulating gene expression in cellular aging of human diploid fibroblasts was studied. Senescent cells at passage 30 were incubated with 70 μM of γ-tocotrienol for 24 h. Gene expression patterns were evaluated using Sentrix HumanRef-8 Expression BeadChip from Illumina, analysed using GeneSpring GX10 software, and validated using quantitative RT-PCR. A total of 100 genes were differentially expressed (P<0.001 by at least 1.5 fold in response to γ-tocotrienol treatment. Amongst the genes were IRAK3, SelS, HSPA5, HERPUD1, DNAJB9, SEPR1, C18orf55, ARF4, RINT1, NXT1, CADPS2, COG6, and GLRX5. Significant gene list was further analysed by Gene Set Enrichment Analysis (GSEA, and the Normalized Enrichment Score (NES showed that biological processes such as inflammation, protein transport, apoptosis, and cell redox homeostasis were modulated in senescent fibroblasts treated with γ-tocotrienol. These findings revealed that γ-tocotrienol may prevent cellular aging of human diploid fibroblasts by modulating gene expression.

  13. Latent physiological factors of complex human diseases revealed by independent component analysis of clinarrays

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    Chen David P

    2010-10-01

    Full Text Available Abstract Background Diagnosis and treatment of patients in the clinical setting is often driven by known symptomatic factors that distinguish one particular condition from another. Treatment based on noticeable symptoms, however, is limited to the types of clinical biomarkers collected, and is prone to overlooking dysfunctions in physiological factors not easily evident to medical practitioners. We used a vector-based representation of patient clinical biomarkers, or clinarrays, to search for latent physiological factors that underlie human diseases directly from clinical laboratory data. Knowledge of these factors could be used to improve assessment of disease severity and help to refine strategies for diagnosis and monitoring disease progression. Results Applying Independent Component Analysis on clinarrays built from patient laboratory measurements revealed both known and novel concomitant physiological factors for asthma, types 1 and 2 diabetes, cystic fibrosis, and Duchenne muscular dystrophy. Serum sodium was found to be the most significant factor for both type 1 and type 2 diabetes, and was also significant in asthma. TSH3, a measure of thyroid function, and blood urea nitrogen, indicative of kidney function, were factors unique to type 1 diabetes respective to type 2 diabetes. Platelet count was significant across all the diseases analyzed. Conclusions The results demonstrate that large-scale analyses of clinical biomarkers using unsupervised methods can offer novel insights into the pathophysiological basis of human disease, and suggest novel clinical utility of established laboratory measurements.

  14. Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles* #

    Science.gov (United States)

    Li, Ping; Li, Xuan; Gu, Qing; Lou, Xiu-yu; Zhang, Xiao-mei; Song, Da-feng; Zhang, Chen

    2016-01-01

    Objective: In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. Methods: The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). Results: We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. Conclusions: Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate. PMID:27487802

  15. Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

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    Roberto Rosini

    Full Text Available The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

  16. Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

    Science.gov (United States)

    Rosini, Roberto; Campisi, Edmondo; De Chiara, Matteo; Tettelin, Hervé; Rinaudo, Daniela; Toniolo, Chiara; Metruccio, Matteo; Guidotti, Silvia; Sørensen, Uffe B Skov; Kilian, Mogens; Ramirez, Mario; Janulczyk, Robert; Donati, Claudio; Grandi, Guido; Margarit, Immaculada

    2015-01-01

    The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.

  17. Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species

    Directory of Open Access Journals (Sweden)

    Hieu X. Cao

    2015-07-01

    Full Text Available In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea’s leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps versus the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g. proteases, phosphatases, lipases, mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-versus-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome.

  18. Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species.

    Science.gov (United States)

    Cao, Hieu X; Schmutzer, Thomas; Scholz, Uwe; Pecinka, Ales; Schubert, Ingo; Vu, Giang T H

    2015-01-01

    In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome. PMID:26236284

  19. RNA sequencing analysis of human podocytes reveals glucocorticoid regulated gene networks targeting non-immune pathways

    Science.gov (United States)

    Jiang, Lulu; Hindmarch, Charles C. T.; Rogers, Mark; Campbell, Colin; Waterfall, Christy; Coghill, Jane; Mathieson, Peter W.; Welsh, Gavin I.

    2016-01-01

    Glucocorticoids are steroids that reduce inflammation and are used as immunosuppressive drugs for many diseases. They are also the mainstay for the treatment of minimal change nephropathy (MCN), which is characterised by an absence of inflammation. Their mechanisms of action remain elusive. Evidence suggests that immunomodulatory drugs can directly act on glomerular epithelial cells or ‘podocytes’, the cell type which is the main target of injury in MCN. To understand the nature of glucocorticoid effects on non-immune cell functions, we generated RNA sequencing data from human podocyte cell lines and identified the genes that are significantly regulated in dexamethasone-treated podocytes compared to vehicle-treated cells. The upregulated genes are of functional relevance to cytoskeleton-related processes, whereas the downregulated genes mostly encode pro-inflammatory cytokines and growth factors. We observed a tendency for dexamethasone-upregulated genes to be downregulated in MCN patients. Integrative analysis revealed gene networks composed of critical signaling pathways that are likely targeted by dexamethasone in podocytes. PMID:27774996

  20. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts.

    Science.gov (United States)

    Turowski, Tomasz W; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-07-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5' peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential "housekeeping" roles. Many tRNA genes were found to generate long, 3'-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3'-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5'-exonuclease Rat1. PMID:27206856

  1. Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

    Science.gov (United States)

    Ginglinger, Jean-François; Boachon, Benoit; Höfer, René; Paetz, Christian; Köllner, Tobias G; Miesch, Laurence; Lugan, Raphael; Baltenweck, Raymonde; Mutterer, Jérôme; Ullmann, Pascaline; Beran, Franziska; Claudel, Patricia; Verstappen, Francel; Fischer, Marc J C; Karst, Francis; Bouwmeester, Harro; Miesch, Michel; Schneider, Bernd; Gershenzon, Jonathan; Ehlting, Jürgen; Werck-Reichhart, Danièle

    2013-11-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

  2. Solutions to Peto's paradox revealed by mathematical modelling and cross-species cancer gene analysis.

    Science.gov (United States)

    Caulin, Aleah F; Graham, Trevor A; Wang, Li-San; Maley, Carlo C

    2015-07-19

    Whales have 1000-fold more cells than humans and mice have 1000-fold fewer; however, cancer risk across species does not increase with the number of somatic cells and the lifespan of the organism. This observation is known as Peto's paradox. How much would evolution have to change the parameters of somatic evolution in order to equalize the cancer risk between species that differ by orders of magnitude in size? Analysis of previously published models of colorectal cancer suggests that a two- to three-fold decrease in the mutation rate or stem cell division rate is enough to reduce a whale's cancer risk to that of a human. Similarly, the addition of one to two required tumour-suppressor gene mutations would also be sufficient. We surveyed mammalian genomes and did not find a positive correlation of tumour-suppressor genes with increasing body mass and longevity. However, we found evidence of the amplification of TP53 in elephants, MAL in horses and FBXO31 in microbats, which might explain Peto's paradox in those species. Exploring parameters that evolution may have fine-tuned in large, long-lived organisms will help guide future experiments to reveal the underlying biology responsible for Peto's paradox and guide cancer prevention in humans. PMID:26056366

  3. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells.

    Science.gov (United States)

    Kim, Daesik; Kim, Jungeun; Hur, Junho K; Been, Kyung Wook; Yoon, Sun-Heui; Kim, Jin-Soo

    2016-08-01

    Programmable clustered regularly interspaced short palindromic repeats (CRISPR) Cpf1 endonucleases are single-RNA-guided (crRNA) enzymes that recognize thymidine-rich protospacer-adjacent motif (PAM) sequences and produce cohesive double-stranded breaks (DSBs). Genome editing with CRISPR-Cpf1 endonucleases could provide an alternative to CRISPR-Cas9 endonucleases, but the determinants of targeting specificity are not well understood. Using mismatched crRNAs we found that Cpf1 could tolerate single or double mismatches in the 3' PAM-distal region, but not in the 5' PAM-proximal region. Genome-wide analysis of cleavage sites in vitro for eight Cpf1 nucleases using Digenome-seq revealed that there were 6 (LbCpf1) and 12 (AsCpf1) cleavage sites per crRNA in the human genome, fewer than are present for Cas9 nucleases (>90). Most Cpf1 off-target cleavage sites did not produce mutations in cells. We found mismatches in either the 3' PAM-distal region or in the PAM sequence of 12 off-target sites that were validated in vivo. Off-target effects were completely abrogated by using preassembled, recombinant Cpf1 ribonucleoproteins.

  4. Highly distinct chromosomal structures in cowpea (Vigna unguiculata), as revealed by molecular cytogenetic analysis.

    Science.gov (United States)

    Iwata-Otsubo, Aiko; Lin, Jer-Young; Gill, Navdeep; Jackson, Scott A

    2016-05-01

    Cowpea (Vigna unguiculata (L.) Walp) is an important legume, particularly in developing countries. However, little is known about its genome or chromosome structure. We used molecular cytogenetics to characterize the structure of pachytene chromosomes to advance our knowledge of chromosome and genome organization of cowpea. Our data showed that cowpea has highly distinct chromosomal structures that are cytologically visible as brightly DAPI-stained heterochromatic regions. Analysis of the repetitive fraction of the cowpea genome present at centromeric and pericentromeric regions confirmed that two retrotransposons are major components of pericentromeric regions and that a 455-bp tandem repeat is found at seven out of 11 centromere pairs in cowpea. These repeats likely evolved after the divergence of cowpea from common bean and form chromosomal structure unique to cowpea. The integration of cowpea genetic and physical chromosome maps reveals potential regions of suppressed recombination due to condensed heterochromatin and a lack of pairing in a few chromosomal termini. This study provides fundamental knowledge on cowpea chromosome structure and molecular cytogenetics tools for further chromosome studies. PMID:26758200

  5. Temporal proteomic analysis reveals continuous impairment of intestinal development in neonatal piglets with intrauterine growth restriction.

    Science.gov (United States)

    Wang, Xiaoqiu; Wu, Weizong; Lin, Gang; Li, Defa; Wu, Guoyao; Wang, Junjun

    2010-02-01

    Efficiency of nutrient utilization is reduced in neonates with intrauterine growth restriction (IUGR) compared with those with a normal birth weight (NBW). However, the underlying mechanisms are largely unknown. In this study, we applied temporal proteomic approach, coupled with histological and biochemical analyses, to study dynamic changes of the proteome in the small intestinal mucosa of IUGR piglets during the nursing period (Days 1, 7 and 21). We identified 56 differentially expressed protein spots between IUGR and NBW piglets. These proteins participate in key biological processes, including (1) absorption, digestion and transport of nutrients; (2) cell structure and motility; (3) glucose and energy metabolism; (4) lipid metabolism; (5) amino acid metabolism; (6) mineral and vitamin metabolism; (7) cellular redox homeostasis; (8) stress response; and (9) apoptosis. The results of our temporal proteomics analysis reveal continuous impairment of intestinal development in neonatal piglets with IUGR. The findings have important implications for understanding metabolic defects in the small intestine of IUGR neonates and are expected to provide new strategies to improve their survival and growth.

  6. Retrospective stable isotope analysis reveals ecosystem responses to river regulation over the last century.

    Science.gov (United States)

    Turner, Thomas F; Krabbenhoft, Trevor I; Collyer, Michael L; Krabbenhoft, Corey A; Edwards, Melanie S; Sharp, Zachary D

    2015-12-01

    Disruption of natural flow regimes, nutrient pollution, and other consequences of human population growth and development have impacted most major rivers of the world. Alarming losses of aquatic biodiversity coincide with human-caused river alteration, but effects of biotic homogenization on aquatic ecosystem processes are not as well documented. This is because unaltered systems for comparison are scarce, and some ecosystem-wide effects may take decades to manifest. We evaluated aquatic ecosystem responses to extensive river- floodplain engineering and nutrient addition in the Rio Grande of southwestern North America as revealed by changes in trophic structure of, and resource availability to, the fish community. Stable Isotope Analysis (SIA) was conducted on museum-preserved fishes collected over a 70-year period of intensive river management and exponential human population growth. Trophic complexity and resource heterogeneity for fish consumers (measured as "isotopic niche breadth") decreased following sediment deprivation and channelization, and these effects persist into the present. Increased nutrient inputs led to δ15N enrichment in the entire fish community at all affected sites, and a shift to autochthonous sources of carbon at the most proximal site downstream of wastewater release, probably via bottom-up transfer. Overall, retrospective SIA of apex consumers suggests radical change and functional impairment of a floodplain river ecosystem already marked by significant biodiversity loss.

  7. The complement system of elasmobranches revealed by liver transcriptome analysis of a hammerhead shark, Sphyrna zygaena.

    Science.gov (United States)

    Goshima, Masayuki; Sekiguchi, Reo; Matsushita, Misao; Nonaka, Masaru

    2016-08-01

    Comprehensive studies of the complement genes in basal vertebrates have revealed that cyclostomes have apparently primitive complement systems whereas bony fish have well-developed complement systems comparable to those of mammals. Here we have performed liver transcriptome analysis of a hammerhead shark, Sphyrna zygaeana, to elucidate the early history of vertebrate complement evolution. Identified genes were; one C1qB, one C1r, one C1s, one MASP-1/-3, one MASP-2, two factor B/C2, one C3, three C4, one C5, one C6, one C7, one C8A, three C8B, one C8G, one C9, two factor I and one S protein. No MBL, ficolin, C1qA or C1qC were found. These results indicate that the lectin, classical, alternative and lytic pathways were established in the common ancestor of jawed vertebrates. In addition to the absence of MBL and ficolin, the MASP transcripts lacked the serine protease domain, suggesting that the lectin pathway was lost in the hammerhead shark lineage. PMID:26987526

  8. Genome-Wide Analysis Revealed the Complex Regulatory Network of Brassinosteroid Effects in Photomorphogenesis

    Institute of Scientific and Technical Information of China (English)

    Li Song; Xiao-Yi Zhou; Li Li; Liang-Jiao Xue; Xi Yang; Hong-Wei Xue

    2009-01-01

    Light and brassinosteroids (BRs) have been proved to be crucial in regulating plant growth and development;however,the mechanism of how they synergistically function is still largely unknown.To explore the underlying mechanisms in photomorphogenesis,genome-wide analyses were carried out through examining the gene expressions of the dark-grown WT or BR biosynthesis-defective mutant det2 seedlings in the presence of light stimuli or exogenous Brassinolide (BL).Results showed that BR deficiency stimulates,while BL treatment suppresses,the expressions of lightresponsive genes and photomorphogenesis,confirming the negative effects of BR in photomorphogenesis.This is consistent with the specific effects of BR on the expression of genes involved in cell wall modification,cellular metabolism and energy utilization during dark-light transition.Further analysis revealed that hormone biosynthesis and signaling-related genes,especially those of auxin,were altered under BL treatment or light stimuli,indicating that BR may modulate photomorphogenesis through synergetic regulation with other hormones.Additionally,suppressed ubiquitin-cycle pathway during light-dark transition hinted the presence of a complicated network among light,hormone,and protein degradation.The study provides the direct evidence of BR effects in photomorphogenesis and identified the genes involved in BR and light signaling pathway,which will help to elucidate the molecular mechanism of plant photomorphogenesis.

  9. Modular kinetic analysis of the adenine nucleotide translocator-mediated effects of palmitoyl-CoA on the oxidative phosphorylation in isolated rat liver mitochondria

    NARCIS (Netherlands)

    Ciapaite, J; Van Eikenhorst, G; Bakker, SJL; Diamant, M; Heine, RJ; Wagner, MJ; Westerhoff, HV; Krab, K

    2005-01-01

    To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Delta psi) and the fraction of matri

  10. Characterization and functional analysis of eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L.

    Science.gov (United States)

    Renu, Indu Kumari; Haque, Inamul; Kumar, Manish; Poddar, Raju; Bandopadhyay, Rajib; Rai, Amit; Mukhopadhyay, Kunal

    2014-03-01

    Eugenol-O-methyltransferase (EOMT) catalyzes the conversion of eugenol to methyleugenol in one of the final steps of phenylpropanoid pathway. There are no comprehensive reports on comparative EOMT gene expression and developmental stage specific accumulation of phenylpropenes in Ocimum tenuiflorum. Seven chemotypes, rich in eugenol and methyleugenol, were selected by assessment of volatile metabolites through multivariate data analysis. Isoeugenol accumulated in higher levels during juvenile stage (36.86 ng g(-1)), but reduced sharply during preflowering (8.04 ng g(-1)), flowering (2.29 ng g(-1)) and postflowering stages (0.17 ng g(-1)), whereas methyleugenol content gradually increased from juvenile (12.25 ng g(-1)) up to preflowering (16.35 ng g(-1)) and then decreased at flowering (7.13 ng g(-1)) and post flowering (5.95 ng g(-1)) from fresh tissue. Extreme variations of free intracellular and alkali hydrolysable cell wall released phenylpropanoid compounds were observed at different developmental stages. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90 bp intron. The translated proteins had eight catalytic domains, the major two being dimerisation superfamily and methyltransferase_2 superfamily. A validated 3D structure of EOMT protein was also determined. The chemotype Ot7 had a reduced reading frame that lacked both dimerisation domains and one of the two protein-kinase-phosphorylation sites; this was also reflected in reduced accumulation of methyleugenol compared to other chemotypes. EOMT transcripts showed enhanced expression in juvenile stage that increased further during preflowering but decreased at flowering and further at postflowering. The expression patterns may possibly be compared and correlated to the amounts of eugenol/isoeugenol and methyleugenol in different developmental stages of all chemotypes.

  11. Correlation-Based Network Analysis of Metabolite and Enzyme Profiles Reveals a Role of Citrate Biosynthesis in Modulating N and C Metabolism in Zea mays.

    Science.gov (United States)

    Toubiana, David; Xue, Wentao; Zhang, Nengyi; Kremling, Karl; Gur, Amit; Pilosof, Shai; Gibon, Yves; Stitt, Mark; Buckler, Edward S; Fernie, Alisdair R; Fait, Aaron

    2016-01-01

    To investigate the natural variability of leaf metabolism and enzymatic activity in a maize inbred population, statistical and network analyses were employed on metabolite and enzyme profiles. The test of coefficient of variation showed that sugars and amino acids displayed opposite trends in their variance within the population, consistently with their related enzymes. The overall higher CV values for metabolites as compared to the tested enzymes are indicative for their greater phenotypic plasticity. H(2) tests revealed galactinol (1) and asparagine (0.91) as the highest scorers among metabolites and nitrate reductase (0.73), NAD-glutamate dehydrogenase (0.52), and phosphoglucomutase (0.51) among enzymes. The overall low H(2) scores for metabolites and enzymes are suggestive for a great environmental impact or gene-environment interaction. Correlation-based network generation followed by community detection analysis, partitioned the network into three main communities and one dyad, (i) reflecting the different levels of phenotypic plasticity of the two molecular classes as observed for the CV values and (ii) highlighting the concerted changes between classes of chemically related metabolites. Community 1 is composed mainly of enzymes and specialized metabolites, community 2' is enriched in N-containing compounds and phosphorylated-intermediates. The third community contains mainly organic acids and sugars. Cross-community linkages are supported by aspartate, by the photorespiration amino acids glycine and serine, by the metabolically related GABA and putrescine, and by citrate. The latter displayed the strongest node-betweenness value (185.25) of all nodes highlighting its fundamental structural role in the connectivity of the network by linking between different communities and to the also strongly connected enzyme aldolase. PMID:27462343

  12. The Immersive Virtual Reality Experience: A Typology of Users Revealed Through Multiple Correspondence Analysis Combined with Cluster Analysis Technique.

    Science.gov (United States)

    Rosa, Pedro J; Morais, Diogo; Gamito, Pedro; Oliveira, Jorge; Saraiva, Tomaz

    2016-03-01

    Immersive virtual reality is thought to be advantageous by leading to higher levels of presence. However, and despite users getting actively involved in immersive three-dimensional virtual environments that incorporate sound and motion, there are individual factors, such as age, video game knowledge, and the predisposition to immersion, that may be associated with the quality of virtual reality experience. Moreover, one particular concern for users engaged in immersive virtual reality environments (VREs) is the possibility of side effects, such as cybersickness. The literature suggests that at least 60% of virtual reality users report having felt symptoms of cybersickness, which reduces the quality of the virtual reality experience. The aim of this study was thus to profile the right user to be involved in a VRE through head-mounted display. To examine which user characteristics are associated with the most effective virtual reality experience (lower cybersickness), a multiple correspondence analysis combined with cluster analysis technique was performed. Results revealed three distinct profiles, showing that the PC gamer profile is more associated with higher levels of virtual reality effectiveness, that is, higher predisposition to be immersed and reduced cybersickness symptoms in the VRE than console gamer and nongamer. These findings can be a useful orientation in clinical practice and future research as they help identify which users are more predisposed to benefit from immersive VREs. PMID:26985781

  13. The Immersive Virtual Reality Experience: A Typology of Users Revealed Through Multiple Correspondence Analysis Combined with Cluster Analysis Technique.

    Science.gov (United States)

    Rosa, Pedro J; Morais, Diogo; Gamito, Pedro; Oliveira, Jorge; Saraiva, Tomaz

    2016-03-01

    Immersive virtual reality is thought to be advantageous by leading to higher levels of presence. However, and despite users getting actively involved in immersive three-dimensional virtual environments that incorporate sound and motion, there are individual factors, such as age, video game knowledge, and the predisposition to immersion, that may be associated with the quality of virtual reality experience. Moreover, one particular concern for users engaged in immersive virtual reality environments (VREs) is the possibility of side effects, such as cybersickness. The literature suggests that at least 60% of virtual reality users report having felt symptoms of cybersickness, which reduces the quality of the virtual reality experience. The aim of this study was thus to profile the right user to be involved in a VRE through head-mounted display. To examine which user characteristics are associated with the most effective virtual reality experience (lower cybersickness), a multiple correspondence analysis combined with cluster analysis technique was performed. Results revealed three distinct profiles, showing that the PC gamer profile is more associated with higher levels of virtual reality effectiveness, that is, higher predisposition to be immersed and reduced cybersickness symptoms in the VRE than console gamer and nongamer. These findings can be a useful orientation in clinical practice and future research as they help identify which users are more predisposed to benefit from immersive VREs.

  14. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

    OpenAIRE

    1987-01-01

    Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the hea...

  15. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  16. Metabolomic Analysis Reveals Cyanidins in Black Raspberry as Candidates for Suppression of Lipopolysaccharide-Induced Inflammation in Murine Macrophages.

    Science.gov (United States)

    Jo, Young-Hee; Park, Hyun-Chang; Choi, Seulgi; Kim, Sugyeong; Bao, Cheng; Kim, Hyung Woo; Choi, Hyung-Kyoon; Lee, Hong Jin; Auh, Joong-Hyuck

    2015-06-10

    The extracts produced by multisolvent extraction and subfractionation with preparative liquid chromatography of black raspberry (Rubus coreanus Miquel) cultivated in Gochang, South Korea, were tested for their anti-inflammatory effects. The metabolomic profiling and analysis by orthogonal partial least-squares discriminant analysis (OLPS-DA) suggested that cyanidin, cyanidin-3-glucoside (C3G), and cyanidin-3-rutinoside (C3R) were key components for the anti-inflammatory responses in the most active fraction BF3-1, where they were present at 0.44, 1.26, and 0.56 μg/mg of BF3-1, respectively. Both BF3-1 and mixture of these cyanidins at the same ratio reduced lipopolysaccharide (LPS)-induced protein level of iNOS expression and suppressed mRNA and protein expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β through inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) and STAT3 in murine macrophage RAW264.7 cells. Overall, the results suggested that co-administration of cyanidin, C3G, and C3R is more effective than that of cyanidin alone and that the coexistence of these anthocyanin components in black raspberry plays a vital role in regulating LPS-induced inflammation even at submicromolar concentrations, making it possible to explain the health beneficial activity of its extracts.

  17. The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.

    Directory of Open Access Journals (Sweden)

    Moritz Treeck

    2014-06-01

    Full Text Available Calcium-dependent protein kinases (CDPKs are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC. This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1, as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  18. Phosphorylated Smad2 in Advanced Stage Gastric Carcinoma

    International Nuclear Information System (INIS)

    Transforming growth factor β (TGFβ) receptor signaling is closely associated with the invasion ability of gastric cancer cells. Although Smad signal is a critical integrator of TGFβ receptor signaling transduction systems, not much is known about the role of Smad2 expression in gastric carcinoma. The aim of the current study is to clarify the role of phosphorylated Smad2 (p-Smad2) in gastric adenocarcinomas at advanced stages. Immunohistochemical staining with anti-p-Smad2 was performed on paraffin-embedded specimens from 135 patients with advanced gastric adenocarcinomas. We also evaluated the relationship between the expression levels of p-Smad2 and clinicopathologic characteristics of patients with gastric adenocarcinomas. The p-Smad2 expression level was high in 63 (47%) of 135 gastric carcinomas. The p-Smad2 expression level was significantly higher in diffuse type carcinoma (p = 0.007), tumours with peritoneal metastasis (p = 0.017), and tumours with lymph node metastasis (p = 0.047). The prognosis for p-Smad2-high patients was significantly (p = 0.035, log-rank) poorer than that of p-Smad2-low patients, while a multivariate analysis revealed that p-Smad2 expression was not an independence prognostic factor. The expression of p-Smad2 is associated with malignant phenotype and poor prognosis in patients with advanced gastric carcinoma

  19. O-GlcNAc modification: why so intimately associated with phosphorylation?

    Directory of Open Access Journals (Sweden)

    Ande Sudharsana R

    2011-01-01

    Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

  20. Prebiotic phosphorylation of nucleosides in formamide

    Science.gov (United States)

    Schoffstall, A. M.

    1976-01-01

    Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

  1. Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

    Directory of Open Access Journals (Sweden)

    Tang Vivian W

    2006-12-01

    Full Text Available Abstract Background Zonula occludens, also known as the tight junction, is a specialized cell-cell interaction characterized by membrane "kisses" between epithelial cells. A cytoplasmic plaque of ~100 nm corresponding to a meshwork of densely packed proteins underlies the tight junction membrane domain. Due to its enormous size and difficulties in obtaining a biochemically pure fraction, the molecular composition of the tight junction remains largely unknown. Results A novel biochemical purification protocol has been developed to isolate tight junction protein complexes from cultured human epithelial cells. After identification of proteins by mass spectroscopy and fingerprint analysis, candidate proteins are scored and assessed individually. A simple algorithm has been devised to incorporate transmembrane domains and protein modification sites for scoring membrane proteins. Using this new scoring system, a total of 912 proteins have been identified. These 912 hits are analyzed using a bioinformatics approach to bin the hits in 4 categories: configuration, molecular function, cellular function, and specialized process. Prominent clusters of proteins related to the cytoskeleton, cell adhesion, and vesicular traffic have been identified. Weaker clusters of proteins associated with cell growth, cell migration, translation, and transcription are also found. However, the strongest clusters belong to synaptic proteins and signaling molecules. Localization studies of key components of synaptic transmission have confirmed the presence of both presynaptic and postsynaptic proteins at the tight junction domain. To correlate proteomics data with structure, the tight junction has been examined using electron microscopy. This has revealed many novel structures including end-on cytoskeletal attachments, vesicles fusing/budding at the tight junction membrane domain, secreted substances encased between the tight junction kisses, endocytosis of tight junction

  2. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    Science.gov (United States)

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  3. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  4. Tarantula myosin free head regulatory light chain phosphorylation stiffens N-terminal extension, releasing it and blocking its docking back.

    Science.gov (United States)

    Alamo, Lorenzo; Li, Xiaochuan Edward; Espinoza-Fonseca, L Michel; Pinto, Antonio; Thomas, David D; Lehman, William; Padrón, Raúl

    2015-08-01

    Molecular dynamics simulations of smooth and striated muscle myosin regulatory light chain (RLC) N-terminal extension (NTE) showed that diphosphorylation induces a disorder-to-order transition. Our goal here was to further explore the effects of mono- and diphosphorylation on the straightening and rigidification of the tarantula myosin RLC NTE. For that we used MD simulations followed by persistence length analysis to explore the consequences of secondary and tertiary structure changes occurring on RLC NTE following phosphorylation. Static and dynamic persistence length analysis of tarantula RLC NTE peptides suggest that diphosphorylation produces an important 24-fold straightening and a 16-fold rigidification of the RLC NTE, while monophosphorylation has a less profound effect. This new information on myosin structural mechanics, not fully revealed by previous EM and MD studies, add support to a cooperative phosphorylation-dependent activation mechanism as proposed for the tarantula thick filament. Our results suggest that the RLC NTE straightening and rigidification after Ser45 phosphorylation leads to a release of the constitutively Ser35 monophosphorylated free head swaying away from the thick filament shaft. This is so because the stiffened diphosphorylated RLC NTE would hinder the docking back of the free head after swaying away, becoming released and mobile and unable to recover its original interacting position on activation. PMID:26038302

  5. Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers

    Science.gov (United States)

    Treangen, Todd J.; Koren, Sergey; Pop, Mihai; Bhaya, Devaki

    2016-01-01

    The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2–13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics. PMID:27611571

  6. Multivariate pattern analysis reveals anatomical connectivity differences between the left and right mesial temporal lobe epilepsy

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    Peng Fang

    2015-01-01

    Full Text Available Previous studies have demonstrated differences of clinical signs and functional brain network organizations between the left and right mesial temporal lobe epilepsy (mTLE, but the anatomical connectivity differences underlying functional variance between the left and right mTLE remain uncharacterized. We examined 43 (22 left, 21 right mTLE patients with hippocampal sclerosis and 39 healthy controls using diffusion tensor imaging. After the whole-brain anatomical networks were constructed for each subject, multivariate pattern analysis was applied to classify the left mTLE from the right mTLE and extract the anatomical connectivity differences between the left and right mTLE patients. The classification results reveal 93.0% accuracy for the left mTLE versus the right mTLE, 93.4% accuracy for the left mTLE versus controls and 90.0% accuracy for the right mTLE versus controls. Compared with the right mTLE, the left mTLE exhibited a different connectivity pattern in the cortical-limbic network and cerebellum. The majority of the most discriminating anatomical connections were located within or across the cortical-limbic network and cerebellum, thereby indicating that these disease-related anatomical network alterations may give rise to a portion of the complex of emotional and memory deficit between the left and right mTLE. Moreover, the orbitofrontal gyrus, cingulate cortex, hippocampus and parahippocampal gyrus, which exhibit high discriminative power in classification, may play critical roles in the pathophysiology of mTLE. The current study demonstrated that anatomical connectivity differences between the left mTLE and the right mTLE may have the potential to serve as a neuroimaging biomarker to guide personalized diagnosis of the left and right mTLE.

  7. Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation

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    Rismani-Yazdi Hamid

    2012-09-01

    Full Text Available Abstract Background The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG biofuel precursor. Results After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Conclusions Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and

  8. Computational bacterial genome-wide analysis of phylogenetic profiles reveals potential virulence genes of Streptococcus agalactiae.

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    Frank Po-Yen Lin

    Full Text Available The phylogenetic profile of a gene is a reflection of its evolutionary history and can be defined as the differential presence or absence of a gene in a set of reference genomes. It has been employed to facilitate the prediction of gene functions. However, the hypothesis that the application of this concept can also facilitate the discovery of bacterial virulence factors has not been fully examined. In this paper, we test this hypothesis and report a computational pipeline designed to identify previously unknown bacterial virulence genes using group B streptococcus (GBS as an example. Phylogenetic profiles of all GBS genes across 467 bacterial reference genomes were determined by candidate-against-all BLAST searches,which were then used to identify candidate virulence genes by machine learning models. Evaluation experiments with known GBS virulence genes suggested good functional and model consistency in cross-validation analyses (areas under ROC curve, 0.80 and 0.98 respectively. Inspection of the top-10 genes in each of the 15 virulence functional groups revealed at least 15 (of 119 homologous genes implicated in virulence in other human pathogens but previously unrecognized as potential virulence genes in GBS. Among these highly-ranked genes, many encode hypothetical proteins with possible roles in GBS virulence. Thus, our approach has led to the identification of a set of genes potentially affecting the virulence potential of GBS, which are potential candidates for further in vitro and in vivo investigations. This computational pipeline can also be extended to in silico analysis of virulence determinants of other bacterial pathogens.

  9. An evolutionary analysis of flightin reveals a conserved motif unique and widespread in Pancrustacea.

    Science.gov (United States)

    Soto-Adames, Felipe N; Alvarez-Ortiz, Pedro; Vigoreaux, Jim O

    2014-01-01

    Flightin is a thick filament protein that in Drosophila melanogaster is uniquely expressed in the asynchronous, indirect flight muscles (IFM). Flightin is required for the structure and function of the IFM and is indispensable for flight in Drosophila. Given the importance of flight acquisition in the evolutionary history of insects, here we study the phylogeny and distribution of flightin. Flightin was identified in 69 species of hexapods in classes Collembola (springtails), Protura, Diplura, and insect orders Thysanura (silverfish), Dictyoptera (roaches), Orthoptera (grasshoppers), Pthiraptera (lice), Hemiptera (true bugs), Coleoptera (beetles), Neuroptera (green lacewing), Hymenoptera (bees, ants, and wasps), Lepidoptera (moths), and Diptera (flies and mosquitoes). Flightin was also found in 14 species of crustaceans in orders Anostraca (water flea), Cladocera (brine shrimp), Isopoda (pill bugs), Amphipoda (scuds, sideswimmers), and Decapoda (lobsters, crabs, and shrimps). Flightin was not identified in representatives of chelicerates, myriapods, or any species outside Pancrustacea (Tetraconata, sensu Dohle). Alignment of amino acid sequences revealed a conserved region of 52 amino acids, referred herein as WYR, that is bound by strictly conserved tryptophan (W) and arginine (R) and an intervening sequence with a high content of tyrosines (Y). This motif has no homologs in GenBank or PROSITE and is unique to flightin and paraflightin, a putative flightin paralog identified in decapods. A third motif of unclear affinities to pancrustacean WYR was observed in chelicerates. Phylogenetic analysis of amino acid sequences of the conserved motif suggests that paraflightin originated before the divergence of amphipods, isopods, and decapods. We conclude that flightin originated de novo in the ancestor of Pancrustacea > 500 MYA, well before the divergence of insects (~400 MYA) and the origin of flight (~325 MYA), and that its IFM-specific function in Drosophila is a more

  10. Diversity in a Polymicrobial Community Revealed by Analysis of Viromes, Endolysins and CRISPR Spacers.

    Science.gov (United States)

    Davison, Michelle; Treangen, Todd J; Koren, Sergey; Pop, Mihai; Bhaya, Devaki

    2016-01-01

    The polymicrobial biofilm communities in Mushroom and Octopus Spring in Yellowstone National Park (YNP) are well characterized, yet little is known about the phage populations. Dominant species, Synechococcus sp. JA-2-3B'a(2-13), Synechococcus sp. JA-3-3Ab, Chloroflexus sp. Y-400-fl, and Roseiflexus sp. RS-1, contain multiple CRISPR-Cas arrays, suggesting complex interactions with phage predators. To analyze phage populations from Octopus Spring biofilms, we sequenced a viral enriched fraction. To assemble and analyze phage metagenomic data, we developed a custom module, VIRITAS, implemented within the MetAMOS framework. This module bins contigs into groups based on tetranucleotide frequencies and CRISPR spacer-protospacer matching and ORF calling. Using this pipeline we were able to assemble phage sequences into contigs and bin them into three clusters that corroborated with their potential host range. The virome contained 52,348 predicted ORFs; some were clearly phage-like; 9319 ORFs had a recognizable Pfam domain while the rest were hypothetical. Of the recognized domains with CRISPR spacer matches, was the phage endolysin used by lytic phage to disrupt cells. Analysis of the endolysins present in the thermophilic cyanophage contigs revealed a subset of characterized endolysins as well as a Glyco_hydro_108 (PF05838) domain not previously associated with sequenced cyanophages. A search for CRISPR spacer matches to all identified phage endolysins demonstrated that a majority of endolysin domains were targets. This strategy provides a general way to link host and phage as endolysins are known to be widely distributed in bacteriophage. Endolysins can also provide information about host cell wall composition and have the additional potential to be used as targets for novel therapeutics. PMID:27611571

  11. Molecular determinants of juvenile hormone action as revealed by 3D QSAR analysis in Drosophila.

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    Denisa Liszeková

    Full Text Available BACKGROUND: Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH. While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 A or longer than 13.5 A, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. CONCLUSIONS/SIGNIFICANCE: The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions.

  12. Analysis of newly established EST databases reveals similarities between heart regeneration in newt and fish

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    Weis Patrick

    2010-01-01

    Full Text Available Abstract Background The newt Notophthalmus viridescens possesses the remarkable ability to respond to cardiac damage by formation of new myocardial tissue. Surprisingly little is known about changes in gene activities that occur during the course of regeneration. To begin to decipher the molecular processes, that underlie restoration of functional cardiac tissue, we generated an EST database from regenerating newt hearts and compared the transcriptional profile of selected candidates with genes deregulated during zebrafish heart regeneration. Results A cDNA library of 100,000 cDNA clones was generated from newt hearts 14 days after ventricular injury. Sequencing of 11520 cDNA clones resulted in 2894 assembled contigs. BLAST searches revealed 1695 sequences with potential homology to sequences from the NCBI database. BLAST searches to TrEMBL and Swiss-Prot databases assigned 1116 proteins to Gene Ontology terms. We also identified a relatively large set of 174 ORFs, which are likely to be unique for urodele amphibians. Expression analysis of newt-zebrafish homologues confirmed the deregulation of selected genes during heart regeneration. Sequences, BLAST results and GO annotations were visualized in a relational web based database followed by grouping of identified proteins into clusters of GO Terms. Comparison of data from regenerating zebrafish hearts identified biological processes, which were uniformly overrepresented during cardiac regeneration in newt and zebrafish. Conclusion We concluded that heart regeneration in newts and zebrafish led to the activation of similar sets of genes, which suggests that heart regeneration in both species might follow similar principles. The design of the newly established newt EST database allows identification of molecular pathways important for heart regeneration.

  13. Particle Motion Analysis Reveals Nanoscale Bond Characteristics and Enhances Dynamic Range for Biosensing.

    Science.gov (United States)

    Visser, Emiel W A; van IJzendoorn, Leo J; Prins, Menno W J

    2016-03-22

    Biofunctionalized colloidal particles are widely used as labels in bioanalytical assays, lab-on-chip devices, biophysical research, and in studies on live biological systems. With detection resolution going down to the level of single particles and single molecules, understanding the nature of the interaction of the particles with surfaces and substrates becomes of paramount importance. Here, we present a comprehensive study of motion patterns of colloidal particles maintained in close proximity to a substrate by short molecular tethers (40 nm). The motion of the particles (500-1000 nm) was optically tracked with a very high localization accuracy (below 3 nm). A surprisingly large variation in motion patterns was observed, which can be attributed to properties of the particle-molecule-substrate system, namely the bond number, the nature of the bond, particle protrusions, and substrate nonuniformities. Experimentally observed motion patterns were compared to numerical Monte Carlo simulations, revealing a close correspondence between the observed motion patterns and properties of the molecular system. Particles bound via single tethers show distinct disc-, ring-, and bell-shaped motion patterns, where the ring- and bell-shaped patterns are caused by protrusions on the particle in the direct vicinity of the molecular attachment point. Double and triple tethered particles exhibit stripe-shaped and triangular-shaped motion patterns, respectively. The developed motion pattern analysis allows for discrimination between particles bound by different bond types, which opens the possibility to improve the limit of detection and the dynamic range of bioanalytical assays, with a projected increase of dynamic range by nearly 2 orders of magnitude.

  14. Comparative methylome analysis in solid tumors reveals aberrant methylation at chromosome 6p in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10−9), but was less obvious in other types of solid tumors except for prostate and Epstein–Barr virus (EBV)-positive gastric cancer (FDR<10−3). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection

  15. Sr Isotope Analysis of Lacustrine Fossils Reveals Paleohydrological Reorganisation in the Turkana Basin Through the Holocene.

    Science.gov (United States)

    Vonhof, H.; Lubbe, J. V. D.; Joordens, J. J.; Feibel, C. S.; Junginger, A.; Garcin, Y.; Krause-Nehring, J.; Beck, C.; Johnson, T. C.

    2015-12-01

    Lake Turkana in northern Kenya is one of the largest lakes in the East African Rift System (EARS) that experienced significant climate-driven lake level variation over the Holocene. Arguably the most important feature of Holocene climate change in the EARS is the termination of the African Humid Period (AHP), that caused a ~70 meter lake level drop in Lake Turkana. The precise hydrological response to the termination of the AHP is potentially complex, because Lake Turkana lies at the cross roads of two large atmospheric convection systems; the Intertropical Convergence Zone (ITCZ) and the Congo Air Boundary (CAB). Shifting of these atmospheric systems around the end of the AHP dramatically rearranged spatial rainfall patterns in the Turkana Basin catchment, causing changes in relative runoff contributions from the different sub-catchments in the Turkana Basin. We here present a Holocene Turkana lake water Sr-isotope reconstruction, based on the analysis of well-dated lacustrine ostracods and shells. This reconstruction reveals consistently high Sr isotope values for the early Holocene, followed by a remarkable drop of Sr isotope ratios around the AHP termination. We interpret this pattern to represent a westward shift in the location of the CAB, leading to the reduction and eventual shutdown of runoff contribution from the Chew Bahir Basin to the Turkana Basin at the end of the AHP. The record demonstrates the exceptional suitability of Sr isotope data for this type of paleohydrological reconstructions. This is mainly due to the chemically conservative Sr-isotope mass balance in EARS lake systems, which is insensitive to environmental change at seasonal timescales that so often overprints the longer term climate signal in stable (oxygen and carbon) isotope records of these lakes. Furthermore, when Sr-isotope signatures of the contributing sub-catchments are known, the observed Sr isotope trends can be interpreted in terms of spatial shifts in climate driven runoff

  16. Proteomic analysis reveals metabolic and regulatory systems involved the syntrophic and axenic lifestyle of Syntrophomonas wolfei.

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    Jessica Rhea Sieber

    2015-02-01

    Full Text Available Microbial syntrophy is a vital metabolic interaction necessary for the complete oxidation of organic biomass to methane in all-anaerobic ecosystems. However, this process is thermodynamically constrained and represents an ecosystem-level metabolic bottleneck. To gain insight into the physiology of this process, a shotgun proteomic approach was used to quantify the protein landscape of the model syntrophic metabolizer, Syntrophomonas wolfei, grown axenically and syntrophically with Methanospirillum hungatei. Remarkably, the abundance of most proteins as represented by normalized spectral abundance factor (NSAF value changed very little between the pure and coculture growth conditions. Among the most abundant proteins detected were GroEL and GroES chaperonins, a small heat shock protein, and proteins involved in electron transfer, beta-oxidation, and ATP synthesis. Several putative energy conservation enzyme systems that utilize NADH and ferredoxin were present. The abundance of an EtfAB2 and the membrane-bound iron-sulfur oxidoreductase (Swol_0698 gene product delineated a potential conduit for electron transfer between acyl-CoA dehydrogenases and membrane redox carriers. Proteins detected only when S. wolfei was grown with M. hungatei included a zinc-dependent dehydrogenase with a GroES domain, whose gene is present in genomes in many organisms capable of syntrophy, and transcriptional regulators responsive to environmental stimuli or the physiological status of the cell. The proteomic analysis revealed an emphasis macromolecular stability and energy metabolism to S. wolfei and presence of regulatory mechanisms responsive to external stimuli and cellular physiological status.

  17. Evolutionary history of barley cultivation in Europe revealed by genetic analysis of extant landraces

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    Jones Huw

    2011-11-01

    Full Text Available Abstract Background Understanding the evolution of cultivated barley is important for two reasons. First, the evolutionary relationships between different landraces might provide information on the spread and subsequent development of barley cultivation, including the adaptation of the crop to new environments and its response to human selection. Second, evolutionary information would enable landraces with similar traits but different genetic backgrounds to be identified, providing alternative strategies for the introduction of these traits into modern germplasm. Results The evolutionary relationships between 651 barley landraces were inferred from the genotypes for 24 microsatellites. The landraces could be divided into nine populations, each with a different geographical distribution. Comparisons with ear row number, caryopsis structure, seasonal growth habit and flowering time revealed a degree of association between population structure and phenotype, and analysis of climate variables indicated that the landraces are adapted, at least to some extent, to their environment. Human selection and/or environmental adaptation may therefore have played a role in the origin and/or maintenance of one or more of the barley landrace populations. There was also evidence that at least some of the population structure derived from geographical partitioning set up during the initial spread of barley cultivation into Europe, or reflected the later introduction of novel varieties. In particular, three closely-related populations were made up almost entirely of plants with the daylength nonresponsive version of the photoperiod response gene PPD-H1, conferring adaptation to the long annual growth season of northern Europe. These three populations probably originated in the eastern Fertile Crescent and entered Europe after the initial spread of agriculture. Conclusions The discovery of population structure, combined with knowledge of associated phenotypes and

  18. Transcriptome analysis of an endoparasitoid wasp Cotesia chilonis (Hymenoptera: Braconidae) reveals genes involved in successful parasitism.

    Science.gov (United States)

    Qi, Yixiang; Teng, Ziwen; Gao, Lingfeng; Wu, Shunfan; Huang, Jia; Ye, Gongyin; Fang, Qi

    2015-04-01

    For successful parasitization, parasitiods usually depend on the chemosensory cues for the selection of hosts, as well as a variety of virulence factors introduced into their hosts to overcome host immunity and prevent rejection of progeny development. In bracovirus-carrying wasps, the symbiotic polydnaviruses act in manipulating development and immunity of hosts. The endoparasitoid Cotesia chilonis carrying bracovirus as a key host immunosuppressive factor is a superior endoparasitoid of rice stem borer, Chilo suppressalis. So far, genomic information for C. chilonis is not available and transcriptomic data may provide valuable resources for global studying on physiological processes of C. chilonis, including chemosensation and parasitism at molecular level. Here, we performed RNA-seq to characterize the transcriptome of C. chilonis adults. We obtained 27,717,892 reads, assembled into 38,318 unigenes with a mean size of 690 bp. Approximately, 62.1% of the unigenes were annotated using NCBI databases. A large number of chemoreception-related genes encoding proteins including odorant receptors, gustatory receptors, odorant-binding proteins, chemosensory proteins, transient receptor potential ion channels, and sensory neuron membrane proteins were identified in silico. Totally, 72 transcripts possessing high identities with the bracovirus-related genes were identified. We investigated the mRNA expression levels of several transcripts at different developmental stages (including egg, larva, pupae, and adult) by quantitative real-time PCR analysis. The results revealed that some genes had adult-specific expression, indicating their potential significance for mating and parasitism. Overall, these results provide comprehensive insights into transcriptomic data of a polydnavirus-carrying parasitoid of a rice pest. PMID:25336406

  19. Comparative genome analysis reveals metabolic versatility and environmental adaptations of Sulfobacillus thermosulfidooxidans strain ST.

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    Xue Guo

    Full Text Available The genus Sulfobacillus is a cohort of mildly thermophilic or thermotolerant acidophiles within the phylum Firmicutes and requires extremely acidic environments and hypersalinity for optimal growth. However, our understanding of them is still preliminary partly because few genome sequences are available. Here, the draft genome of Sulfobacillus thermosulfidooxidans strain ST was deciphered to obtain a comprehensive insight into the genetic content and to understand the cellular mechanisms necessary for its survival. Furthermore, the expressions of key genes related with iron and sulfur oxidation were verified by semi-quantitative RT-PCR analysis. The draft genome sequence of Sulfobacillus thermosulfidooxidans strain ST, which encodes 3225 predicted coding genes on a total length of 3,333,554 bp and a 48.35% G+C, revealed the high degree of heterogeneity with other Sulfobacillus species. The presence of numerous transposases, genomic islands and complete CRISPR/Cas defence systems testifies to its dynamic evolution consistent with the genome heterogeneity. As expected, S. thermosulfidooxidans encodes a suit of conserved enzymes required for the oxidation of inorganic sulfur compounds (ISCs. The model of sulfur oxidation in S. thermosulfidooxidans was proposed, which showed some different characteristics from the sulfur oxidation of Gram-negative A. ferrooxidans. Sulfur oxygenase reductase and heterodisulfide reductase were suggested to play important roles in the sulfur oxidation. Although the iron oxidation ability was observed, some key proteins cannot be identified in S. thermosulfidooxidans. Unexpectedly, a predicted sulfocyanin is proposed to transfer electrons in the iron oxidation. Furthermore, its carbon metabolism is rather flexible, can perform the transformation of pentose through the oxidative and non-oxidative pentose phosphate pathways and has the ability to take up small organic compounds. It encodes a multitude of heavy metal

  20. Properties of galaxies around AGNs with the most massive supermassive black holes revealed by clustering analysis

    Science.gov (United States)

    Shirasaki, Yuji; Komiya, Yutaka; Ohishi, Masatoshi; Mizumoto, Yoshihiko

    2016-04-01

    We present results of the clustering analysis between active galactic nuclei (AGNs) and galaxies at redshift 0.1-1.0, which was performed to investigate the properties of galaxies associated with the AGNs and reveal the nature of the fueling mechanism of supermassive black holes (SMBHs). We used 8059 AGNs/quasi-stellar objects (QSOs) for which virial masses of individual SMBHs were measured, and divided them into four mass groups.Cross-correlation analysis was performed to reconfirm our previous result that cross-correlation length increases with SMBH mass MBH; we obtained consistent results. A linear bias of AGN for each mass group was measured as 1.47 for MBH = 107.5-108.2 M⊙ and 3.08 for MBH = 109-1010 M⊙. The averaged color and luminosity distributions of galaxies around the AGNs/QSOs were also derived for each mass group. The galaxy color Dopt-IR was estimated from a spectral energy distribution (SED) constructed from a catalog derived by merging the Sloan Digital Sky Survey (SDSS) and the UKIRT Infrared Deep Sky Survey (UKIDSS) catalogs. The distributions of color and luminosity were derived by a subtraction method, which does not require redshift information of galaxies. The main results of this work are as follows. (1) A linear bias increases by a factor of two from the lower-mass group to the highest-mass group. (2) The environment around AGNs with the most massive SMBHs (MBH > 109 M⊙) is dominated by red sequence galaxies. (3) Marginal indication of decline in luminosity function at dimmer side of MIR > -19.5 is found for galaxies around AGNs with MBH = 108.2-109 M⊙ and nearest redshift group (z = 0.1-0.3). These results indicate that AGNs with the most massive SMBHs reside in haloes where a large fraction of galaxies have been transited to the red sequence. The accretion of hot halo gas as well as recycled gas from evolving stars can be one of the plausible mechanisms to fuel the SMBHs above ˜ 109 M⊙.

  1. SKY analysis revealed recurrent numerical and structural chromosome changes in BDII rat endometrial carcinomas

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    Behboudi Afrouz

    2011-06-01

    Full Text Available Abstract Background Genomic alterations are common features of cancer cells, and some of these changes are proven to be neoplastic-specific. Such alterations may serve as valuable tools for diagnosis and classification of tumors, prediction of clinical outcome, disease monitoring, and choice of therapy as well as for providing clues to the location of crucial cancer-related genes. Endometrial carcinoma (EC is the most frequently diagnosed malignancy of the female genital tract, ranking fourth among all invasive tumors affecting women. Cytogenetic studies of human ECs have not produced very conclusive data, since many of these studies are based on karyotyping of limited number of cases and no really specific karyotypic changes have yet been identified. As the majority of the genes are conserved among mammals, the use of inbred animal model systems may serve as a tool for identification of underlying genes and pathways involved in tumorigenesis in humans. In the present work we used spectral karyotyping (SKY to identify cancer-related aberrations in a well-characterized experimental model for spontaneous endometrial carcinoma in the BDII rat tumor model. Results Analysis of 21 experimental ECs revealed specific nonrandom numerical and structural chromosomal changes. The most recurrent numerical alterations were gains in rat chromosome 4 (RNO4 and losses in RNO15. The most commonly structural changes were mainly in form of chromosomal translocations and were detected in RNO3, RNO6, RNO10, RNO11, RNO12, and RNO20. Unbalanced chromosomal translocations involving RNO3p was the most commonly observed structural changes in this material followed by RNO11p and RNO10 translocations. Conclusion The non-random nature of these events, as documented by their high frequencies of incidence, is suggesting for dynamic selection of these changes during experimental EC tumorigenesis and therefore for their potential contribution into development of this malignancy

  2. Platelet-activating factor (PAF)-dependent biochemical, morphologic, and physiologic responses of human platelets: Demonstration of translocation of protein kinase C associated with protein phosphorylation

    International Nuclear Information System (INIS)

    Platelet-activating factor (PAF) is a potent stimulus for platelet aggregation and secretion. PAF has been shown to stimulate the phosphatidylinositol (PI) pathway in platelets, which implies that PAF should activate protein kinase C. In this study, measurements of PI metabolites, the elevation of intracellular free calcium concentration, (Ca2+)i, the activation of protein kinase C, and the phosphorylation of platelet proteins (using a two-dimensional gel electrophoretic technique) were performed before and after the addition of 10(-8) M PAF to human platelets. These findings were correlated with morphologic changes in the platelets as determined by immunoelectron microscopic studies on the cytoskeleton and by X-ray analysis of dense bodies. The results show that PAF stimulates the production of PI metabolites and causes an increase in the membrane-associated activity of protein kinase C. These changes are accompanied by a rise in the (Ca2+)i and protein phosphorylation. The increase in protein kinase C activity reaches a maximum at approximately 60 s, a time frame that is consistent with the protein phosphorylation and the subsequent morphologic and secretory events. X-ray analysis revealed two types of dense bodies containing various amounts of calcium which appeared to be released sequentially after PAF activation. These results suggest that the protein phosphorylation that controls the physiologic events resulting from PAF activation of human platelets is catalyzed by protein kinase C

  3. Transcriptome analysis of murine thymocytes reveals age-associated changes in thymic gene expression

    Directory of Open Access Journals (Sweden)

    Ana Lustig, Arnell Carter, Dorothy Bertak, Divya Enika, Bolormaa Vandanmagsar, William Wood, Kevin G. Becker, Ashani T. Weeraratna, Dennis D. Taub

    2009-01-01

    Full Text Available The decline in adaptive immunity, naïve T-cell output and a contraction in the peripheral T cell receptor (TCR repertoire with age are largely attributable to thymic involution and the loss of critical cytokines and hormones within the thymic microenvironment. To assess the molecular changes associated with this loss of thymic function, we used cDNA microarray analyses to examine the transcriptomes of thymocytes from mice of various ages ranging from very young (1 month to very old (24 months. Genes associated with various biological and molecular processes including oxidative phosphorylation, T- and B- cell receptor signaling and antigen presentation were observed to significantly change with thymocyte age. These include several immunoglobulin chains, chemokine and ribosomal proteins, annexin A2, vav 1 and several S100 signaling proteins. The increased expression of immunoglobulin genes in aged thymocytes could be attributed to the thymic B cells which were found to be actively producing IgG and IgM antibodies. Upon further examination, we found that purified thymic T cells derived from aged but not young thymi also exhibited IgM on their cell surface suggesting the possible presence of auto-antibodies on the surface thymocytes with advancing age. These studies provide valuable insight into the cellular and molecular mechanisms associated with thymic aging.

  4. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    Directory of Open Access Journals (Sweden)

    Noriko Hamada-Kawaguchi

    Full Text Available Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150 and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  5. Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

    Science.gov (United States)

    Hamada-Kawaguchi, Noriko; Nishida, Yasuyoshi; Yamamoto, Daisuke

    2015-01-01

    Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

  6. The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site

    Directory of Open Access Journals (Sweden)

    Lilischkis Richard

    2006-03-01

    Full Text Available Abstract Background The U2 small nuclear ribonucleoprotein particle (snRNP component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. Results Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1. Conclusion The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.

  7. Phytophthora infestans specific phosphorylation patterns and new putative control targets.

    Science.gov (United States)

    Frades, Itziar; Andreasson, Erik

    2016-04-01

    In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation. PMID:27020162

  8. Symposia on Plant (Protein Phosphorylation

    Directory of Open Access Journals (Sweden)

    Sacco C. De Vries

    2012-08-01

    Full Text Available From September 14-16, 2011 the twelfth symposium on Plant Protein Phosphorylation was held in Tübingen, Germany. The topic is as broad as the name suggests and covers all aspects of this important means of protein modification in plants. I have had the pleasure of attending the 2007 and the 2011 symposia. The interesting concept behind these meetings is to hear about the same biochemical mechanism operative in a multitude of experimental systems. The meetings are quite informal and present an excellent mix ranging from technology to biochemical experience and novel findings and tools.The two-and-a-half-day program was divided into five double sessions: biotic interactions, hormone signaling, abiotic interactions, Mitogen Activated Protein Kinase (MAPK and Ca++ pathways and phosphoproteomics. It was hosted by the Zentrum für Molekularbiologie der Pflanzen (ZMBP and the organizing committee chaired by Klaus Harter.

  9. Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

    Directory of Open Access Journals (Sweden)

    Henry D Priest

    Full Text Available Brachypodium distachyon is a close relative of many important cereal crops. Abiotic stress tolerance has a significant impact on productivity of agriculturally important food and feedstock crops. Analysis of the transcriptome of Brachypodium after chilling, high-salinity, drought, and heat stresses revealed diverse differential expression of many transcripts. Weighted Gene Co-Expression Network Analysis revealed 22 distinct gene modules with specific profiles of expression under each stress. Promoter analysis implicated short DNA sequences directly upstream of module members in the regulation of 21 of 22 modules. Functional analysis of module members revealed enrichment in functional terms for 10 of 22 network modules. Analysis of condition-specific correlations between differentially expressed gene pairs revealed extensive plasticity in the expression relationships of gene pairs. Photosynthesis, cell cycle, and cell wall expression modules were down-regulated by all abiotic stresses. Modules which were up-regulated by each abiotic stress fell into diverse and unique gene ontology GO categories. This study provides genomics resources and improves our understanding of abiotic stress responses of Brachypodium.

  10. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  11. Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

    KAUST Repository

    Chandramouli, Kondethimmanahalli

    2013-03-01

    Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

  12. Evaluation of phosphorylated psyllium seed polysaccharide as a release retardant

    Directory of Open Access Journals (Sweden)

    Monica R P Rao

    2015-01-01

    Full Text Available The aim of the present study was to modify psyllium seed polysaccharide and evaluate the modified polysaccharide as release retardant in tablets employing ciprofloxacin hydrochloride as model drug. Studies on polysaccharide from psyllium husk has been reported but no work has been reported on characterization and modification of the polysaccharide present in the psyllium (Plantago ovata seed and the use of the modified polysaccharide as a release retardant in tablets. In this study, the seed gum was modified using sodium trimetaphosphate as crosslinking agent. Sustained release matrix tablets of ciprofloxacin hydrochloride were prepared by wet granulation using various drug-polymer ratios. The polymers investigated were psyllium polysaccharide, phosphorylated psyllium polysaccharide and widely used release retardant hydroxypropyl methylcellulose K100M. The tablets were evaluated for hardness, friability, drug content, swelling profile and in vitro dissolution studies. The matrix tablets containing 1:3 proportion of drug-phosphorylated psyllium polysaccharide was found to have higher hardness as compared to tablets containing 1:1 and 1:2 proportions. The results of swelling behavior in water showed that the tablets containing 1:3 drug:phosphorylated psyllium polysaccharide ratio had swelling comparable to that of tablets containing 1:3 drug:hydroxypropyl methylcellulose ratio. The in vitro dissolution studies shows that the dissolution rate was retarded from 98.41 to 37.6% in 6 h with increase in concentration of phosphorylated psyllium polysaccharide from 100 to 300 mg. Formulations containing psyllium polysaccharide showed complete drug release in 8 h whereas those formulated with phosphorylated psyllium polysaccharide exhibited extended drug release over the 12 h period. Drug release kinetic studies revealed that drug release followed Korsmeyer-Peppas model.

  13. Evaluation of Phosphorylated Psyllium Seed Polysaccharide as a Release Retardant.

    Science.gov (United States)

    Rao, Monica R P; Warrier, Deepa U; Rao, Shivani H

    2015-01-01

    The aim of the present study was to modify psyllium seed polysaccharide and evaluate the modified polysaccharide as release retardant in tablets employing ciprofloxacin hydrochloride as model drug. Studies on polysaccharide from psyllium husk has been reported but no work has been reported on characterization and modification of the polysaccharide present in the psyllium (Plantago ovata) seed and the use of the modified polysaccharide as a release retardant in tablets. In this study, the seed gum was modified using sodium trimetaphosphate as crosslinking agent. Sustained release matrix tablets of ciprofloxacin hydrochloride were prepared by wet granulation using various drug-polymer ratios. The polymers investigated were psyllium polysaccharide, phosphorylated psyllium polysaccharide and widely used release retardant hydroxypropyl methylcellulose K100M. The tablets were evaluated for hardness, friability, drug content, swelling profile and in vitro dissolution studies. The matrix tablets containing 1:3 proportion of drug-phosphorylated psyllium polysaccharide was found to have higher hardness as compared to tablets containing 1:1 and 1:2 proportions. The results of swelling behavior in water showed that the tablets containing 1:3 drug:phosphorylated psyllium polysaccharide ratio had swelling comparable to that of tablets containing 1:3 drug:hydroxypropyl methylcellulose ratio. The in vitro dissolution studies shows that the dissolution rate was retarded from 98.41 to 37.6% in 6 h with increase in concentration of phosphorylated psyllium polysaccharide from 100 to 300 mg. Formulations containing psyllium polysaccharide showed complete drug release in 8 h whereas those formulated with phosphorylated psyllium polysaccharide exhibited extended drug release over the 12 h period. Drug release kinetic studies revealed that drug release followed Korsmeyer-Peppas model. PMID:26798177

  14. Three-dimensional Reconstruction of Tarantula Myosin Filaments Suggests How Phosphorylation May Regulate Myosin Activity

    OpenAIRE

    Alamo, Lorenzo; Wriggers, Willy; Pinto, Antonio; Bártoli, Fulvia; Salazar, Leiría; Zhao, Fa-Qing; Craig, Roger; Padrón, Raúl

    2008-01-01

    Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLC). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that my...

  15. Carbon sources in the Beaufort Sea revealed by molecular lipid biomarkers and compound specific isotope analysis

    Directory of Open Access Journals (Sweden)

    I. Tolosa

    2012-10-01

    Full Text Available Molecular lipid biomarkers (hydrocarbons, alcohols, sterols and fatty acids and compound specific isotope analysis of suspended particulate organic matter (SPM and surface sediments of the Mackenzie Shelf and slope (Southeast Beaufort Sea, Arctic Ocean, were studied in summer 2009. The concentrations of the molecular lipid markers, characteristic of known organic matter sources, were grouped and used as proxies to evaluate the relative importance of fresh algal, detrital algal, fossil, C3 terrestrial plants, bacterial and zooplankton material in the sedimentary organic matter (OM.

    Fossil and detrital algal contributions were the major fractions of the freshwater SPM from the Mackenzie River with ~34% each of the total molecular biomarkers. Fresh algal, C3 terrestrial, bacterial and zooplanktonic components represented much lower percentages, 17, 10, 4 and < 1%, respectively. In marine SPM from the Mackenzie slope, the major contributions were fresh and detrital algal components (> 80% with a minor contribution of fossil and C3 terrestrial biomarkers. Characterization of the sediments revealed a major sink of refractory algal material mixed with some fresh algal material, fossil hydrocarbons and a small input of C3 terrestrial sources. In particular, the sediments from the shelf and at the mouth of the Amundsen Gulf presented the highest contribution of detrital algal material (60–75% whereas those from the slope contained the highest proportion of fossil (40% and C3 terrestrial plant material (10%. Overall, considering that the detrital algal material is marine derived, autochthonous sources contributed more than allochthonous sources to the OM lipid pool. Using the ratio of an allochthonous biomarker (normalized to total organic carbon, TOC found in the sediments to those measured at the river mouth water, we estimated that the fraction of terrestrial material preserved in the

  16. Quantitative cell signalling analysis reveals down-regulation of MAPK pathway activation in colorectal cancer.

    LENUS (Irish Health Repository)

    Gulmann, Christian

    2009-08-01

    Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes\\' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes\\' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series.

  17. A comparative phylogenetic analysis of medicinal plant Tribulus terrestris in Northwest India revealed by RAPD and ISSR markers

    OpenAIRE

    ASHWANI KUMAR; NEELAM VERMA

    2012-01-01

    Kumar A, Verma N. 2012. A comparative phylogenetic analysis of medicinal plant Tribulus terrestris in Northwest India revealed by RAPD and ISSR markers. Biodiversitas 13: 107-113. Several DNA marker systems and associated techniques are available today for fingerprinting of plant varieties. A total of 5 RAPD and 8 ISSR primers were used. Amplification of genomic DNA of the 6 genotypes, using RAPD analysis, yielded 164 fragments that could be scored, of which 47 were polymorphic, with an avera...

  18. Characterizing the Microenvironment Surrounding Phosphorylated Protein Sites

    Institute of Scientific and Technical Information of China (English)

    Shi-Cai Fan; Xue-Gong Zhang

    2005-01-01

    Protein phosphorylation plays an important role in various cellular processes. Due to its high complexity, the mechanism needs to be further studied. In the last few years, many methods have been contributed to this field, but almost all of them investigated the mechanism based on protein sequences around protein sites. In this study, we implement an exploration by characterizing the microenvironment surrounding phosphorylated protein sites with a modified shell model, and obtain some significant properties by the rank-sum test, such as the lack of some classes of residues, atoms, and secondary structures. Furthermore, we find that the depletion of some properties affects protein phosphorylation remarkably. Our results suggest that it is a meaningful direction to explore the mechanism of protein phosphorylation from microenvironment and we expect further findings along with the increasing size of phosphorylation and protein structure data.

  19. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta;

    2013-01-01

    protein, in which both EPLYA/EGLYA tyrosines were converted to phenylalanine, was no longer tyrosine phosphorylated, despite the presence of other tyrosine residues in p140Cap sequence. Moreover, this mutant lost its ability to bind the C-terminal Src kinase (Csk), previously shown to interact with p140...... phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

  20. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  1. Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina: characteristics of the phosphorylated proteins and their dephosphorylation

    International Nuclear Information System (INIS)

    To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, the cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein was studied. cGMP or cAMP with [γ-32P]ATP in the dark enhanced the phosphorylation of two ROS proteins with M/sub r/ = 10,500 (Band 1) and 8500 (Band 2) according to sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg2+. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. Both 32P-phosphorylated Bands 1 and 2 were solubilized during preparation and the molecular weight of each was 19,000. Their isoelectric point was 5.2. The sites of phosphorylation were the serine residue(s). Dephosphorylation of 37P-Bands 1 and 2 in dark-adapted ROS suspension required Mn2+ or Mg2+. Both phosphorylation and dephosphorylation were inhibited by Zn2+

  2. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.

    Science.gov (United States)

    Van Ness, Lori K; Jayaraman, Dhileepkumar; Maeda, Junko; Barrett-Wilt, Gregory A; Sussman, Michael R; Ané, Jean-Michel

    2016-01-01

    Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn. PMID:27203723

  3. Phosphorylation-Dependent PIH1D1 Interactions Define Substrate Specificity of the R2TP Cochaperone Complex

    Directory of Open Access Journals (Sweden)

    Zuzana Hořejší

    2014-04-01

    Full Text Available The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs, RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

  4. Analysis of mitochondrial DNA sequences in childhood encephalomyopathies reveals new disease-associated variants.

    Directory of Open Access Journals (Sweden)

    Aijaz A Wani

    Full Text Available BACKGROUND: Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA or nuclear genes encoding oxidative phosphorylation (OXPHOS. We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease. METHODOLOGY/PRINCIPLE FINDINGS: Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e.g. G5783A and A10149T. CONCLUSIONS AND SIGNIFICANCE: The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role

  5. Oxidative phosphorylation in cancer cells.

    Science.gov (United States)

    Solaini, Giancarlo; Sgarbi, Gianluca; Baracca, Alessandra

    2011-06-01

    Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

  6. Phylogeographic analysis reveals significant spatial genetic structure of Incarvillea sinensis as a product of mountain building

    Directory of Open Access Journals (Sweden)

    Chen Shaotian

    2012-04-01

    Full Text Available Abstract Background Incarvillea sinensis is widely distributed from Southwest China to Northeast China and in the Russian Far East. The distribution of this species was thought to be influenced by the uplift of the Qinghai-Tibet Plateau and Quaternary glaciation. To reveal the imprints of geological events on the spatial genetic structure of Incarvillea sinensis, we examined two cpDNA segments ( trnH- psbA and trnS- trnfM in 705 individuals from 47 localities. Results A total of 16 haplotypes was identified, and significant genetic differentiation was revealed (GST =0.843, NST = 0.975, P  Conclusions The results revealed that the uplift of the Qinghai-Tibet Plateau likely resulted in the significant divergence between the lineage in the eastern Qinghai-Tibet Plateau and the other one outside this area. The diverse niches in the eastern Qinghai-Tibet Plateau created a wide spectrum of habitats to accumulate and accommodate new mutations. The features of genetic diversity of populations outside the eastern Qinghai-Tibet Plateau seemed to reveal the imprints of extinction during the Glacial and the interglacial and postglacial recolonization. Our study is a typical case of the significance of the uplift of the Qinghai-Tibet Plateau and the Quaternary Glacial in spatial genetic structure of eastern Asian plants, and sheds new light on the evolution of biodiversity in the Qinghai-Tibet Plateau at the intraspecies level.

  7. Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

    Institute of Scientific and Technical Information of China (English)

    Guosheng Fu; Xia Ding; Kai Yuan; Felix Aikhionbare; Jianhui Yao; Xin Cai; Kai Jiang; Xuebiao Yao

    2007-01-01

    Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phosphorylation sites were mapped to Ser14 and Ser507 as judged by the incorporation of 32P. Although such phosphorylation is not required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgol provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

  8. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    Science.gov (United States)

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-01

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  9. Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes.

    Science.gov (United States)

    Wu, Ronghu; Dephoure, Noah; Haas, Wilhelm; Huttlin, Edward L; Zhai, Bo; Sowa, Mathew E; Gygi, Steven P

    2011-08-01

    The interpretation of quantitative phosphoproteomics studies is complicated because each differential phosphorylation event integrates both changes in protein expression and phosphorylation. Here we investigated this phenomenon by performing parallel comparisons of protein expression and phosphorylation in S. cerevisiae. In each of two experiments comparing yeast mutants bearing deletions in FUS3 or STE7 with their wild-type counterparts, we quantified over 4100 proteins, including all members of the yeast mating pathway. We also identified 12,499 unique phosphorylation sites in this work. We demonstrate the critical importance of controlling the protein-level false-discovery rate and provide a novel method to assess the accuracy of protein false-discovery rate estimates. For the first time, 96% of nonredundant phosphopeptide ratios could be calibrated by protein levels, allowing truly differential phosphorylation to be distinguished from altered protein expression. This revealed a starkly different view, with 25% of seemingly differential phosphopeptides now attributed to changes in protein expression. Combined protein expression and phosphorylation surveys uncovered both independent and concerted changes in protein expression and phosphorylation, while highlighting the partially redundant role of a second MAPK (Kss1) in the mating pathway. PMID:21551504

  10. Cardiac mitochondria in heart failure: normal cardiolipin profile and increased threonine phosphorylation of complex IV.

    Science.gov (United States)

    Rosca, Mariana; Minkler, Paul; Hoppel, Charles L

    2011-11-01

    Mitochondrial dysfunction is a major contributor in heart failure (HF). We investigated whether the decrease in respirasome organization reported by us previously in cardiac mitochondria in HF is due to changes in the phospholipids of the mitochondrial inner membrane or modifications of the subunits of the electron transport chain (ETC) complexes. The contents of the main phospholipid species, including cardiolipin, as well as the molecular species of cardiolipin were unchanged in cardiac mitochondria in HF. Oxidized cardiolipin molecular species were not observed. In heart mitochondria isolated from HF, complex IV not incorporated into respirasomes exhibits increased threonine phosphorylation. Since HF is associated with increased adrenergic drive to cardiomyocytes, this increased protein phosphorylation might be explained by the involvement of cAMP-activated protein kinase. Does the preservation of cAMP-induced phosphorylation changes of mitochondrial proteins or the addition of exogenous cAMP have similar effects on oxidative phosphorylation? The usage of phosphatase inhibitors revealed a specific decrease in complex I-supported respiration with glutamate. In saponin-permeabilized cardiac fibers, pre-incubation with cAMP decreases oxidative phosphorylation due to a defect localized at complex IV of the ETC inter alia. We propose that phosphorylation of specific complex IV subunits decreases oxidative phosphorylation either by limiting the incorporation of complex IV in supercomplexes or by decreasing supercomplex stability.

  11. Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

    Directory of Open Access Journals (Sweden)

    Gongjun Tan

    2014-11-01

    Full Text Available N,N'-dinitrosopiperazine (DNP with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567, vimentin (serine 55, stathmin (serine 25 and STAT3 (serine 727. Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors.

  12. CD133 promotes gallbladder carcinoma cell migration through activating Akt phosphorylation

    Science.gov (United States)

    Zhen, Jiaojiao; Ai, Zhilong

    2016-01-01

    Gallbladder carcinoma (GBC) is the fifth most common malignancy of gastrointestinal tract. The prognosis of gallbladder carcinoma is extremely terrible partially due to metastasis. However, the mechanisms underlying gallbladder carcinoma metastasis remain largely unknown. CD133 is a widely used cancer stem cell marker including in gallbladder carcinoma. Here, we found that CD133 was highly expressed in gallbladder carcinoma as compared to normal tissues. CD133 was located in the invasive areas in gallbladder carcinoma. Down-regulation expression of CD133 inhibited migration and invasion of gallbladder carcinoma cell without obviously reducing cell proliferation. Mechanism analysis revealed that down-regulation expression of CD133 inhibited Akt phosphorylation and increased PTEN protein level. The inhibitory effect of CD133 down-regulation on gallbladder carcinoma cell migration could be rescued by Akt activation. Consistent with this, addition of Akt inhibitor Wortmannin markedly inhibited the migration ability of CD133-overexpressing cells. Thus, down-regulation of CD133 inhibits migration of gallbladder carcinoma cells through reducing Akt phosphorylation. These findings explore the fundamental biological aspect of CD133 in gallbladder carcinoma progression, providing insights into gallbladder carcinoma cell migration. PMID:26910892

  13. PR65A phosphorylation regulates PP2A complex signaling.

    Directory of Open Access Journals (Sweden)

    Kumar Kotlo

    Full Text Available Serine-threonine Protein phosphatase 2 A (PP2A, a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac; a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa; and one of at least 18 associated variable regulatory proteins (B subunits classified into 3 families. In the present study, three in vivo sites of phosphorylation in cardiac PR65A are identified (S303, T268, S314. Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A and non-phosphorylated (N-PR65A amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A, elongation factor 2, heat shock protein 60 (HSP60, NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.

  14. Force-clamp analysis techniques reveal stretched exponential unfolding kinetics in ubiquitin

    CERN Document Server

    Lannon, Herbert; Brujic, Jasna

    2012-01-01

    Force-clamp spectroscopy reveals the unfolding and disulfide bond rupture times of single protein molecules as a function of the stretching force, point mutations and solvent conditions. The statistics of these times reveal whether the protein domains are independent of one another, the mechanical hierarchy in the polyprotein chain, and the functional form of the probability distribution from which they originate. It is therefore important to use robust statistical tests to decipher the correct theoretical model underlying the process. Here we develop multiple techniques to compare the well-established experimental data set on ubiquitin with existing theoretical models as a case study. We show that robustness against filtering, agreement with a maximum likelihood function that takes into account experimental artifacts, the Kuiper statistic test and alignment with synthetic data all identify the Weibull or stretched exponential distribution as the best fitting model. Our results are inconsistent with recently ...

  15. Single molecule analysis reveals three phases of DNA degradation by an exonuclease

    OpenAIRE

    Lee, Gwangrog; Yoo, Jungmin; Leslie, Benjamin J.; Ha, Taekjip

    2011-01-01

    λ exonuclease degrades one strand of duplex DNA in the 5’-3’ direction to generate a 3’ overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure and most studies have focused on the processive phase. Here, we use single molecule FRET to reveal three phases of λ exonuclease reactions: initiation, distributive and processive phases. The distributive phase occurs at early reactions where the 3’ overhang is too short f...

  16. Revealed comparative advantage index: an analysis of export trade in the Austrian district of Burgerland

    OpenAIRE

    Granabetter, Doris

    2016-01-01

    The Austrian District of Burgenland is located in the eastern part of the country. In the last decade, the region has benefited from different subsidy programs from the European Union. The aim of this paper is to analyze and evaluate the export competitiveness of Burgenland in relation to the foreign trade of Austria as a whole from 2010 to 2014, which is the period showing the highest increase in exports since the Iron Curtain fell. The Revealed Comparative Advantage index (RCA) was used ...

  17. Visual gene-network analysis reveals the cancer gene co-expression in human endometrial cancer

    OpenAIRE

    Chou, Wei-Chun; Cheng, An-Lin; Brotto, Marco; Chuang, Chun-Yu

    2014-01-01

    Background Endometrial cancers (ECs) are the most common form of gynecologic malignancy. Recent studies have reported that ECs reveal distinct markers for molecular pathogenesis, which in turn is linked to the various histological types of ECs. To understand further the molecular events contributing to ECs and endometrial tumorigenesis in general, a more precise identification of cancer-associated molecules and signaling networks would be useful for the detection and monitoring of malignancy,...

  18. Quantitative analysis of proteome and lipidome dynamics reveals functional regulation of global lipid metabolism

    DEFF Research Database (Denmark)

    Casanovas, Albert; Sprenger, Richard R; Tarasov, Kirill;

    2015-01-01

    architecture and processes during physiological adaptations in yeast. Our results reveal that activation of cardiolipin synthesis and remodeling supports mitochondrial biogenesis in the transition from fermentative to respiratory metabolism, that down-regulation of de novo sterol synthesis machinery prompts...... differential turnover of lipid droplet-associated triacylglycerols and sterol esters during respiratory growth, that sphingolipid metabolism is regulated in a previously unrecognized growth stage-specific manner, and that endogenous synthesis of unsaturated fatty acids constitutes an in vivo upstream activator...

  19. Functional proteomic analysis revealed ground-base ion radiations cannot reflect biological effects of space radiations of rice

    Science.gov (United States)

    Wang, Wei; Sun, Yeqing; Zhao, Qian; Han, Lu

    2016-07-01

    Highly ionizing radiation (HZE) in space is considered as main factor causing biological effects. Radiobiological studies during space flights are unrepeatable due to the variable space radiation environment, ground-base ion radiations are usually performed to simulate of the space biological effect. Spaceflights present a low-dose rate (0.1˜~0.3mGy/day) radiation environment inside aerocrafts while ground-base ion radiations present a much higher dose rate (100˜~500mGy/min). Whether ground-base ion radiation can reflect effects of space radiation is worth of evaluation. In this research, we compared the functional proteomic profiles of rice plants between on-ground simulated HZE particle radiation and spaceflight treatments. Three independent ground-base seed ionizing radiation experiments with different cumulative doses (dose range: 2˜~20000mGy) and different liner energy transfer (LET) values (13.3˜~500keV/μμm) and two independent seed spaceflight experiments onboard Chinese 20th satellite and SZ-6 spacecraft were carried out. Alterations in the proteome were analyzed by two-dimensional difference gel electrophoresis (2-D DIGE) with MALDI-TOF/TOF mass spectrometry identifications. 45 and 59 proteins showed significant (pmetabolic process, protein folding and phosphorylation. The results implied that ground-base radiations cannot truly reflect effects of spaceflight radiations, ground-base radiation was a kind of indirect effect to rice causing oxidation and metabolism stresses, but space radiation was a kind of direct effect leading to macromolecule (DNA and protein) damage and signal pathway disorders. This functional proteomic analysis work might provide a new evaluation method for further on-ground simulated HZE radiation experiments.

  20. Multilocus analysis reveals large genetic diversity in Kluyveromyces marxianus strains isolated from Parmigiano Reggiano and Pecorino di Farindola cheeses.

    Science.gov (United States)

    Fasoli, Giuseppe; Barrio, Eladio; Tofalo, Rosanna; Suzzi, Giovanna; Belloch, Carmela

    2016-09-16

    In the present study, we have analysed the genetic diversity in Kluyveromyces marxianus isolated from Parmigiano Reggiano and Pecorino di Farindola cheesemaking environment. Molecular typing methods inter-RTL fingerprint and mtDNA RFLPs, as well as, sequence diversity and heterozygosity in the intergenic region between KmSSB1 and KmRIO2 genes and analysis of the mating locus were applied to 54 K. marxianus strains. Inter-RTL fingerprint revealed a large degree of genetic heterogeneity and clustering allowed differentiation of K. marxianus strains from different geographical origins. In general, inter-LTR profiles were more discriminating than RFLPs of mtDNA; however our results also indicate that both techniques could be complementary unveiling different degrees of genetic diversity. Sequence analysis of the intergenic region between KmSSB1 and KmRIO2 genes revealed 26 variable positions in which a double peak could be observed in the sequence chromatogram. Further analysis revealed the presence of heterozygous strains in the K. marxianus population isolated from Parmigiano Reggiano. On the other hand, all strains isolated from Pecorino di Farindola were homozygous. Two very different groups of haplotypes could be observed as well as mixtures between them. Phylogenetic reconstruction divided K. marxianus dairy strains into two separate populations. A few heterozygous strains in an intermediate position between them could also be observed. Mating type locus analysis revealed a large population of diploid strains containing both MATa and MATα alleles and few haploid strains, most of them presenting the MATα allele. Different scenarios explaining the presence and maintaining of homozygous and heterozygous diploids as well as hybrids between them in the Parmigiano Reggiano K. marxianus population are proposed. A principal component analysis supported the large differences between K. marxianus isolated from Parmigiano Reggiano and Pecorino di Farindola. PMID:27294555

  1. Phosphoryl-rich flame-retardant ions (FRIONs): towards safer lithium-ion batteries.

    Science.gov (United States)

    Rectenwald, Michael F; Gaffen, Joshua R; Rheingold, Arnold L; Morgan, Alexander B; Protasiewicz, John D

    2014-04-14

    The functionalized catecholate, tetraethyl (2,3-dihydroxy-1,4-phenylene)bis(phosphonate) (H2 -DPC), has been used to prepare a series of lithium salts Li[B(DPC)(oxalato)], Li[B(DPC)2], Li[B(DPC)F2], and Li[P(DPC)3]. The phosphoryl-rich character of these anions was designed to impart flame-retardant properties for their use as potential flame-retardant ions (FRIONs), additives, or replacements for other lithium salts for safer lithium-ion batteries. The new materials were fully characterized, and the single-crystal structures of Li[B(DPC)(oxalato)] and Li[P(DPC)3] have been determined. Thermogravimetric analysis of the four lithium salts show that they are thermally stable up to around 200 °C. Pyrolysis combustion flow calorimetry reveals that these salts produce high char yields upon combustion. PMID:24615890

  2. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes.

    Science.gov (United States)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-12-01

    Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  3. Responses of cyclic phosphorylation of MAPK-like proteins in intertidal macroalgae after environmental stress.

    Science.gov (United States)

    Parages, María L; Capasso, Juan M; Niell, F Xavier; Jiménez, Carlos

    2014-02-15

    The presence and activation of MAPK-like proteins in intertidal macroalgae is described in the current study. Two MAPK-like proteins of 40 and 42 kDa in size similar to p38 and JNK, of mammalian cells have been identified in six representative species of intertidal macroalgae from the Strait of Gibraltar (Southern Spain), namely in the chlorophytes Ulva rigida and Chaetomorpha aerea, the rhodophytes Corallina elongata and Jania rubens, and the phaeophytes Dictyota dichotoma and Dilophus spiralis. Phosphorylation of MAPK-like proteins was studied during semi-tidal cycles. Analysis of p38-like and JNK-like MAPKs in macroalgae protein extracts was carried out by using specific antibodies against the phosphorylated forms of both MAPKs. Protein blot analysis of samples collected from 2009 to 2011 in natural growing sites on days when either low or high tide occurred at midday, indicated that MAPK-like proteins in all species were highly phosphorylated in response to desiccation imposed by low tide or high irradiance. Phosphorylation of p38-like MAPK always preceded that of JNK-like MAPK. In addition, phosphorylation of MAPKs was fastest in rhodophytes, followed by chlorophytes and then finally phaeophytes. In the first group, phosphorylation was mostly dependent on desiccation, whereas both high irradiance and desiccation were responsible for p38-like and JNK-like phosphorylation in chlorophytes. In phaeophytes, high irradiance was mostly responsible for MAPK-like activation. PMID:24120533

  4. Mitochondrial DNA analysis reveals three stocks of yellowfin tuna Thunnus albacares (Bonnaterre, 1788) in Indian waters

    Digital Repository Service at National Institute of Oceanography (India)

    Kunal, S.P.; GirishKumar; Menezes, M.R.; Meena, R.M.

    .001). In addition, spatial analysis of molecular variance identified three genetically heterogeneous groups of yellowfin tuna in Indian waters. Results were further corroborated by significant value of nearest neighbour statistic (S sub(nn)=0.261, P less than...

  5. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

    Science.gov (United States)

    Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, we performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and...

  6. Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation.

    Science.gov (United States)

    Shen, Qi; Chen, Yue; Jin, Danfeng; Lin, Hui; Wang, Qun; Zhao, Yu-Hua

    2016-11-01

    In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3-4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production. PMID:27664738

  7. Differential proteomic analysis reveals novel links between primary metabolism and antibiotic production in Amycolatopsis balhimycina

    DEFF Research Database (Denmark)

    Gallo, G.; Renzone, G.; Alduina, R.;

    2010-01-01

    A differential proteomic analysis, based on 2-DE and MS procedures, was performed on Amycolatopsis balhimycina DSM5908, the actinomycete producing the vancomycin-like antibiotic balhimycin. A comparison of proteomic profiles before and during balhimycin production characterized differentially...

  8. Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways

    DEFF Research Database (Denmark)

    Thøgersen, Juliane Charlotte; Mørup, Morten; Pedersen, Søren Damkiær;

    2013-01-01

    BACKGROUND: Analysis of global gene expression by DNA microarrays is widely used in experimental molecular biology. However, the complexity of such high-dimensional data sets makes it difficult to fully understand the underlying biological features present in the data.The aim of this study...... is to introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first “Archetypal Analysis” of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas...... expression between different groups identified adaptive changes of the bacteria residing in the cystic fibrosis lung. The analysis suggests a similar gene expression pattern between isolates with a high mutation rate (hypermutators) despite accumulation of different mutations for these isolates...

  9. Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Christopher L Brett

    Full Text Available Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v (5.27±0.13 was resistant to acid stress (5.28±0.14 but shifted significantly in response to alkali stress (5.83±0.13. Of 107 mutants that displayed aberrant pH(v under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v dysregulation in a neo1(ts mutant restored viability whereas cholesterol accumulation in human NPC1(-/- fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

  10. Genetic analysis reveals the complex structure of HIV-1 transmission within defined risk groups

    OpenAIRE

    HUE,S; Pillay, D; Clewley, J. P.; Pybus, O. G.

    2005-01-01

    We explored the epidemic history of HIV-1 subtype B in the United Kingdom using statistical methods that infer the population history of pathogens from sampled gene sequence data. Phylogenetic analysis of HIV-1 pol gene sequences from Britain showed at least six large transmission chains, indicating a genetically variable, but epidemiologically homogeneous, epidemic among men having sex with men. Through coalescent-based analysis we showed that these chains arose through separa...

  11. Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice.

    Directory of Open Access Journals (Sweden)

    Joeli Marrero

    2013-01-01

    Full Text Available Mycobacterium tuberculosis (Mtb is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. (13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb's central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

  12. Whole genome sequence analysis of Cryptococcus gattii from the Pacific Northwest reveals unexpected diversity.

    Directory of Open Access Journals (Sweden)

    John D Gillece

    Full Text Available A recent emergence of Cryptococcus gattii in the Pacific Northwest involves strains that fall into three primarily clonal molecular subtypes: VGIIa, VGIIb and VGIIc. Multilocus sequence typing (MLST and variable number tandem repeat analysis appear to identify little diversity within these molecular subtypes. Given the apparent expansion of these subtypes into new geographic areas and their ability to cause disease in immunocompetent individuals, differentiation of isolates belonging to these subtypes could be very important from a public health perspective. We used whole genome sequence typing (WGST to perform fine-scale phylogenetic analysis on 20 C. gattii isolates, 18 of which are from the VGII molecular type largely responsible for the Pacific Northwest emergence. Analysis both including and excluding (289,586 SNPs and 56,845 SNPs, respectively molecular types VGI and VGIII isolates resulted in phylogenetic reconstructions consistent, for the most part, with MLST analysis but with far greater resolution among isolates. The WGST analysis presented here resulted in identification of over 100 SNPs among eight VGIIc isolates as well as unique genotypes for each of the VGIIa, VGIIb and VGIIc isolates. Similar levels of genetic diversity were found within each of the molecular subtype isolates, despite the fact that the VGIIb clade is thought to have emerged much earlier. The analysis presented here is the first multi-genome WGST study to focus on the C. gattii molecular subtypes involved in the Pacific Northwest emergence and describes the tools that will further our understanding of this emerging pathogen.

  13. Comparative Genome Sequence Analysis Reveals the Extent of Diversity and Conservation for Glycan-Associated Proteins in Burkholderia spp.

    OpenAIRE

    Ong, Hui San; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2012-01-01

    Members of the Burkholderia family occupy diverse ecological niches. In pathogenic family members, glycan-associated proteins are often linked to functions that include virulence, protein conformation maintenance, surface recognition, cell adhesion, and immune system evasion. Comparative analysis of available Burkholderia genomes has revealed a core set of 178 glycan-associated proteins shared by all Burkholderia of which 68 are homologous to known essential genes. The genome sequence compari...

  14. Studies on the effect of phosphorylation on the dipeptides actions by radiation chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Manwei; Wang Zhiyong; Chu Gaosheng; Zhang Zhicheng [Univ. of Science and Technology of China, Hefei (China)

    2000-03-01

    The electron transfer within several dipeptides and their corresponding phosphorylated dipeptides was studied by electron pulse radiolysis, laser photolysis and electron spin resonance. The electron transfer rate constants were calculated by data modeling and kinetic analysis. It is found that the phosphoryl group in peptides participates the electron transfer process, and reduces the electron transfer rate in all cases. These are very important in life science since every biological process refers to the phosphorylation and nonphosphorylation of protein. It may be concerned in personalities and individualities of the personae. (author)

  15. Phosphorylation of Recombinant Tristetraprolin in Vitro

    OpenAIRE

    Cao, Heping; Lin, Rui

    2008-01-01

    Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some proinflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of proinflammatory cytokines. TTP gene expression is induced by various factors including insulin, cinnamon, and green tea extracts. Previous studies have shown that TTP is highly phosphorylated in vivo and multiple phosphorylation sites are identified in human TTP. This study evaluated the potent...

  16. In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex

    Science.gov (United States)

    Cao, Bo; Zheng, Xiaoqing; Cheng, Qiuxiang; Yao, Fen; Zheng, Tao; Ramesh Babu, I.; Zhou, Huchen; Dedon, Peter; You, Delin

    2015-01-01

    A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex. PMID:26213215

  17. Comparative genomic analysis reveals a distant liver enhancer upstream of the COUP-TFII gene

    Energy Technology Data Exchange (ETDEWEB)

    Baroukh, Nadine; Ahituv, Nadav; Chang, Jessie; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Pennacchio, Len A.

    2004-08-20

    COUP-TFII is a central nuclear hormone receptor that tightly regulates the expression of numerous target lipid metabolism genes in vertebrates. However, it remains unclear how COUP-TFII itself is transcriptionally controlled since studies with its promoter and upstream region fail to recapitulate the genes liver expression. In an attempt to identify liver enhancers in the vicinity of COUP-TFII, we employed a comparative genomic approach. Initial comparisons between humans and mice of the 3,470kb gene poor region surrounding COUP-TFII revealed 2,023 conserved non-coding elements. To prioritize a subset of these elements for functional studies, we performed further genomic comparisons with the orthologous pufferfish (Fugu rubripes) locus and uncovered two anciently conserved non-coding sequences (CNS) upstream of COUP-TFII (CNS-62kb and CNS-66kb). Testing these two elements using reporter constructs in liver (HepG2) cells revealed that CNS-66kb, but not CNS-62kb, yielded robust in vitro enhancer activity. In addition, an in vivo reporter assay using naked DNA transfer with CNS-66kb linked to luciferase displayed strong reproducible liver expression in adult mice, further supporting its role as a liver enhancer. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the substrates to better understand the regulation and expression of COUP-TFII.

  18. Adaptations to a Subterranean Environment and Longevity Revealed by the Analysis of Mole Rat Genomes

    Directory of Open Access Journals (Sweden)

    Xiaodong Fang

    2014-09-01

    Full Text Available Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber. Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.

  19. Metagenomic and whole-genome analysis reveals new lineages of gokushoviruses and biogeographic separation in the sea

    Directory of Open Access Journals (Sweden)

    Jessica Myriam Labonté

    2013-12-01

    Full Text Available Much remains to be learned about single-stranded (ss DNA viruses in natural systems, and the evolutionary relationships among them. One of the eight recognized families of ssDNA viruses is the Microviridae, a group of viruses infecting bacteria. In this study we used metagenomic analysis, genome assembly and amplicon sequencing of purified ssDNA to show that bacteriophages belonging to the subfamily Gokushovirinae within the Microviridae are genetically diverse and widespread members of marine microbial communities. Metagenomic analysis of coastal samples from the Gulf of Mexico and British Columbia, Canada, revealed numerous sequences belonging to gokushoviruses and allowed the assembly of five putative genomes with an organization similar to chlamydiamicroviruses. Fragment recruitment to these genomes from different metagenomic data sets is consistent with gokushovirus genotypes being restricted to specific oceanic regions. Conservation among the assembled genomes allowed the design of degenerate primers that target an 800 bp fragment from the gene encoding the major capsid protein. Sequences could be amplified from coastal temperate and subtropical waters, but not from samples collected from the Arctic Ocean, or freshwater lakes. Phylogenetic analysis revealed that most sequences were distantly related to those from cultured representatives. Moreover, the sequences fell into at least seven distinct evolutionary groups, most of which were represented by one of the assembled metagenomes. Our results greatly expand the known sequence space for gokushoviruses, and reveal biogeographic separation and new evolutionary lineages of gokushoviruses in the oceans.

  20. Mitotic phosphorylation of SOX2 mediated by Aurora kinase A is critical for the stem-cell like cell maintenance in PA-1 cells.

    Science.gov (United States)

    Qi, Dandan; Wang, Qianqian; Yu, Min; Lan, Rongfeng; Li, Shuiming; Lu, Fei

    2016-08-01

    Transcription factor SOX2 is multiple phosphorylated. However, the kinase and the timing regulating SOX2 phosphorylation remains poorly understood. Here we reported mitotic phosphorylation of SOX2 by Aurora kinase A (AURKA). AURKA inhibitors (VX680, Aurora kinase Inhibitor I) but not PLK1 inhibitors (BI2536, CBB2001) eliminate the mitotic phosphorylation of SOX2. Consistently, siRNA inhibition of AURKA can eliminate mitotic SOX2 phosphorylation. Ser220 and Ser251 are two sites that identified for mitotic phosphorylation on SOX2. Moreover, SOX2 mutants (S220A and S251A) can promote SOX2 induced OCT4 re-expression in differentiated cells. These findings reveal a novel regulation mechanism of SOX2 phosphorylation mediated by AURKA in mitosis and its function in stem cell pluripotency maintenance in cancer cells. PMID:27249336

  1. Statistical strategies to reveal potential vibrational markers for in vivo analysis by confocal Raman spectroscopy

    Science.gov (United States)

    Oliveira Mendes, Thiago de; Pinto, Liliane Pereira; Santos, Laurita dos; Tippavajhala, Vamshi Krishna; Téllez Soto, Claudio Alberto; Martin, Airton Abrahão

    2016-07-01

    The analysis of biological systems by spectroscopic techniques involves the evaluation of hundreds to thousands of variables. Hence, different statistical approaches are used to elucidate regions that discriminate classes of samples and to propose new vibrational markers for explaining various phenomena like disease monitoring, mechanisms of action of drugs, food, and so on. However, the technical statistics are not always widely discussed in applied sciences. In this context, this work presents a detailed discussion including the various steps necessary for proper statistical analysis. It includes univariate parametric and nonparametric tests, as well as multivariate unsupervised and supervised approaches. The main objective of this study is to promote proper understanding of the application of various statistical tools in these spectroscopic methods used for the analysis of biological samples. The discussion of these methods is performed on a set of in vivo confocal Raman spectra of human skin analysis that aims to identify skin aging markers. In the Appendix, a complete routine of data analysis is executed in a free software that can be used by the scientific community involved in these studies.

  2. The relationships among bovine αS-casein phosphorylation isoforms suggest different phosphorylation pathways.

    Science.gov (United States)

    Fang, Z H; Visker, M H P W; Miranda, G; Delacroix-Buchet, A; Bovenhuis, H; Martin, P

    2016-10-01

    Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins. PMID:27522420

  3. Compartment-Specific Phosphorylation of Squid Neurofilaments.

    Science.gov (United States)

    Grant, Philip; Pant, Harish C

    2016-01-01

    Studies of the giant axon and synapse of third-order neurons in the squid stellate ganglion have provided a vast literature on neuronal physiology and axon transport. Large neuronal size also lends itself to comparative biochemical studies of cell body versus axon. These have focused on the regulation of synthesis, assembly, posttranslational modification and function of neuronal cytoskeletal proteins (microtubules (MTs) and neurofilaments (NFs)), the predominant proteins in axoplasm. These contribute to axonal organization, stability, transport, and impulse transmission responsible for rapid contractions of mantle muscles underlying jet propulsion. Studies of vertebrate NFs have established an extensive literature on NF structure, organization, and function; studies of squid NFs, however, have made it possible to compare compartment-specific regulation of NF synthesis, assembly, and function in soma versus axoplasm. Since NFs contain over 100 eligible sites for phosphorylation by protein kinases, the compartment-specific patterns of phosphorylation have been a primary focus of biochemical studies. We have learned that NF phosphorylation is tightly compartmentalized; extensive phosphorylation occurs only in the axonal compartment in squid and in vertebrate neurons. This extensive phosphorylation plays a key role in organizing NFs, in association with microtubules (MTs), into a stable, dynamic functional lattice that supports axon growth, diameter, impulse transmission, and synaptic activity. To understand how cytoskeletal phosphorylation is topographically regulated, the kinases and phosphatases, bound to NFs isolated from cell bodies and axoplasm, have also been studied.

  4. Network Analysis Reveals the Recognition Mechanism for Mannose-binding Lectins

    Science.gov (United States)

    Zhao, Yunjie; Jian, Yiren; Zeng, Chen; Computational Biophysics Lab Team

    The specific carbohydrate binding of mannose-binding lectin (MBL) protein in plants makes it a very useful molecular tool for cancer cell detection and other applications. The biological states of most MBL proteins are dimeric. Using dynamics network analysis on molecular dynamics (MD) simulations on the model protein of MBL, we elucidate the short- and long-range driving forces behind the dimer formation. The results are further supported by sequence coevolution analysis. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

  5. Genome-wide analysis of the Arabidopsis leaf transcriptome reveals interaction of phosphate and sugar metabolism

    DEFF Research Database (Denmark)

    Muller, Renate; Morant, Marc; Jarmer, Hanne Østergaard;

    2007-01-01

    factors individually. The genes exhibiting interactions form three main clusters with different response patterns and functionality of genes. One cluster (cluster 1) most likely represents a regulatory program to support increased growth and development when both P and carbohydrates are ample. Another...... cluster (cluster 3) represents genes induced to alleviate P starvation and these are further induced by carbohydrate accumulation. Thus, interactions between P and Suc reveal two different signaling programs and novel interactions in gene regulation in response to environmental factors. cis......-Regulatory elements were analyzed for each factor and for interaction clusters. PHR1 binding sites were more frequent in promoters of P-regulated genes as compared to the entire Arabidopsis genome, and E2F and PHR1 binding sites were more frequent in interaction clusters 1 and 3, respectively....

  6. Comparative analysis reveals loss of the appetite-regulating peptide hormone ghrelin in falcons.

    Science.gov (United States)

    Seim, Inge; Jeffery, Penny L; Herington, Adrian C; Chopin, Lisa K

    2015-05-15

    Ghrelin and leptin are key peripherally secreted appetite-regulating hormones in vertebrates. Here we consider the ghrelin gene (GHRL) of birds (class Aves), where it has been reported that ghrelin inhibits rather than augments feeding. Thirty-one bird species were compared, revealing that most species harbour a functional copy of GHRL and the coding region for its derived peptides ghrelin and obestatin. We provide evidence for loss of GHRL in saker and peregrine falcons, and this is likely to result from the insertion of an ERVK retrotransposon in intron 0. We hypothesise that the loss of anorexigenic ghrelin is a predatory adaptation that results in increased food-seeking behaviour and feeding in falcons.

  7. Comprehensive molecular pathology analysis of small bowel adenocarcinoma reveals novel targets with potential for clinical utility.

    Science.gov (United States)

    Alvi, Muhammad A; McArt, Darragh G; Kelly, Paul; Fuchs, Marc-Aurel; Alderdice, Matthew; McCabe, Clare M; Bingham, Victoria; McGready, Claire; Tripathi, Shailesh; Emmert-Streib, Frank; Loughrey, Maurice B; McQuaid, Stephen; Maxwell, Perry; Hamilton, Peter W; Turkington, Richard; James, Jacqueline A; Wilson, Richard H; Salto-Tellez, Manuel

    2015-08-28

    Small bowel accounts for only 0.5% of cancer cases in the US but incidence rates have been rising at 2.4% per year over the past decade. One-third of these are adenocarcinomas but little is known about their molecular pathology and no molecular markers are available for clinical use. Using a retrospective 28 patient matched normal-tumor cohort, next-generation sequencing, gene expression arrays and CpG methylation arrays were used for molecular profiling. Next-generation sequencing identified novel mutations in IDH1, CDH1, KIT, FGFR2, FLT3, NPM1, PTEN, MET, AKT1, RET, NOTCH1 and ERBB4. Array data revealed 17% of CpGs and 5% of RNA transcripts assayed to be differentially methylated and expressed respectively (p clinically exploitable markers.

  8. Integrated analysis of multiple data sources reveals modular structure of biological networks

    International Nuclear Information System (INIS)

    It has been a challenging task to integrate high-throughput data into investigations of the systematic and dynamic organization of biological networks. Here, we presented a simple hierarchical clustering algorithm that goes a long way to achieve this aim. Our method effectively reveals the modular structure of the yeast protein-protein interaction network and distinguishes protein complexes from functional modules by integrating high-throughput protein-protein interaction data with the added subcellular localization and expression profile data. Furthermore, we take advantage of the detected modules to provide a reliably functional context for the uncharacterized components within modules. On the other hand, the integration of various protein-protein association information makes our method robust to false-positives, especially for derived protein complexes. More importantly, this simple method can be extended naturally to other types of data fusion and provides a framework for the study of more comprehensive properties of the biological network and other forms of complex networks

  9. Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

    Directory of Open Access Journals (Sweden)

    Oleg M Ganichkin

    Full Text Available BACKGROUND: Selenocysteine tRNAs (tRNA(Sec exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec-interacting factors are incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec. tRNA(Sec lacking the single-stranded 3'-acceptor end ((ΔGCCARNA(Sec yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCARNA(Sec resembles the structure of human tRNA(Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Secin vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec. CONCLUSIONS/SIGNIFICANCE: We provide the most highly resolved structure of a tRNA(Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec support its interaction with proteins.

  10. Gene Coexpression Analysis Reveals Complex Metabolism of the Monoterpene Alcohol Linalool in Arabidopsis FlowersW

    NARCIS (Netherlands)

    Ginglinger, J.F.; Boachon, B.; Hofer, R.; Paetz, C.; Kollner, T.G.; Miesch, L.; Lugan, R.; Baltenweck, R.; Mutterer, J.; Ullman, P.; Verstappen, F.W.A.; Bouwmeester, H.J.

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus

  11. Multiplatform analysis of 12 cancer types reveals molecular classification within and across tissues of origin

    DEFF Research Database (Denmark)

    Hoadley, Katherine A; Yau, Christina; Wolf, Denise M;

    2014-01-01

    Recent genomic analyses of pathologically defined tumor types identify "within-a-tissue" disease subtypes. However, the extent to which genomic signatures are shared across tissues is still unclear. We performed an integrative analysis using five genome-wide platforms and one proteomic platform o...

  12. A Bayesian multidimensional scaling procedure for the spatial analysis of revealed choice data

    NARCIS (Netherlands)

    DeSarbo, WS; Kim, Y; Fong, D

    1999-01-01

    We present a new Bayesian formulation of a vector multidimensional scaling procedure for the spatial analysis of binary choice data. The Gibbs sampler is gainfully employed to estimate the posterior distribution of the specified scalar products, bilinear model parameters. The computational procedure

  13. Meta-analysis reveals a critical period for management of powdery mildew on Hop cones

    Science.gov (United States)

    Results of 28 field trials conducted over a 12-year period investigating management of hop powdery mildew caused by Podosphaera macularis were quantitatively summarized by meta-analysis to compare product efficacy and use patterns by mode of action as defined by Fungicide Resistance Action Committe...

  14. Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis.

    NARCIS (Netherlands)

    Sergeeva, L.I.; Keurentjes, J.J.B.; Bentsink, L.; Vonk, J.; Plas, van der L.H.W.; Koornneef, M.; Vreugdenhil, D.

    2006-01-01

    The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase

  15. Proteomic analysis reveals that iron availability alters the metabolic status of the pathogenic fungus Paracoccidioides brasiliensis.

    Directory of Open Access Journals (Sweden)

    Ana F A Parente

    Full Text Available Paracoccidioides brasiliensis is a thermodimorphic fungus and the causative agent of paracoccidioidomycosis (PCM. The ability of P. brasiliensis to uptake nutrients is fundamental for growth, but a reduction in the availability of iron and other nutrients is a host defense mechanism many pathogenic fungi must overcome. Thus, fungal mechanisms that scavenge iron from host may contribute to P. brasiliensis virulence. In order to better understand how P. brasiliensis adapts to iron starvation in the host we compared the two-dimensional (2D gel protein profile of yeast cells during iron starvation to that of iron rich condition. Protein spots were selected for comparative analysis based on the protein staining intensity as determined by image analysis. A total of 1752 protein spots were selected for comparison, and a total of 274 out of the 1752 protein spots were determined to have changed significantly in abundance due to iron depletion. Ninety six of the 274 proteins were grouped into the following functional categories; energy, metabolism, cell rescue, virulence, cell cycle, protein synthesis, protein fate, transcription, cellular communication, and cell fate. A correlation between protein and transcript levels was also discovered using quantitative RT-PCR analysis from RNA obtained from P. brasiliensis under iron restricting conditions and from yeast cells isolated from infected mouse spleens. In addition, western blot analysis and enzyme activity assays validated the differential regulation of proteins identified by 2-D gel analysis. We observed an increase in glycolytic pathway protein regulation while tricarboxylic acid cycle, glyoxylate and methylcitrate cycles, and electron transport chain proteins decreased in abundance under iron limiting conditions. These data suggest a remodeling of P. brasiliensis metabolism by prioritizing iron independent pathways.

  16. Phosphorylation by Dyrk1A of clathrin coated vesicle-associated proteins: identification of the substrate proteins and the effects of phosphorylation.

    Directory of Open Access Journals (Sweden)

    Noriko Murakami

    Full Text Available Dyrk1A phosphorylated multiple proteins in the clathrin-coated vesicle (CCV preparations obtained from rat brains. Mass spectrometric analysis identified MAP1A, MAP2, AP180, and α- and β-adaptins as the phosphorylated proteins in the CCVs. Each protein was subsequently confirmed by [(32P]-labeling and immunological methods. The Dyrk1A-mediated phosphorylation released the majority of MAP1A and MAP2 and enhanced the release of AP180 and adaptin subunits from the CCVs. Furthermore, Dyrk1A displaced adaptor proteins physically from CCVs in a kinase-concentration dependent manner. The clathrin heavy chain release rate, in contrast, was not affected by Dyrk1A. Surprisingly, the Dyrk1A-mediated phosphorylation of α- and β-adaptins led to dissociation of the AP2 complex, and released only β-adaptin from the CCVs. AP180 was phosphorylated by Dyrk1A also in the membrane-free fractions, but α- and β-adaptins were not. Dyrk1A was detected in the isolated CCVs and was co-localized with clathrin in neurons from mouse brain sections and from primary cultured rat hippocampus. Previously, we proposed that Dyrk1A inhibits the onset of clathrin-mediated endocytosis in neurons by phosphorylating dynamin 1, amphiphysin 1, and synaptojanin 1. Current results suggest that besides the inhibition, Dyrk1A promotes the uncoating process of endocytosed CCVs.

  17. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes

    International Nuclear Information System (INIS)

    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H3PO4/P2O5/Et3PO4 followed by acid–base reaction with Ca(OAc)2 to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for 1H, and 31P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2 w/v%) with NaAlg solution (2 w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO4 or CaCl2 were added externally. The gelation was completed within about 3–40 min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ≤ 6.7 kPa for compressive strength at break and about 8.4 kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100–800 μm. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. - Highlights: • Preparation of water-soluble alginic acid complexes with calcium phosphate • Self-assembly of the phosphorylated alginic acid calcium complexes with sodium alginate • Preparation of injectable hydrogels with diverse gelation times within about 3–40 min

  18. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Mark R. Winter

    2015-10-01

    Full Text Available Time-lapse microscopy can capture patterns of development through multiple divisions for an entire clone of proliferating cells. Images are taken every few minutes over many days, generating data too vast to process completely by hand. Computational analysis of this data can benefit from occasional human guidance. Here we combine improved automated algorithms with minimized human validation to produce fully corrected segmentation, tracking, and lineaging results with dramatic reduction in effort. A web-based viewer provides access to data and results. The improved approach allows efficient analysis of large numbers of clones. Using this method, we studied populations of progenitor cells derived from the anterior and posterior embryonic mouse cerebral cortex, each growing in a standardized culture environment. Progenitors from the anterior cortex were smaller, less motile, and produced smaller clones compared to those from the posterior cortex, demonstrating cell-intrinsic differences that may contribute to the areal organization of the cerebral cortex.

  19. Potential analysis reveals changing number of climate states during the last 60 kyr

    Directory of Open Access Journals (Sweden)

    V. N. Livina

    2010-02-01

    Full Text Available We develop and apply a new statistical method of potential analysis for detecting the number of states of a geophysical system, from its recorded time series. Estimation of the degree of a polynomial potential allows us to derive the number of potential wells in a system. The method correctly detects changes in the number of wells in artificial data. In ice-core proxy records of Greenland paleotemperature, a reduction in the number of climate states from two to one is detected sometime prior to the last glacial maximum (LGM, 23–19 kyr BP. This result is also found in analysis of Greenland Ca data. The bifurcation can be interpreted as loss of stability of the warm interstadial state of the Dansgaard-Oeschger (DO events. The proposed method can be applied to a wide range of geophysical time series exhibiting bifurcations.

  20. Comparative proteomic analysis reveals mite (Varroa destructor) resistance-related proteins in Eastern honeybees (Apis cerana).

    Science.gov (United States)

    Ji, T; Shen, F; Liu, Z; Yin, L; Shen, J; Liang, Q; Luo, Y X

    2015-08-21

    The mite (Varroa destructor) has become the greatest threat to apiculture worldwide. As the original host of the mite, Apis cerana can effectively resist the mite. An increased understanding of the resistance mechanisms of Eastern honeybees against V. destructor may help researchers to protect other species against these parasites. In this study, the proteomes of 4 Apis cerana colonies were analyzed using an isobaric tag for relative and absolute quantitation technology. We determined the differences in gene and protein expression between susceptible and resistant colonies that were either unchallenged or challenged by V. destructor. The results showed that a total of 1532 proteins were identified. Gene Ontology enrichment analysis suggested that the transcription factors and basic metabolic and respiratory processes were efficient and feasible factors controlling this resistance, and 12 differentially expressed proteins were identified in Venn analysis. The results were validated by quantitative polymerase chain reaction. This study may provide insight into the genetic mechanisms underlying the resistance of honeybee to mites.

  1. Phylogenetic analysis of 48 gene families revealing relationships between Hagfishes, Lampreys, and Gnathostomata

    Institute of Scientific and Technical Information of China (English)

    Shuiyan Yu; Weiwei Zhang; Ling Li; Huifang Huang; Fei Ma; Qingwei Li

    2008-01-01

    It has become clear that the extant vertebrates are divided into three major groups, that is, hagfishes, lampreys, and jawed vertebrates.Morphological and molecular studies, however, have resulted in conflicting views with regard m their interrelationships. To clarify the phylogenetic relationships between them, 48 orthologous protein-coding gene families were analyzed. Even as the analysis of 34 nuclear gene families supported the monophyly of cyclostomes, the analysis of 14 mitochondrial gene families suggested a closer relationship between lampreys and gnathostomes compared to hagfishes. Lampreys were sister group of gnathostomes. The results of this study sup-ported the eyclostomes. Choice of outgroup, tree-making methods, and software may affect the phylogenetic prediction, which may have caused much debate over the subject. Development of new methods for tackling such problems is still necessary.

  2. Histopathological and ultrastructural analysis of vestibular endorgans in Meniere's disease reveals basement membrane pathology

    Directory of Open Access Journals (Sweden)

    McCall Andrew A

    2009-06-01

    Full Text Available Abstract Background We report the systematic analysis of the ultrastructural and cytological histopathology of vestibular endorgans acquired from labyrinthectomy in Meniere's disease. Methods 17 subjects with intractable Meniere's disease and ipsilateral non-serviceable hearing presenting to the Neurotology Clinic from 1997 to 2006 who chose ablative labyrinthectomy (average age = 62 years; range 29–83 years participated. The average duration of symptoms prior to surgery was 7 years (range 1–20 years. Results Nearly all vestibular endorgans demonstrated varying degrees of degeneration. A monolayer of epithelial cells occurred significantly more frequently in the horizontal cristae (12/13 = 92% (p Conclusion Systematic histopathological analysis of the vestibular endorgans from Meniere's disease demonstrated neuroepithelial degeneration which was highly correlated with an associated BM thickening. Other findings included hair cell and supporting cell microvessicles, increased intercellular clear spaces in the stroma, and endothelial cell vacuolization and stromal perivascular BM thickening.

  3. Proteomic analysis of regenerated rabbit lenses reveal crystallin expression characteristic of adult rabbits

    OpenAIRE

    Liu, Xialin; Min ZHANG; Liu, Yuhua; Challa, Pratap; Gonzalez, Pedro; Liu, Yizhi

    2008-01-01

    Purpose To explore lens crystallin characteristics and morphology of rabbit regenerated lenses in comparison with wild type natural lenses by means of proteomic analysis and histological assay. Methods The lens regeneration model of the New Zealand rabbit was established, and lens regeneration was observed by slit lamp examination and photography. A histological assay was evaluated under light microscopy and transmission electron microscopy (TEM). Protein samples of regenerated lenses were co...

  4. Analysis of the matrix metalloproteinase family reveals that MMP8 is often mutated in melanoma

    OpenAIRE

    Palavalli, Lavanya H.; Prickett, Todd D.; Wunderlich, John R.; Wei, Xiaomu; Burrell, Allison S.; Porter-Gill, Patricia; Davis, Sean; Wang, Chenwei; Cronin, Julia C.; Agrawal, Neena S.; Lin, Jimmy C.; Westbroek, Wendy; Hoogstraten-Miller, Shelley; Molinolo, Alfredo A; Fetsch, Patricia

    2009-01-01

    A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

  5. Balkanized Research in Ecological Engineering Revealed by a Bibliometric Analysis of Earthworms and Ecosystem Services

    OpenAIRE

    Blouin, Manuel; Sery, Nicolas; Cluzeau, Daniel; Brun, Jean-Jacques; Bédécarrats, Alain

    2013-01-01

    International audience Energy crisis, climate changes, and biodiversity losses have reinforced the drive for more ecologically based approaches for environmental management. Such approaches are characterized by the use of organisms rather than energy-consuming technologies. Although earthworms are believed to be potentially useful organisms for managing ecosystem services, there is actually no quantification of such a trend in literature. This bibliometric analysis aimed to measure the evo...

  6. Stable isotope analysis of vertebrae reveals ontogenetic changes in habitat in an endothermic pelagic shark

    OpenAIRE

    Carlisle, Aaron B.; Goldman, Kenneth J.; Litvin, Steven Y.; Madigan, Daniel J; Bigman, Jennifer S.; Alan M Swithenbank; Kline, Thomas C.; Barbara A. Block

    2015-01-01

    Ontogenetic changes in habitat are driven by shifting life-history requirements and play an important role in population dynamics. However, large portions of the life history of many pelagic species are still poorly understood or unknown. We used a novel combination of stable isotope analysis of vertebral annuli, Bayesian mixing models, isoscapes and electronic tag data to reconstruct ontogenetic patterns of habitat and resource use in a pelagic apex predator, the salmon shark (Lamna ditropis...

  7. Analysis of image versus position, scale and direction reveals pattern texture anisotropy

    Directory of Open Access Journals (Sweden)

    Roland eLehoucq

    2015-01-01

    Full Text Available Pattern heterogeneities and anisotropies often carry significant physical information. We provide a toolbox which: (i cumulates analysis in terms of position, direction and scale; (ii is as general as possible; (iii is simple and fast to understand, implement, execute and exploit.It consists in dividing the image into analysis boxes at a chosen scale; in each box an ellipse (the inertia tensor is fitted to the signal and thus determines the direction in which the signal is more present. This tensor can be averaged in position and/or be used to study the dependence with scale. This choice is formally linked with Leray transforms and anisotropic wavelet analysis. Such protocol is intutively interpreted and consistent with what the eye detects: relevant scales, local variations in space, priviledged directions. It is fast and parallelizable.Its several variants are adaptable to the user's data and needs. It is useful to statistically characterize anisotropies of 2D or 3D patterns in which individual objects are not easily distinguished, with only minimal pre-processing of the raw image, and more generally applies to data in higher dimensions.It is less sensitive to edge effects, and thus better adapted for a multiscale analysis down to small scale boxes, than pair correlation function or Fourier transform.Easy to understand and implement,it complements more sophisticated methods such as Hough transform or diffusion tensor imaging.We use it on various fracture patterns (sea ice cover, thin sections of granite, granular materials, to pinpoint the maximal anisotropy scales. The results are robust to noise and to user choices. This toolbox could turn also useful for granular materials, hard condensed matter, geophysics, thin films, statistical mechanics, characterisation of networks, fluctuating amorphous systems, inhomogeneous and disordered systems, or medical imaging, among others.

  8. Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling

    OpenAIRE

    Brand Andrea H; Egger Boris; von Trotha Jakob W

    2009-01-01

    Abstract Background The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain. Results We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU) labelling, that cell proliferation does occur in the Droso...

  9. Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice

    OpenAIRE

    Garg, Rohini; Tyagi, Akhilesh K.; Jain, Mukesh

    2012-01-01

    Hormones exert pleiotropic effects on plant growth and development throughout the life cycle. Many of these effects are mediated at molecular level via altering gene expression. In this study, we investigated the exogenous effect of plant hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jasmonic acid, on the transcription of rice genes at whole genome level using microarray. Our analysis identified a total of 4171 genes involved in several biological processes...

  10. A Simple Force-Motion Relation for Migrating Cells Revealed by Multipole Analysis of Traction Stress

    OpenAIRE

    Tanimoto, Hirokazu; Sano, Masaki

    2014-01-01

    For biophysical understanding of cell motility, the relationship between mechanical force and cell migration must be uncovered, but it remains elusive. Since cells migrate at small scale in dissipative circumstances, the inertia force is negligible and all forces should cancel out. This implies that one must quantify the spatial pattern of the force instead of just the summation to elucidate the force-motion relation. Here, we introduced multipole analysis to quantify the traction stress dyna...

  11. Time-series analysis reveals genetic responses to intensive management of razorback sucker (Xyrauchen texanus)

    OpenAIRE

    Thomas E Dowling; Turner, Thomas F.; Carson, Evan W; Saltzgiver, Melody J; Adams, Deborah; Kesner, Brian; Paul C Marsh

    2013-01-01

    Time-series analysis is used widely in ecology to study complex phenomena and may have considerable potential to clarify relationships of genetic and demographic processes in natural and exploited populations. We explored the utility of this approach to evaluate population responses to management in razorback sucker, a long-lived and fecund, but declining freshwater fish species. A core population in Lake Mohave (Arizona-Nevada, USA) has experienced no natural recruitment for decades and is m...

  12. Vacuolar invertase regulates elongation of Arabidopsis thaliana roots as revealed by QTL and mutant analysis

    OpenAIRE

    Sergeeva, L.I.; Keurentjes, J. J. B.; Bentsink, L.; Vonk, J.; Plas, van der, M..; Koornneef, M; Vreugdenhil, D.

    2006-01-01

    The possible role of the sucrose-splitting enzymes sucrose synthase and invertase in elongating roots and hypocotyls of Arabidopsis was tested by using a combination of histochemical methods and quantitative trait locus (QTL) analysis. Lengths of roots and hypocotyls correlated better with invertase activities than with sucrose synthase activities. The highest correlations were observed with activities in the elongating zones of roots. The genetic basis of these correlations was studied by us...

  13. The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

    Science.gov (United States)

    Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

    2001-05-01

    cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on

  14. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    Science.gov (United States)

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  15. BAHAMAS: New Analysis of Type Ia Supernovae Reveals Inconsistencies with Standard Cosmology

    Science.gov (United States)

    Shariff, Hikmatali; Jiao, Xiyun; Trotta, Roberto; van Dyk, David A.

    2016-08-01

    We present results obtained by applying our BAyesian HierArchical Modeling for the Analysis of Supernova cosmology (BAHAMAS) software package to the 740 spectroscopically confirmed supernovae of type Ia (SNe Ia) from the “Joint Light-curve Analysis” (JLA) data set. We simultaneously determine cosmological parameters and standardization parameters, including corrections for host galaxy mass, residual scatter, and object-by-object intrinsic magnitudes. Combining JLA and Planck data on the cosmic microwave background, we find significant discrepancies in cosmological parameter constraints with respect to the standard analysis: we find {{{Ω }}}{{m}}=0.399+/- 0.027, 2.8σ higher than previously reported, and w=-0.910+/- 0.045, 1.6σ higher than the standard analysis. We determine the residual scatter to be {σ }{{res}}=0.104+/- 0.005. We confirm (at the 95% probability level) the existence of two subpopulations segregated by host galaxy mass, separated at {{log}}10(M/{M}ȯ )=10, differing in mean intrinsic magnitude by 0.055 ± 0.022 mag, lower than previously reported. Cosmological parameter constraints, however, are unaffected by the inclusion of corrections for host galaxy mass. We find ˜ 4σ evidence for a sharp drop in the value of the color correction parameter, β (z), at a redshift {z}t=0.662+/- 0.055. We rule out some possible explanations for this behavior, which remains unexplained.

  16. BAHAMAS: New Analysis of Type Ia Supernovae Reveals Inconsistencies with Standard Cosmology

    Science.gov (United States)

    Shariff, Hikmatali; Jiao, Xiyun; Trotta, Roberto; van Dyk, David A.

    2016-08-01

    We present results obtained by applying our BAyesian HierArchical Modeling for the Analysis of Supernova cosmology (BAHAMAS) software package to the 740 spectroscopically confirmed supernovae of type Ia (SNe Ia) from the “Joint Light-curve Analysis” (JLA) data set. We simultaneously determine cosmological parameters and standardization parameters, including corrections for host galaxy mass, residual scatter, and object-by-object intrinsic magnitudes. Combining JLA and Planck data on the cosmic microwave background, we find significant discrepancies in cosmological parameter constraints with respect to the standard analysis: we find {{{Ω }}}{{m}}=0.399+/- 0.027, 2.8σ higher than previously reported, and w=-0.910+/- 0.045, 1.6σ higher than the standard analysis. We determine the residual scatter to be {σ }{{res}}=0.104+/- 0.005. We confirm (at the 95% probability level) the existence of two subpopulations segregated by host galaxy mass, separated at {{log}}10(M/{M}⊙ )=10, differing in mean intrinsic magnitude by 0.055 ± 0.022 mag, lower than previously reported. Cosmological parameter constraints, however, are unaffected by the inclusion of corrections for host galaxy mass. We find ˜ 4σ evidence for a sharp drop in the value of the color correction parameter, β (z), at a redshift {z}t=0.662+/- 0.055. We rule out some possible explanations for this behavior, which remains unexplained.

  17. Microsatellite analysis reveals genetically distinct populations of red pine (Pinus resinosa, Pinaceae).

    Science.gov (United States)

    Boys, Jacquelyn; Cherry, Marilyn; Dayanandan, Selvadurai

    2005-05-01

    Red pine (Pinus resinosa Ait.) is an ecologically and economically important forest tree species of northeastern North America and is considered one of the most genetically depauperate conifer species in the region. We have isolated and characterized 13 nuclear microsatellite loci by screening a partial genomic library with di-, tri-, and tetranucleotide repeat oligonucleotide probes. In an analysis of over 500 individuals representing 17 red pine populations from Manitoba through Newfoundland, five polymorphic microsatellite loci with an average of nine alleles per locus were identified. The mean expected and observed heterozygosity values were 0.508 and 0.185, respectively. Significant departures from Hardy-Weinberg equilibrium with excess homozygosity indicating high levels of inbreeding were evident in all populations studied. The population differentiation was high with 28-35% of genetic variation partitioned among populations. The genetic distance analysis showed that three northeastern (two Newfoundland and one New Brunswick) populations are genetically distinct from the remaining populations. The coalescence-based analysis suggests that "northeastern" and "main" populations likely became isolated during the most recent Pleistocene glacial period, and severe population bottlenecks may have led to the evolution of a highly selfing mating system in red pine. PMID:21652464

  18. Metabolomic analysis reveals mechanism of antioxidant butylated hydroxyanisole on lipid accumulation in Crypthecodinium cohnii.

    Science.gov (United States)

    Sui, Xiao; Niu, Xiangfeng; Shi, Mengliang; Pei, Guangsheng; Li, Jinghan; Chen, Lei; Wang, Jiangxin; Zhang, Weiwen

    2014-12-24

    The heterotrophic dinoflagellate alga Crypthecodinium cohnii is known to accumulate lipids with a high fraction of docosahexaenoic acid (DHA). In this study, we first evaluated two antioxidant compounds, butylated hydroxyanisole (BHA) and propyl gallate (PG), for their effects on lipid accumulation in C. cohnii. The results showed that antioxidant BHA could increase lipid accumulation in C. cohnii by 8.80% at a final concentration of 30 μM, while PG had no obvious effect on lipid accumulation at the tested concentrations. To decipher the molecular mechanism responsible for the increased lipid accumulation by BHA, we employed an integrated GC-MS and LC-MS metabolomic approach to determine the time-series metabolic profiles with or without BHA, and then subjected the metabolomic data to a principal component analysis (PCA) and a weighted gene coexpression network analysis (WGCNA) network analyses to identify the key metabolic modules and metabolites possibly relevant to the increased lipid accumulation. LC-MS analysis showed that several metabolites, including NADPH, could be important for the stimulation role of BHA on lipid accumulation. Meanwhile GC-MS and network analyses allowed identification of eight metabolic modules and nine hub metabolites possibly relevant to the stimulation role of BHA in C. cohnii. The study provided a metabolomics view of the BHA mode of action on lipid accumulation in C. cohnii, and the information could be valuable for a better understanding of antioxidant effects on lipid accumulation in other microalgae as well. PMID:25436856

  19. Dynamic Bcl-xL (S49) and (S62) Phosphorylation/Dephosphorylation during Mitosis Prevents Chromosome Instability and Aneuploidy in Normal Human Diploid Fibroblasts

    Science.gov (United States)

    Baruah, Prasamit Saurav; Beauchemin, Myriam; Hébert, Josée; Bertrand, Richard

    2016-01-01

    Bcl-xL proteins undergo dynamic phosphorylation/dephosphorylation on Ser49 and Ser62 residues during mitosis. The expression of Bcl-xL(S49A), (S62A) and dual (S49/62A) phosphorylation mutants in tumor cells lead to severe mitotic defects associated with multipolar spindle, chromosome lagging and bridging, and micro-, bi- and multi-nucleated cells. Because the above observations were made in tumor cells which already display genomic instability, we now address the question: will similar effects occur in normal human diploid cells? We studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL (wild type), (S49A), (S49D), (S62A), (S62D) and the dual-site (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 (IL-6) secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL(S49) and (S62) phosphorylation/dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. They could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy. PMID:27398719

  20. Phosphorylation of Streptococcus salivarius Lactose Permease (LacS) by HPr(His∼P) and HPr(Ser-P)(His∼P) and Effects on Growth

    Science.gov (United States)

    Lessard, Christian; Cochu, Armelle; Lemay, Jean-Dominique; Roy, Denis; Vaillancourt, Katy; Frenette, Michel; Moineau, Sylvain; Vadeboncoeur, Christian

    2003-01-01

    The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His∼P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIALacS) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIALacS was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His∼P) but also by HPr(Ser-P)(His∼P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIABLMan and IIABHMan, were unable to phosphorylate IIALacS. The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His∼P) or HPr(Ser-P)(His∼P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose. PMID:14617640

  1. The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation.

    Science.gov (United States)

    Zündorf, Gregor; Reiser, Georg

    2011-12-01

    Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (•-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration. PMID:21988180

  2. ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Yan Chunhong; Han Rui

    1998-01-01

    Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cell-ECM interaction and its role. Methods:To interrupt the cell-ECM interaction by suppression of adhesion-induced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16-B16mouse melanoma cells. Results: When B16-BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein inresponse to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesion-induced protein tyrosine phosphorylation the genistein-treated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genistein-treated cells.Conclusion: It was suggested that protein tyrosine phosphorylation in cell-ECM interaction might be associated with invasive and metastatic potentials in cancer cells.

  3. Antennal RNA-sequencing analysis reveals evolutionary aspects of chemosensory proteins in the carpenter ant, Camponotus japonicus.

    Science.gov (United States)

    Hojo, Masaru K; Ishii, Kenichi; Sakura, Midori; Yamaguchi, Katsushi; Shigenobu, Shuji; Ozaki, Mamiko

    2015-01-01

    Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants. PMID:26310137

  4. Antennal RNA-sequencing analysis reveals evolutionary aspects of chemosensory proteins in the carpenter ant, Camponotus japonicus.

    Science.gov (United States)

    Hojo, Masaru K; Ishii, Kenichi; Sakura, Midori; Yamaguchi, Katsushi; Shigenobu, Shuji; Ozaki, Mamiko

    2015-08-27

    Chemical communication is essential for the coordination of complex organisation in ant societies. Recent comparative genomic approaches have revealed that chemosensory genes are diversified in ant lineages, and suggest that this diversification is crucial for social organisation. However, how such diversified genes shape the peripheral chemosensory systems remains unknown. In this study, we annotated and analysed the gene expression profiles of chemosensory proteins (CSPs), which transport lipophilic compounds toward chemosensory receptors in the carpenter ant, Camponotus japonicus. Transcriptome analysis revealed 12 CSP genes and phylogenetic analysis showed that 3 of these are lineage-specifically expanded in the clade of ants. RNA sequencing and real-time quantitative polymerase chain reaction revealed that, among the ant specific CSP genes, two of them (CjapCSP12 and CjapCSP13) were specifically expressed in the chemosensory organs and differentially expressed amongst ant castes. Furthermore, CjapCSP12 and CjapCSP13 had a ratio of divergence at non-synonymous and synonymous sites (dN/dS) greater than 1, and they were co-expressed with CjapCSP1, which is known to bind cuticular hydrocarbons. Our results suggested that CjapCSP12 and CjapCSP13 were functionally differentiated for ant-specific chemosensory events, and that CjapCSP1, CjapCSP12, and CjapCSP13 work cooperatively in the antennal chemosensilla of worker ants.

  5. Novel protein phosphorylation site identification in spinach stroma membranes by titanium dioxide microcolumns and tandem mass spectrometry

    DEFF Research Database (Denmark)

    Rinalducci, Sara; Larsen, Martin Røssel; Mohammed, Shabaz;

    2006-01-01

    In this work, spinach stroma membrane, instead of thylakoid, has been investigated for the presence of phosphorylated proteins. We identified seven previously unknown phosphorylation sites by taking advantage of TiO(2) phosphopeptides enrichment coupled to mass spectrometric analysis. Upon illumi...

  6. Chicken genome analysis reveals novel genes encoding biotin-binding proteins related to avidin family

    Directory of Open Access Journals (Sweden)

    Nordlund Henri R

    2005-03-01

    Full Text Available Abstract Background A chicken egg contains several biotin-binding proteins (BBPs, whose complete DNA and amino acid sequences are not known. In order to identify and characterise these genes and proteins we studied chicken cDNAs and genes available in the NCBI database and chicken genome database using the reported N-terminal amino acid sequences of chicken egg-yolk BBPs as search strings. Results Two separate hits showing significant homology for these N-terminal sequences were discovered. For one of these hits, the chromosomal location in the immediate proximity of the avidin gene family was found. Both of these hits encode proteins having high sequence similarity with avidin suggesting that chicken BBPs are paralogous to avidin family. In particular, almost all residues corresponding to biotin binding in avidin are conserved in these putative BBP proteins. One of the found DNA sequences, however, seems to encode a carboxy-terminal extension not present in avidin. Conclusion We describe here the predicted properties of the putative BBP genes and proteins. Our present observations link BBP genes together with avidin gene family and shed more light on the genetic arrangement and variability of this family. In addition, comparative modelling revealed the potential structural elements important for the functional and structural properties of the putative BBP proteins.

  7. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies.

    Directory of Open Access Journals (Sweden)

    Masakazu Kohda

    2016-01-01

    Full Text Available Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (MRPS23, QRSL1, and PNPLA4 as novel causative genes. We also identified 2 genes known to cause monogenic diseases (MECP2 and TNNI3 and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21 as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.

  8. Regulation of BZR1 in fruit ripening revealed by iTRAQ proteomics analysis

    Science.gov (United States)

    Liu, Lihong; Liu, Haoran; Li, Shuo; Zhang, Xin; Zhang, Min; Zhu, Ning; Dufresne, Craig P.; Chen, Sixue; Wang, Qiaomei

    2016-01-01

    Fruit ripening is a complex and genetically programmed process. Brassinosteroids (BRs) play an essential role in plant growth and development, including fruit ripening. As a central component of BR signaling, the transcription factor BZR1 is involved in fruit development in tomato. However, the transcriptional network through which BZR1 regulates fruit ripening is mostly unknown. In this study, we use isobaric tags for relative and absolute quantitation (iTRAQ) labeling technology to explore important proteins regulated by BZR1 in two independent tomato transgenic lines over-expressing BZR1-1D at four ripening stages, identifying 411 differentially expressed proteins. These proteins were implicated in light reaction, plant hormone pathways and cell-wall-related metabolism, etc. The ‘light reaction’ metabolic pathway was identified as a markedly enhanced pathway by BZR1 during tomato fruit ripening. The protein level of a probable 2-oxoglutarate-dependent dioxygenase 2-ODD2, involved in gibberellin biosynthesis was significantly increased at all four developmental and ripening stages. The results reveal molecular links between BR signaling pathway and downstream components involved in multiple ripening-associated events during tomato fruit ripening, which will provide new insights into the molecular mechanisms underlying tomato ripening regulatory networks, and be potential in understanding BR-regulated fruit ripening. PMID:27680870

  9. Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

    Directory of Open Access Journals (Sweden)

    Uta Gey

    Full Text Available The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p. The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p, Lys114 (Kgd2p and Lys102 (Gcv3p, respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

  10. Characterization and phylogenetic analysis of -gliadin gene sequences reveals significant genomic divergence in Triticeae species

    Indian Academy of Sciences (India)

    Guang-Rong Li; Tao Lang; En-Nian Yang; Cheng Liu; Zu-Jun Yang

    2014-12-01

    Although the unique properties of wheat -gliadin gene family are well characterized, little is known about the evolution and genomic divergence of -gliadin gene family within the Triticeae. We isolated a total of 203 -gliadin gene sequences from 11 representative diploid and polyploid Triticeae species, and found 108 sequences putatively functional. Our results indicate that -gliadin genes may have possibly originated from wild Secale species, where the sequences contain the shortest repetitive domains and display minimum variation. A miniature inverted-repeat transposable element insertion is reported for the first time in -gliadin gene sequence of Thinopyrum intermedium in this study, indicating that the transposable element might have contributed to the diversification of -gliadin genes family among Triticeae genomes. The phylogenetic analyses revealed that the -gliadin gene sequences of Dasypyrum, Australopyrum, Lophopyrum, Eremopyrum and Pseudoroengeria species have amplified several times. A search for four typical toxic epitopes for celiac disease within the Triticeae -gliadin gene sequences showed that the -gliadins of wild Secale, Australopyrum and Agropyron genomes lack all four epitopes, while other Triticeae species have accumulated these epitopes, suggesting that the evolution of these toxic epitopes sequences occurred during the course of speciation, domestication or polyploidization of Triticeae.

  11. Service innovation management practices in the telecommunications industry: what does cross country analysis reveal?

    Science.gov (United States)

    Rahman, Syed Abidur; Taghizadeh, Seyedeh Khadijeh; Ramayah, T; Ahmad, Noor Hazlina

    2015-01-01

    Service innovation management practice is currently being widely scrutinized mainly in the developed countries, where it has been initiated. The current study attempts to propose a framework and empirically validate and explain the service innovation practices for successful performance in the telecommunications industry of two developing countries, Malaysia and Bangladesh. The research framework proposes relationships among organisational culture, operating core (innovation process, cross-functional organisation, and implementation of tools/technology), competition-informed pricing, and performance. A total of 176 usable data from both countries are analysed for the purpose of the research. The findings show that organisational culture tends to be more influential on innovation process and cross-functional organisation in Malaysian telecommunication industry. In contrast, implementation of tools/technology plays a more instrumental role in competition-informed pricing practices in Bangladesh. This study revealed few differences in the innovation management practices between two developing countries. The findings have strategic implications for the service sectors in both the developing countries regarding implementation of innovative enterprises, especially in Bangladesh where innovation is the basis for survival. Testing the innovation management practices in the developing countries perhaps contains uniqueness in the field of innovation management. PMID:26722630

  12. Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species.

    Science.gov (United States)

    Lee, Kim-Chung; Tam, Emily W T; Lo, Ka-Ching; Tsang, Alan K L; Lau, Candy C Y; To, Kelvin K W; Chan, Jasper F W; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2015-06-17

    Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu-Glu-Leu-Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu-Glu-Leu-Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species.

  13. Transcriptome analysis reveals novel genes involved in nonhost response to bacterial infection in tobacco.

    Science.gov (United States)

    Daurelio, Lucas Damián; Petrocelli, Silvana; Blanco, Francisca; Holuigue, Loreto; Ottado, Jorgelina; Orellano, Elena Graciela

    2011-03-01

    Plants are continuously exposed to pathogen challenge. The most common defense response to pathogenic microorganisms is the nonhost response, which is usually accompanied by transcriptional changes. In order to identify genes involved in nonhost resistance, we evaluated the tobacco transcriptome profile after infection with Xanthomonas axonopodis pv. citri (Xac), a nonhost phytopathogenic bacterium. cDNA-amplified fragment length polymorphism was used to identify differentially expressed transcripts in tobacco leaves infected with Xac at 2, 8 and 24h post-inoculation. From a total of 2087 transcript-derived fragments (TDFs) screened (approximately 20% of the tobacco transcriptome), 316 TDFs showed differential expression. Based on sequence similarities, 82 differential TDFs were identified and assigned to different functional categories: 56 displayed homology to genes with known functions, 12 to proteins with unknown functions and 14 did not have a match. Real-time PCR was carried out with selected transcripts to confirm the expression pattern obtained. The results reveal novel genes associated with nonhost resistance in plant-pathogen interaction in tobacco. These novel genes could be included in future strategies of molecular breeding for nonhost disease resistance. PMID:20828873

  14. Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species

    Directory of Open Access Journals (Sweden)

    Kim-Chung Lee

    2015-06-01

    Full Text Available Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu–Glu–Leu–Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxybenzoic acid and (sulfooxybenzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxybenzoic acid possesses a structure similar to those of aspirin [2-(acetoxybenzoic acid] and salicylic acid (2-hydroxybenzoic acid. Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu–Glu–Leu–Glu, hydroxy-(sulfooxybenzoic acid and (sulfooxybenzoic acid are virulent factors of the pathogenic Aspergillus species.

  15. A novel genome-wide full- length kinesin prediction analysis reveals additional mammalian kinesins

    Institute of Scientific and Technical Information of China (English)

    XUE Yu; LIU Dan; FU Chuanhai; DOU Zhen; ZHOU Qing; YAO Xuebiao

    2006-01-01

    Kinesin superfamily of microtubule- based motor orchestrates a variety of cellular processes. Recent availability of mammalian genomes has enabled analyses of kinesins on the whole genome. Here we present a novel full-length kinesin prediction program (FKPP) for mammalian kinesin gene discovery based on a comparative genomics approach. Contrary to previous predictions of 94 kinesins, we identify a total of 134 potentially kinesin genes from mammalian genomes, including 45 from mouse, 45 from rat and 44 from human. In addition, FKPP synthesizes 25 potentially full-length mammalian kinesins based on the partial sequences in the database. Surprisingly, FKPP reveals that full-length human CENP-E contains 2701 aa rather than 2663 aa in the database. Experimentation using sequence specific antibody and cDNA sequencing of human CENP-E validates the accuracy of FKPP. Given the remarkable computing efficiency and accuracy of FKPP, we reclassify the mammalian kinesin superfamily. Since current databases contain many incomplete sequences, FKPP may provide a novel approach for molecular delineation of kinesins and other protein families.

  16. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  17. Metagenomic analysis reveals that modern microbialites and polar microbial mats have similar taxonomic and functional potential

    Directory of Open Access Journals (Sweden)

    Richard Allen White III

    2015-09-01

    Full Text Available Within the subarctic climate of Clinton Creek, Yukon, Canada, lies an abandoned and flooded open-pit asbestos mine that harbors rapidly growing microbialites. To understand their formation we completed a metagenomic community profile of the microbialites and their surrounding sediments. Assembled metagenomic data revealed that bacteria within the phylum Proteobacteria numerically dominated this system, although the relative abundances of taxa within the phylum varied among environments. Bacteria belonging to Alphaproteobacteria and Gammaproteobacteria were dominant in the microbialites and sediments, respectively. The microbialites were also home to many other groups associated with microbialite formation including filamentous cyanobacteria and dissimilatory sulfate-reducing Deltaproteobacteria, consistent with the idea of a shared global microbialite microbiome. Other members were present that are typically not associated with microbialites including Gemmatimonadetes and iron-oxidizing Betaproteobacteria, which participate in carbon metabolism and iron cycling. Compared to the sediments, the microbialite microbiome has significantly more genes associated with photosynthetic processes (e.g., photosystem II reaction centers, carotenoid and chlorophyll biosynthesis and carbon fixation (e.g., CO dehydrogenase. The Clinton Creek microbialite communities had strikingly similar functional potentials to non-lithifying microbial mats from the Canadian High Arctic and Antarctica, but are functionally distinct, from non-lithifying mats or biofilms from Yellowstone. Clinton Creek microbialites also share metabolic genes (R2 0.900. These metagenomic profiles from an anthropogenic microbialite-forming ecosystem provide context to microbialite formation on a human-relevant timescale.

  18. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  19. Integrative Genome-wide Analysis Reveals Cooperative Regulation of Alternative Splicing by hnRNP Proteins

    Directory of Open Access Journals (Sweden)

    Stephanie C. Huelga

    2012-02-01

    Full Text Available Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here, we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre-mRNA splicing (AS in human cells. Using splicing-sensitive microarrays, crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveal position-dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross- and autoregulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.

  20. Comparative Transcriptome Analysis to Reveal Genes Involved in Wheat Hybrid Necrosis

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    2014-12-01

    Full Text Available Wheat hybrid necrosis is an interesting genetic phenomenon that is found frequently and results in gradual death or loss of productivity of wheat. However, the molecular basis and mechanisms of this genetic phenomenon are still not well understood. In this study, the transcriptomes of wheat hybrid necrosis F1 and its parents (Neimai 8 and II469 were investigated using digital gene expression (DGE. A total of 1300 differentially expressed genes were identified, indicating that the response to hybrid necrosis in wheat is complicated. The assignments of the annotated genes based on Gene Ontology (GO revealed that most of the up-regulated genes belong to “universal stress related”, “DNA/RNA binding”, “protein degradation” functional groups, while the down-regulated genes belong to “carbohydrate metabolism” and “translation regulation” functional groups. These findings suggest that these pathways were affected by hybrid necrosis. Our results provide preliminarily new insight into the underlying molecular mechanisms of hybrid necrosis and will help to identify important candidate genes involved in wheat hybrid necrosis.

  1. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta "Gold Standard" Leaves.

    Science.gov (United States)

    Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

    2016-01-01

    Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta "Gold Standard" is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta "Gold Standard", as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta "Gold Standard". For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves. PMID:27005614

  2. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves

    Directory of Open Access Journals (Sweden)

    Juanjuan Yu

    2016-03-01

    Full Text Available Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1, heat shock protein 70 (Hsp70, and chloroplastic elongation factor G (cpEF-G were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.

  3. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves

    Science.gov (United States)

    Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

    2016-01-01

    Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves. PMID:27005614

  4. Service innovation management practices in the telecommunications industry: what does cross country analysis reveal?

    Science.gov (United States)

    Rahman, Syed Abidur; Taghizadeh, Seyedeh Khadijeh; Ramayah, T; Ahmad, Noor Hazlina

    2015-01-01

    Service innovation management practice is currently being widely scrutinized mainly in the developed countries, where it has been initiated. The current study attempts to propose a framework and empirically validate and explain the service innovation practices for successful performance in the telecommunications industry of two developing countries, Malaysia and Bangladesh. The research framework proposes relationships among organisational culture, operating core (innovation process, cross-functional organisation, and implementation of tools/technology), competition-informed pricing, and performance. A total of 176 usable data from both countries are analysed for the purpose of the research. The findings show that organisational culture tends to be more influential on innovation process and cross-functional organisation in Malaysian telecommunication industry. In contrast, implementation of tools/technology plays a more instrumental role in competition-informed pricing practices in Bangladesh. This study revealed few differences in the innovation management practices between two developing countries. The findings have strategic implications for the service sectors in both the developing countries regarding implementation of innovative enterprises, especially in Bangladesh where innovation is the basis for survival. Testing the innovation management practices in the developing countries perhaps contains uniqueness in the field of innovation management.

  5. Systematic transcriptome analysis reveals elevated expression of alcohol-metabolizing genes in NAFLD livers.

    Science.gov (United States)

    Zhu, Ruixin; Baker, Susan S; Moylan, Cynthia A; Abdelmalek, Manal F; Guy, Cynthia D; Zamboni, Fausto; Wu, Dingfeng; Lin, Weili; Liu, Wensheng; Baker, Robert D; Govindarajan, Sugantha; Cao, Zhiwei; Farci, Patrizia; Diehl, Anna Mae; Zhu, Lixin

    2016-03-01

    Obese animals and non-alcoholic fatty liver disease (NAFLD) patients exhibit elevated blood alcohol, suggesting potential contributions of alcohol metabolism to the development of NAFLD. Liver gene expression in patients with biopsy-proven mild (N = 40) and severe (N = 32) NAFLD were compared to that in healthy liver donors (N = 7) and alcoholic hepatitis (AH; N = 15) using microarrays. Principal components analyses (PCA) revealed similar gene expression patterns between mild and severe NAFLD which clustered with those of AH but were distinct from those of healthy livers. Differential gene expression between NAFLD and healthy livers was consistent with established NAFLD-associated genes and NAFLD pathophysiology. Alcohol-metabolizing enzymes including ADH, ALDH, CYP2E1, and CAT were up-regulated in NAFLD livers. The expression level of alcohol-metabolizing genes in severe NAFLD was similar to that in AH. The NAFLD gene expression profiles provide new directions for future investigations to identify disease markers and targets for prevention and treatment, as well as to foster our understanding of NAFLD pathogenesis and pathophysiology. Particularly, increased expression of alcohol-metabolizing genes in NAFLD livers supports a role for endogenous alcohol metabolism in NAFLD pathology and provides further support for gut microbiome therapy in NAFLD management. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley © Sons, Ltd.

  6. Proteomic analysis of a segregant population reveals candidate proteins linked to mealiness in peach.

    Science.gov (United States)

    Almeida, Andréa Miyasaka; Urra, Claudio; Moraga, Carol; Jego, Marcela; Flores, Alejandra; Meisel, Lee; González, Mauricio; Infante, Rodrigo; Defilippi, Bruno G; Campos-Vargas, Reinaldo; Orellana, Ariel

    2016-01-10

    Peaches are stored at low temperatures to delay ripening and increase postharvest life. However some varieties are susceptible to chilling injury,which leads to fruit mealiness, browning and flesh bleeding. In order to identify potentialmarkers associated with chilling injury,we performed proteomic analyses on a segregating population with contrasting susceptibility to chilling-induced mealiness. Chilling-induced mealiness was assessed by measuring juiciness in fruits that have been stored in cold and then allowed to ripen. Fruitmesocarp and leaf proteome from contrasting segregants were analyzed using 2-DE gels. Comparison of protein abundance between segregants revealed 133 spots from fruit mesocarp and 36 from leaf. Thirty four fruit mesocarp proteins were identified from these spots. Most of these proteins were related to ethylene synthesis, ABA response and stress response. Leaf protein analyses identified 22 proteins, most of which related to energy metabolism. Some of the genes that code for these proteins have been previously correlated with chilling injury through transcript analyses and co-segregation with mealiness QTLs. The results from this study, further deciphers the molecular mechanisms associated with chilling response in peach fruit, and identifies candidate proteins linked to mealiness in peach which may be used as putative markers for this trait. PMID:26459401

  7. Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability.

    Directory of Open Access Journals (Sweden)

    Mathieu Cayla

    2014-09-01

    Full Text Available Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10. Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

  8. Phosphorylation and inactivation of glycogen synthase kinase 3β (GSK3β) by dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A).

    Science.gov (United States)

    Song, Woo-Joo; Song, Eun-Ah Christine; Jung, Min-Su; Choi, Sun-Hee; Baik, Hyung-Hwan; Jin, Byung Kwan; Kim, Jeong Hee; Chung, Sul-Hee

    2015-01-23

    Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr(356) residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)(356)-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β. PMID:25477508

  9. Behavioral Analysis of Dopaminergic Activation in Zebrafish and Rats Reveals Similar Phenotypes.

    Science.gov (United States)

    Ek, Fredrik; Malo, Marcus; Åberg Andersson, Madelene; Wedding, Christoffer; Kronborg, Joel; Svensson, Peder; Waters, Susanna; Petersson, Per; Olsson, Roger

    2016-05-18

    Zebrafish is emerging as a complement to mammals in behavioral studies; however, there is a lack of comparative studies with rodents and humans to establish the zebrafish as a predictive translational model. Here we present a detailed phenotype evaluation of zebrafish larvae, measuring 300-3000 variables and analyzing them using multivariate analysis to identify the most important ones for further evaluations. The dopamine agonist apomorphine has previously been shown to have a complex U-shaped dose-response relationship in the variable distance traveled. In this study, we focused on breaking down distance traveled into more detailed behavioral phenotypes for both zebrafish and rats and identified in the multivariate analysis low and high dose phenotypes with characteristic behavioral features. Further analysis of single parameters also identified an increased activity at the lowest concentration indicative of a U-shaped dose-response. Apomorphine increased the distance of each swim movement (bout) at both high and low doses, but the underlying behavior of this increase is different; at high dose, both bout duration and frequency increased whereas bout max speed was higher at low dose. Larvae also displayed differences in place preference. The low dose phenotype spent more time in the center, indicative of an anxiolytic effect, while the high-dose phenotype had a wall preference. These dose-dependent effects corroborated findings in a parallel rat study and previous observations in humans. The translational value of pharmacological zebrafish studies was further evaluated by comparing the amino acid sequence of the dopamine receptors (D1-D4), between zebrafish, rats and humans. Humans and zebrafish share 100% of the amino acids in the binding site for D1 and D3 whereas D2 and D4 receptors share 85-95%. Molecular modeling of dopamine D2 and D4 receptors indicated that nonconserved amino acids have limited influence on important ligand-receptor interactions. PMID

  10. Phylogenetic analysis of the Australian rosella parrots (Platycercus) reveals discordance among molecules and plumage.

    Science.gov (United States)

    Shipham, Ashlee; Schmidt, Daniel J; Joseph, Leo; Hughes, Jane M

    2015-10-01

    Relationships and species limits among the colourful Australian parrots known as rosellas (Platycercus) are contentious because of poorly understood patterns of parapatry, sympatry and hybridization as well as complex patterns of geographical replacement of phenotypic forms. Two subgenera are, however, conventionally recognised: Platycercus comprises the blue-cheeked crimson rosella complex (Crimson Rosella P. elegans and Green Rosella P. caledonicus), and Violania contains the remaining four currently recognised species (Pale-headed Rosella P. adscitus, Eastern Rosella P. eximius, Northern Rosella P. venustus, and Western Rosella P. icterotis). We used phylogenetic analysis of ten loci (one mitochondrial, eight autosomal and one z-linked) and several individuals per nominal species primarily to examine relationships within the subgenera, especially the relationships and species limits within Violania. Of these, P. adscitus and P. eximius have long been considered sister species or conspecific due to a morphology-based hybrid zone and an early phylogenetic analysis of mitochondrial DNA restriction fragment length polymorphisms. The multilocus phylogenetic analysis presented here supports an alternative hypothesis aligning P. adscitus and P. venustus as sister species. Using divergence rates published in other avian studies, we estimated the divergence between P. venustus and P. adscitus at 0.0148-0.6124MYA and that between the P. adscitus/P. venustus ancestor and P. eximius earlier at 0.1617-1.0816MYA, both within the Pleistocene. Discordant topologies among gene and species trees are discussed and proposed to be the result of historical gene flow and/or incomplete lineage sorting (ILS). In particular, we suggest that discordance between mitochondrial and nuclear data may be the result of asymmetrical mitochondrial introgression from P. adscitus into P. eximius. The biogeographical implications of our findings are discussed relative to similarly distributed groups

  11. Phylogenetic analysis of the Australian rosella parrots (Platycercus) reveals discordance among molecules and plumage.

    Science.gov (United States)

    Shipham, Ashlee; Schmidt, Daniel J; Joseph, Leo; Hughes, Jane M

    2015-10-01

    Relationships and species limits among the colourful Australian parrots known as rosellas (Platycercus) are contentious because of poorly understood patterns of parapatry, sympatry and hybridization as well as complex patterns of geographical replacement of phenotypic forms. Two subgenera are, however, conventionally recognised: Platycercus comprises the blue-cheeked crimson rosella complex (Crimson Rosella P. elegans and Green Rosella P. caledonicus), and Violania contains the remaining four currently recognised species (Pale-headed Rosella P. adscitus, Eastern Rosella P. eximius, Northern Rosella P. venustus, and Western Rosella P. icterotis). We used phylogenetic analysis of ten loci (one mitochondrial, eight autosomal and one z-linked) and several individuals per nominal species primarily to examine relationships within the subgenera, especially the relationships and species limits within Violania. Of these, P. adscitus and P. eximius have long been considered sister species or conspecific due to a morphology-based hybrid zone and an early phylogenetic analysis of mitochondrial DNA restriction fragment length polymorphisms. The multilocus phylogenetic analysis presented here supports an alternative hypothesis aligning P. adscitus and P. venustus as sister species. Using divergence rates published in other avian studies, we estimated the divergence between P. venustus and P. adscitus at 0.0148-0.6124MYA and that between the P. adscitus/P. venustus ancestor and P. eximius earlier at 0.1617-1.0816MYA, both within the Pleistocene. Discordant topologies among gene and species trees are discussed and proposed to be the result of historical gene flow and/or incomplete lineage sorting (ILS). In particular, we suggest that discordance between mitochondrial and nuclear data may be the result of asymmetrical mitochondrial introgression from P. adscitus into P. eximius. The biogeographical implications of our findings are discussed relative to similarly distributed groups

  12. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Energy Technology Data Exchange (ETDEWEB)

    Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  13. Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

    OpenAIRE

    Ginglinger, J.-F.; Boachon, B.; Hofer, R.; Paetz, C.; Kollner, T. G.; Miesch, L.; Lugan, R.; Baltenweck, R.; Mutterer, J.; Ullmann, P.; Beran, F.; Claudel, P.; Verstappen, F.; Fischer, M. J. C.; Karst, F

    2013-01-01

    The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpe