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Sample records for analysis reveals phosphorylation

  1. 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...... experimental and computational pipeline, we investigated the conservation of human phosphorylation events in distantly related model organisms (fly, worm, and yeast). With a sequence-alignment approach, we identified 479 phosphorylation events in 344 human proteins that appear to be positionally conserved over...... approximately 600 million years of evolution and hence are likely to be involved in fundamental cellular processes. This sequence-alignment analysis suggested that many phosphorylation sites evolve rapidly and therefore do not display strong evolutionary conservation in terms of sequence position in distantly...

  2. 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

  3. 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

    we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation...... sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site...

  4. 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...

  5. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    OpenAIRE

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Bin CHENG; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique p...

  6. 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.

  7. Analysis of the sodium chloride-dependent respiratory kinetics of wheat mitochondria reveals differential effects on phosphorylating and non-phosphorylating electron transport pathways.

    Science.gov (United States)

    Jacoby, R P; Che-Othman, M H; Millar, A H; Taylor, N L

    2016-04-01

    A number of previous studies have documented the gross response of mitochondrial respiration to salinity treatment, but it is unclear how NaCl directly affects the kinetics of plant phosphorylating and non-phosphorylating electron transport pathways. This study investigates the direct effects of NaCl upon different respiratory pathways in wheat, by measuring rates of isolated mitochondrial oxygen consumption across different substrate oxidation pathways in saline media. We also profile the abundance of respiratory proteins by using targeted selected reaction monitoring (SRM) mass spectrometry of mitochondria isolated from control and salt-treated wheat plants. We show that all pathways of electron transport were inhibited by NaCl concentrations above 400 mM; however electron transfer chains showed divergent responses to NaCl concentrations between 0 and 200 mM. Stimulation of oxygen consumption was measured in response to NaCl in scenarios where exogenous NADH was provided as substrate and electron flow was coupled to the generation of a proton gradient across the inner membrane. Protein abundance measurements show that several enzymes with activities less affected by NaCl are induced by salinity, whereas enzymes with activities inhibited by NaCl are depleted. These data deepen our understanding of how plant respiration responds to NaCl, offering new mechanistic explanations for the divergent salinity responses of whole-plant respiratory rate in the literature. PMID:26470009

  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

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skel...

  9. Label-free LC-MS/MS Analysis with Automated Phosphopeptide Enrichment Reveals Dynamic Human Milk Protein Phosphorylation during Lactation

    OpenAIRE

    Froehlich, John W.; Chu, Caroline S.; Tang, Ning; Waddell, Keith; Grimm, Rudolf; Lebrilla, Carlito B.

    2010-01-01

    Protein phosphorylation is a critical post-translational modification that affects cell-cell signaling and protein function. However, quantifying the relative, site-specific changes of phosphorylation occupancies remain a major issue. An on-line enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards, and utilized to det...

  10. Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes*

    OpenAIRE

    Zhao, Xiaolu; León, Ileana R.; Bak, Steffen; Mogensen, Martin; Wrzesinski, Krzysztof; Højlund, Kurt; Jensen, Ole Nørregaard

    2010-01-01

    Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human...

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

    Science.gov (United States)

    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 reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27.

    Science.gov (United States)

    Wu, Wan-Ling; Liao, Jiahn-Haur; Lin, Guang-Huey; Lin, Miao-Hsia; Chang, Ying-Che; Liang, Suh-Yuen; Yang, Feng-Ling; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2013-10-01

    Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production. PMID:23828892

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

    Science.gov (United States)

    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. PMID:23221999

  14. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    Science.gov (United States)

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-01

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs. PMID:26476206

  15. Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag gel analysis.

    Science.gov (United States)

    Madec, Edwige; Bontemps-Gallo, Sébastien; Lacroix, Jean-Marie

    2014-12-01

    Osmoregulated periplasmic glucans (OPGs) are general constituents of many proteobacteria. OPGs are important factors required for full virulence in many pathogens including Dickeya dadantii. D. dadantii causes the soft-rot disease in a wide range of plant species. The pleiotropic phenotype of opg-negative strains includes total loss of virulence and motility, and is linked to the constitutive activation of the RcsCDB phosphorelay, deduced from expression analysis of genes of the RcsCDB regulon. The constitutive activation of the RcsCDB phosphorelay in an opg-negative strain was demonstrated by direct analysis of the phosphorylation level of the RcsB regulator protein in vivo by using a Phos-tag retardation gel approach, and was correlated with the phenotype and the expression of motility genes. Data revealed a low level of RcsB phosphorylated form in the wild-type strain and a slight increase of phosphorylation in opgG mutant strains sufficient to induce the pleiotropic phenotype observed. PMID:25320363

  16. 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....

  17. Analysis of Protein Phosphorylation Using Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Pao-Chi Liao

    2008-06-01

    Full Text Available Protein phosphorylation has been known to be a pivotalmodification regulating many cellular activities and functions.Except for several conventional techniques, mass spectrometry-based strategies are increasingly considered as vital toolsthat can be utilized to characterize phosphorylated peptides orproteins. In this article, we summarized currently availablemass spectrometry-based techniques for the analysis of phosphorylation.Due to the low abundance of phosphopeptides,enrichment steps such as specific antibodies, immobilizedmetal affinity chromatography, and specific tags are crucial fortheir use in detection. Since the non-specific binding of theenrichment techniques are constantly of major concerns, phosphatasetreatment, neutral loss scan, or precursor ion scanenable the recognition of the phosphopeptide signals. In addition,quantitative methods including isotope labeling and masstags are also discussed. Phosphoproteome analysis seems to provide elucidation of signalingnetworks and global decipherment of cell activities, which require powerful analytical methodsfor complete and routine identification of the phosphorylation event. Despite thatnumerous approaches have been exploited, comprehensive analysis of protein phosphorylationremains a challenging task. With the progressively more improvements of instrumentsand methodologies, we can foresee the implementation of a comprehensive approach for theanalysis of phosphorylation states of proteins.

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

    Science.gov (United States)

    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

  19. Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

    OpenAIRE

    la Fuente van Bentem, Sergio de; Anrather, Dorothea; Roitinger, Elisabeth; Djamei, Armin; Hufnagl, Thomas; Barta, Andrea; Csaszar, Edina; Dohnal, Ilse; Lecourieux, David; Hirt, Heribert

    2006-01-01

    Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in...

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

    Science.gov (United States)

    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.

  1. Automatic generation of predictive dynamic models reveals nuclear phosphorylation as the key Msn2 control mechanism.

    Science.gov (United States)

    Sunnåker, Mikael; Zamora-Sillero, Elias; Dechant, Reinhard; Ludwig, Christina; Busetto, Alberto Giovanni; Wagner, Andreas; Stelling, Joerg

    2013-05-28

    Predictive dynamical models are critical for the analysis of complex biological systems. However, methods to systematically develop and discriminate among systems biology models are still lacking. We describe a computational method that incorporates all hypothetical mechanisms about the architecture of a biological system into a single model and automatically generates a set of simpler models compatible with observational data. As a proof of principle, we analyzed the dynamic control of the transcription factor Msn2 in Saccharomyces cerevisiae, specifically the short-term mechanisms mediating the cells' recovery after release from starvation stress. Our method determined that 12 of 192 possible models were compatible with available Msn2 localization data. Iterations between model predictions and rationally designed phosphoproteomics and imaging experiments identified a single-circuit topology with a relative probability of 99% among the 192 models. Model analysis revealed that the coupling of dynamic phenomena in Msn2 phosphorylation and transport could lead to efficient stress response signaling by establishing a rate-of-change sensor. Similar principles could apply to mammalian stress response pathways. Systematic construction of dynamic models may yield detailed insight into nonobvious molecular mechanisms. PMID:23716718

  2. Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks

    OpenAIRE

    Reiland, S; Messerli, G.; Baerenfaller, K.; Gerrits, B.; Endler, A; Grossmann, J.; Gruissem, W; Baginsky, S

    2009-01-01

    We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as w...

  3. 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...

  4. Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3

    OpenAIRE

    Vincent Michel; Lavoie Sébastien B; Albert Alexandra L

    2004-01-01

    Abstract Background The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation. Pin1 isomerizes the peptide bond of specific phosphorylated serine or threonine residues preceding proline in several proteins involved in various cellular events including mitosis, transcription, differentiation and DNA damage response. Many Pin1 substrates are antigens of the phosphodependent monoclonal antibody MPM-2, which reacts with a ...

  5. 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...

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

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max

    2012-01-01

    Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in...... 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....... 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 in...

  7. 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.

  8. Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3

    Directory of Open Access Journals (Sweden)

    Vincent Michel

    2004-06-01

    Full Text Available Abstract Background The peptidyl-prolyl isomerase Pin1 recently revealed itself as a new player in the regulation of protein function by phosphorylation. Pin1 isomerizes the peptide bond of specific phosphorylated serine or threonine residues preceding proline in several proteins involved in various cellular events including mitosis, transcription, differentiation and DNA damage response. Many Pin1 substrates are antigens of the phosphodependent monoclonal antibody MPM-2, which reacts with a subset of proteins phosphorylated at the G2/M transition. Results As MPM-2 is not a general marker of mitotic phosphoproteins, and as most mitotic substrates are phosphorylated more than once, we used a different phosphodependent antibody, mAb CC-3, to identify additional mitotic phosphoproteins and eventual Pin1 substrates by combining affinity purification, MALDI-TOF mass spectrometry and immunoblotting. Most CC-3-reactive phosphoproteins appeared to be known or novel MPM-2 antigens and included the RNA-binding protein p54nrb/nmt55, the spliceosomal protein SAP155, the Ki-67 antigen, MAP-1B, DNA topoisomerases II α and β, the elongation factor hSpt5 and the largest subunit of RNA polymerase II. The CC-3 mitotic antigens were also shown to be Pin1 targets. The fine CC-3- and MPM-2-epitope mapping of the RNA polymerase II carboxy-terminal domain confirmed that the epitopes were different and could be generated in vitro by distinct kinases. Finally, the post-mitotic dephosphorylation of both CC-3 and MPM-2 antigens was prevented when cellular Pin1 activity was blocked by the selective inhibitor juglone. Conclusion These observations indicate that the mitotic phosphoproteins associated with Pin1 are phosphorylated on multiple sites, suggesting combinatorial regulation of substrate recognition and isomerization.

  9. 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.

  10. 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 (

  11. Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun; Meyer, Christian; Geetha, Thangiah; 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 (...

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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. Published 2016. This article is a U.S. Government work and is in the public domain in the

  17. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    Science.gov (United States)

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  18. Analysis of proteins phosphorylated at the early response of DNA double-strand breaks

    International Nuclear Information System (INIS)

    The serine/threonine protein kinase, ATM, recognizes DNA double-strand breaks (DSBs) at a very early stage, and plays a central role in transmitting damage signals to the cell cycle control machinery. To further understand the early events in DSBs recognition, we are undertaking two approaches: 1) identification of novel substrates which are phosphorylated by ATM using phospho-specific antibody, and 2) analysis of phosphorylation profile employing the proteomics-based 2-DE gel electrophoresis technique. There is a well-known consensus sequence for phosphorylation by ATM characterized by serine followed by glutamine (SQ), and their surrounding amino acids. Using one of the consensus sequences, we synthesized a phosphorylated peptide for immunizing rabbit and generated a phospho-specific antibody. This novel antibody recognized phosphorylated proteins in mouse fibroblast cultures after 5 min of exposure to ionizing radiation. This high and constant level of phosphorylation persisted until the 2nd hour and started decreasing to basal levels from the fourth hour. This antibody also detected phospho-proteins in mouse fibroblast cultures exposed to chemical reagents that induce DSBs such as NCS, bleomycin and etoposide, and is thought to be specific for DNA DSBs-related proteins. Furthermore, this antibody did not recognize phospho-proteins in ATM-deficient cells, suggesting that this phosphorylation is dependent on ATM kinase or downstream kinases controlled by ATM. As a second approach, we identified several spots that are phosphorylated in ATM wild type cells but not in ATM-deficient cells by 2-DE proteomic analysis. We are conducting further studies to generate comprehensive, ATM-dependent phosphorylation profiles as a novel approach to understand early events in DNA damage recognition by ATM

  19. Analysis of phosphorylation-dependent protein-protein interactions of histone H3.

    OpenAIRE

    Klingberg, R.; Jost, J.; Schümann, M; Gelato, K.; Fischle, W; Krause, E.; Schwarzer, D

    2015-01-01

    Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. ...

  20. Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 reveals a tight link between tyrosine phosphorylation and virulence.

    Science.gov (United States)

    Lin, Miao-Hsia; Hsu, Tung-Li; Lin, Shu-Yu; Pan, Yi-Jiun; Jan, Jia-Tsrong; Wang, Jin-Town; Khoo, Kay-Hooi; Wu, Shih-Hsiung

    2009-12-01

    Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD(50) increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD(50) increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044. PMID:19696081

  1. Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils.

    Science.gov (United States)

    Shindo, Sawako; Moore, Rick; Negishi, Masahiko

    2016-01-01

    Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with αP-S216 or neutrophil marker Ly6G antibody) and double immunofluorescence staining of mouse uterine sections prepared from C3H/HeNCrIBR females revealed that phosphorylated ERα was expressed in all infiltrating neutrophils during hormonal cycles but not in any other of the other uterine cells. Neutrophils infiltrate the uterus from the blood stream. White blood cells (WBC) were prepared from peripheral blood of C3H/HeNCrIBR females or males and double immunostained. Blood neutrophils also expressed phosphorylated ERα but in only about 20 % of cells in both sexes. Only the neutrophils expressing phosphorylated ERα spontaneously migrated in in vitro Transwell migration assays and infiltrated the uterus in mice. PMID:26585153

  2. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks.

    Science.gov (United States)

    White, Forest M; Wolf-Yadlin, Alejandro

    2016-06-12

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks. PMID:27049636

  3. Automatic Generation of Predictive Dynamic Models Reveals Nuclear Phosphorylation as the Key Msn2 Control Mechanism

    OpenAIRE

    Sunnåker, Mikael; Zamora-Sillero, Elias; Dechant, Reinhard; Ludwig, Christina; Busetto, Alberto Giovanni; Wagner, Andreas; Stelling, Joerg

    2013-01-01

    Predictive dynamical models are critical for the analysis of complex biological systems. However, methods to systematically develop and discriminate among systems biology models are still lacking. Here, we describe a computational method that incorporates all hypothetical mechanisms about the architecture of a biological system into a single model, and automatically generates a set of simpler models compatible with observational data. As a proof-of-principle, we analyzed the dynamic control o...

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

    International Nuclear Information System (INIS)

    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

  11. 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...

  12. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells

    Science.gov (United States)

    Paulo, Joao A.; Gaun, Aleksandr; Gygi, Steven P.

    2016-01-01

    Smoking is a risk factor in pancreatic disease, however, the biochemical mechanisms correlating smoking with pancreatic dysfunction remain poorly understood. Strategies using multiplexed isobaric tag-based mass spectrometry facilitate the study of drug-induced perturbations on biological systems. Here, we present the first large scale analysis of the proteomic and phosphoproteomic alterations in pancreatic stellate cells following treatment with two nicotinic acetylcholine receptor (nAChR) ligands: nicotine and α-bungarotoxin. We treated cells with nicotine or α-bungarotoxin for 12hr in triplicate and compared alterations in protein expression and phosphorylation levels to mock treated cells using a tandem mass tag (TMT9plex)-based approach. Over 8,100 proteins were quantified across all nine samples of which 46 were altered in abundance upon treatment with nicotine. Proteins with increased abundance included those associated with neurons, defense mechanisms, indicators of pancreatic disease and lysosomal proteins. In addition, we measured differences for ∼16,000 phosphorylation sites across all nine samples using a titanium dioxide-based strategy, of which 132 sites were altered with nicotine and 451 with α-bungarotoxin treatment. Many altered phosphorylation sites were involved in nuclear function and transcriptional events. This study supports the development of future targeted investigations to establish a better understanding for the role of nicotine and associated receptors in pancreatic disease. PMID:26265067

  13. Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

    Science.gov (United States)

    Klingberg, Rebecca; Jost, Jan Oliver; Schümann, Michael; Gelato, Kathy Ann; Fischle, Wolfgang; Krause, Eberhard; Schwarzer, Dirk

    2015-01-16

    Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails. PMID:25330109

  14. Analysis of the Phosphorylation Sites of Herpes Simplex Virus Type 1 Regulatory Protein ICP27

    OpenAIRE

    Zhi, Yan; Sandri-Goldin, Rozanne M.

    1999-01-01

    The herpes simplex virus type 1 (HSV-1) regulatory protein ICP27 is a 63-kDa phosphoprotein required for viral replication. ICP27 has been shown to contain both stable phosphate groups and phosphate groups that cycle on and off during infection (K. W. Wilcox, A. Kohn, E. Sklyanskaya, and B. Roizman, J. Virol. 33:167–182, 1980). Despite extensive genetic analysis of the ICP27 gene, there is no information available about the sites of the ICP27 molecule that are phosphorylated during viral infe...

  15. An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

    Science.gov (United States)

    Shih, Heather H; Tu, Chao; Cao, Wei; Klein, Anne; Ramsey, Renee; Fennell, Brian J; Lambert, Matthew; Ní Shúilleabháin, Deirdre; Autin, Bénédicte; Kouranova, Eugenia; Laxmanan, Sri; Braithwaite, Steven; Wu, Leeying; Ait-Zahra, Mostafa; Milici, Anthony J; Dumin, Jo Ann; LaVallie, Edward R; Arai, Maya; Corcoran, Christopher; Paulsen, Janet E; Gill, Davinder; Cunningham, Orla; Bard, Joel; Mosyak, Lydia; Finlay, William J J

    2012-12-28

    Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity. PMID:23148212

  16. 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

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

    International Nuclear Information System (INIS)

    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

  18. 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.

  19. Analysis of Ipl1-Mediated Phosphorylation of the Ndc80 Kinetochore Protein in Saccharomyces cerevisiae

    OpenAIRE

    Akiyoshi, Bungo; Nelson, Christian R.; Ranish, Jeffrey A; Biggins, Sue

    2009-01-01

    Phosphorylation of the Ndc80 kinetochore protein by the Ipl1/Aurora B kinase reduces its microtubule binding activity in vitro. We found that kinetochore-bound Ndc80 is phosphorylated on Ipl1 sites in vivo, but this phosphorylation is not essential. Instead, we show that additional Ipl1 targets contribute to segregation and the spindle checkpoint.

  20. 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...

  1. 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

  2. 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

  3. 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

  4. 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...

  5. 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...

  6. Dynamic Light Scattering Analysis of the Effect of Phosphorylated Osteopontin Peptides on Mineral Formation

    Science.gov (United States)

    Mozaffari, Maryam; Goiko, Maria; de Bruyn, John; Goldberg, Harvey

    2015-03-01

    Biomineralization is the process by which living organisms synthesize minerals. Osteopontin (OPN), a mineral-associated protein, has been shown to be a potent inhibitor of mineral formation, a process that is dependent on phosphorylation. To gain a better understanding of the mechanism of inhibition, dynamic light scattering (DLS) was used to monitor the initial stages of nucleation, providing information about the size and relative concentration of the growing crystals as a function of time. DLS was used to investigate the effect of phosphorylated (P3, pOPAR) and non-phosphorylated (P0, OPAR) OPN peptides on the formation and growth of hydroxyapatite (HA) crystals from supersaturated solutions of calcium and phosphate ions. The non-phosphorylated P0 had a limited effect on HA nucleation and growth, while its thrice-phosphorylated isoform, P3, was a potent inhibitor of HA nucleation. The aspartic acid-rich OPAR was found to moderately inhibit nucleation but not growth, while its singly-phosphorylated isoform, pOPAR, inhibited HA nucleation more effectively, with some effect on HA crystal growth. The order of the inhibitory potential of these peptides was pOPAR>OPAR>P3>P0. This work confirms that highly acidic and phosphorylated peptides can inhibit the nucleation of HA more effectively.

  7. 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

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

    International Nuclear Information System (INIS)

    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. 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. 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. The identification of pTyr proteins and signaling pathways associated

  9. Quantitative Analysis of Cytokine-Induced Hepatocyte Nuclear Factor-4α Phosphorylation by Mass Spectrometry

    OpenAIRE

    Wang, Zhongyan; Salih, Erdjan; Burke, Peter A.

    2011-01-01

    Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative ...

  10. Quantitative analysis of protein phosphorylation on a system-wide scale by mass spectrometry-based proteomics.

    Science.gov (United States)

    Bodenmiller, Bernd; Aebersold, Ruedi

    2010-01-01

    Systems biology at the molecular level is concerned with networks of interacting molecules, their structure, and dynamic response to perturbations that give rise to systems' properties that determine measurable, macroscopic phenotypes. At any time, in any cell, multiple types of molecular networks are concurrently active. One of the most important known regulatory systems in eukaryotic cells is reversible protein phosphorylation catalyzed by protein kinases and phosphatases, respectively. Therefore, it is essential to understand and eventually model the protein phosphorylation-mediated informational fluxes in cells from sensors and signaling systems to effector molecules, to comprehensively analyze the dynamic system of kinases/phosphatases and their substrates and to determine the basic rules of information processing in cells. In this chapter, we describe the protocols necessary to comprehensively and quantitatively measure the phosphorylation-modulated informational networks in cells. The pipeline relies on the selective, quantitative isolation of phosphopeptides generated by the tryptic digestion of complex protein mixtures and their subsequent mass spectrometric and computational analysis. We believe that the protocols and data processing tools described in this chapter will be a valuable resource for biologists interested in the analysis of protein phosphorylation-based signal transduction. PMID:20946816

  11. Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events

    OpenAIRE

    Liao, Lujian; McClatchy, Daniel B.; Park, Sung Kyu; Xu, Tao; Lu, Bingwen; Yates, John R.

    2008-01-01

    Protein phosphorylation is a globally adopted and tightly controlled post-translational modification, and represents one of the most important molecular switching mechanisms that govern the entire spectrum of biological processes. In the central nervous system, it has been demonstrated that phosphorylation of key proteins mediating chromatin remodeling and gene transcription plays an important role controlling brain development, synaptogenesis, learning and memory. Many studies have focused o...

  12. Identification and analysis of phosphorylation status of proteins in dormant terminal buds of poplar

    Directory of Open Access Journals (Sweden)

    Liu Chang-Cai

    2011-11-01

    Full Text Available Abstract Background Although there has been considerable progress made towards understanding the molecular mechanisms of bud dormancy, the roles of protein phosphorylation in the process of dormancy regulation in woody plants remain unclear. Results We used mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies to investigate the phosphoproteome of dormant terminal buds (DTBs in poplar (Populus simonii × P. nigra. There were 161 unique phosphorylated sites in 161 phosphopeptides from 151 proteins; 141 proteins have orthologs in Arabidopsis, and 10 proteins are unique to poplar. Only 34 sites in proteins in poplar did not match well with the equivalent phosphorylation sites of their orthologs in Arabidopsis, indicating that regulatory mechanisms are well conserved between poplar and Arabidopsis. Further functional classifications showed that most of these phosphoproteins were involved in binding and catalytic activity. Extraction of the phosphorylation motif using Motif-X indicated that proline-directed kinases are a major kinase group involved in protein phosphorylation in dormant poplar tissues. Conclusions This study provides evidence about the significance of protein phosphorylation during dormancy, and will be useful for similar studies on other woody plants.

  13. 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.

  14. 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

  15. 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

  16. Irradiation influence on a protein phosphorylation systems singularity functioning analysis using non-traditional methods

    International Nuclear Information System (INIS)

    Molecular mechanisms of functioning of phosphorylation different systems in spleen lymphocytes of rats in control and under ionizing radiation are studied as well as their participation in cell adaptation reaction in response to irradiation. Experiments were performed using rats and RUM-17 X-ray plant, irradiation was conducted at 0.5 and 1 Gy. It is shown that the exposure at 0.5 Gy dose does not disturb the functional relations between kinetic indices of protein phosphorylation while the irradiation at 1 Gy dose leads to their total distortion

  17. 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.

  18. Two new organotin(IV) phosphoryl complexes: crystal structure and Hirshfeld surface analysis.

    Czech Academy of Sciences Publication Activity Database

    Pourayoubi, M.; Saneei, A.; Dušek, Michal; Rostami, S.A.; Crochet, A.; Kučeráková, Monika

    2015-01-01

    Roč. 12, č. 12 (2015), s. 2093-2103. ISSN 1735-207X R&D Projects: GA ČR(CZ) GA14-03276S Institutional support: RVO:68378271 Keywords : Hirshfeld surfaces * tin * organotin(IV)-phosphoryl complex * crystal structure Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 1.087, year: 2014

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

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Wagner, Sebastian A; Beli, Petra; Gupta, Rajat; Kristiansen, Trine A; Malinova, Dessislava; Francavilla, Chiara; Tolar, Pavel; Bishop, Gail A; Hostager, Bruce S; Choudhary, Chuna Ram

    2015-01-01

    ubiquitylation 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...... provided datasets can serve as a valuable resource for further understanding BCR signaling networks....

  20. 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.

  1. 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

  2. Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes.

    OpenAIRE

    Chevet, E.; Wong, H N; Gerber, D.; Cochet, C; A Fazel; Cameron, P H; Gushue, J N; Thomas, D. Y.; Bergeron, J J

    1999-01-01

    Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two-dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isola...

  3. Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

    Science.gov (United States)

    Iliuk, Anton; Li, Li; Melesse, Michael; Hall, Mark C; Tao, W Andy

    2016-05-17

    Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. PMID:27037847

  4. 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.

  5. 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. PMID:26841317

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

    OpenAIRE

    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 string of dimeric ATP synthase (complex V(2)). A row-like organization of OXPHOS complexes 1, III and IV into respiratory strings has also been proposed. These transient strings cannot be purified...

  7. 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...

  8. 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.

  9. 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

  10. Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7-induced signaling.

    Science.gov (United States)

    Rochman, Yrina; Kashyap, Mohit; Robinson, Gertraud W; Sakamoto, Kazuhito; Gomez-Rodriguez, Julio; Wagner, Kay-Uwe; Leonard, Warren J

    2010-11-01

    Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays essential roles in allergic/inflammatory skin and airway disorders, in helminth infections, and in regulating intestinal immunity. TSLP signals via IL-7Rα and a specific TSLPR subunit that is highly related to the common cytokine receptor γ chain, γ(c). Although TSLP has effects on a broad range of hematopoetic cells and can induce STAT5 phosphorylation, TSLP was reported to not signal via JAK kinases, and the mechanism by which TSLP regulates STAT5 phosphorylation has been unclear. We now demonstrate the role of JAK1 and JAK2 in TSLP-mediated STAT5 phosphorylation in mouse and human primary CD4(+) T cells, in contrast to the known activation of JAK1 and JAK3 by the related cytokine, IL-7. We also show that just as JAK1 interacts with IL-7Rα, JAK2 is associated with TSLPR protein. Moreover, we demonstrate the importance of STAT5 activation for TSLP-mediated survival and proliferation of CD4(+) T cells. These findings clarify the basis for TSLP-mediated signaling and provide an example wherein a cytokine uses JAK1 and JAK2 to mediate the activation of STAT5. PMID:20974963

  11. RPPA-based protein profiling reveals eIF4G overexpression and 4E-BP1 serine 65 phosphorylation as molecular events that correspond with a pro-survival phenotype in chronic lymphocytic leukemia.

    Science.gov (United States)

    Shull, Austin Y; Noonepalle, Satish K; Awan, Farrukh T; Liu, Jimei; Pei, Lirong; Bollag, Roni J; Salman, Huda; Ding, Zhiyong; Shi, Huidong

    2015-06-10

    Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival. PMID:25999352

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

    International Nuclear Information System (INIS)

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (32P) PO43- 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

  13. 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.

  14. Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7–induced signaling

    OpenAIRE

    Rochman, Yrina; Kashyap, Mohit; Robinson, Gertraud W; Sakamoto, Kazuhito; Gomez-Rodriguez, Julio; Wagner, Kay-Uwe; Leonard, Warren J.

    2010-01-01

    Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays essential roles in allergic/inflammatory skin and airway disorders, in helminth infections, and in regulating intestinal immunity. TSLP signals via IL-7Rα and a specific TSLPR subunit that is highly related to the common cytokine receptor γ chain, γc. Although TSLP has effects on a broad range of hematopoetic cells and can induce STAT5 phosphorylation, TSLP was reported to not signal via JAK kinases, and the mechanism by whic...

  15. 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...

  16. 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.

  17. The Use of Solid Supports to Facilitate Peptide Chemistry for Analytical Purposes Including Phosphorylation Analysis

    OpenAIRE

    Willis, Ian; Angeletti, Ruth Hogue; Nika, Heinz; Hawke, David

    2012-01-01

    We have been exploring the use of sample immobilization on reversed-phase supports to modify peptides prior to mass spectrometric analysis. An immediate consequence is that the products are eluted from the support after washing, in essentially the same way peptides are often “cleaned up” for analysis. The priciple is that we can both modify and purify peptides on the same support. For example we have adapted the Ba2+ ion-catalyzed concurrent Michael addition reaction to solid-phase derivatiza...

  18. 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.

  19. 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...

  20. Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

    Science.gov (United States)

    Lanucara, Francesco; Chi Hoo Lee, Dave; Eyers, Claire E.

    2013-12-01

    A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

  1. 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

  2. Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

    Science.gov (United States)

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima; Fisher-Wellman, Kelsey H; Kleinert, Maximilian; Humphrey, Sean J; Yang, Pengyi; Holliday, Mira; Trefely, Sophie; Fazakerley, Daniel J; Stöckli, Jacqueline; Burchfield, James G; Jensen, Thomas E; Jothi, Raja; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; James, David E

    2015-11-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry. PMID:26437602

  3. 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....

  4. 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.

  5. 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.

  6. AKAP signaling in reinstated cocaine seeking revealed by iTRAQ proteomic analysis.

    Science.gov (United States)

    Reissner, Kathryn J; Uys, Joachim D; Schwacke, John H; Comte-Walters, Susanna; Rutherford-Bethard, Jennifer L; Dunn, Thomas E; Blumer, Joe B; Schey, Kevin L; Kalivas, Peter W

    2011-04-13

    To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc. PMID:21490206

  7. 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.

  8. 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.

  9. 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

  10. Salt stress-induced protein phosphorylation

    International Nuclear Information System (INIS)

    Protein phosphorylation induced by salt stress in tomato germinating seeds were investigated by two-dimensional polyacrilamide gel electrophoresis of proteins labeled in vivo with (32P)-Phosphate. NaCl induced the phosphorylation of a 14 Kd polypeptide. Pulse-chase experiments revealed that the phosphorylated molecules of this polypeptide are only stable while the stress is present. Phosphorylated 14 Kd polypeptides could be detected in radicles of salt-shocked seedlings after 6 hours stress period. 14 Kd polypeptide phosphorylation was also observed in seeds germinating in the presence of abscisic acid (ABA). The amount of phosphorylated 14 Kd polypeptide was significantly increased in seeds treated simultaneously with NaCl and ABA

  11. 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

  12. 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

  13. 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

  14. 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 explan...

  15. Functional Analysis of Dishevelled-3 Phosphorylation Identifies Distinct Mechanisms Driven by Casein Kinase 1 epsilon and Frizzled5

    Czech Academy of Sciences Publication Activity Database

    Bernatík, Ondřej; Šedová, K.; Schille, C.; Ganji, R.S.; Červenka, I.; Trantírek, L.; Schambony, A.; Zdráhal, Z.; Bryja, Vítězslav

    2014-01-01

    Roč. 289, č. 34 (2014), s. 23520-23533. ISSN 0021-9258 R&D Projects: GA ČR(CZ) GP13-31488P Institutional support: RVO:68081707 Keywords : Cell Signaling * Mass Spectrometry * Phosphorylation Subject RIV: BO - Biophysics Impact factor: 4.573, year: 2014

  16. 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...

  17. 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

  18. 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.

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

    International Nuclear Information System (INIS)

    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

  20. Transcriptome meta-analysis reveals dysregulated pathways in nasopharyngeal carcinoma.

    Science.gov (United States)

    Tulalamba, Warut; Larbcharoensub, Noppadol; Sirachainan, Ekaphop; Tantiwetrueangdet, Aunchalee; Janvilisri, Tavan

    2015-08-01

    Nasopharyngeal carcinoma (NPC) is a malignant cancer arising from the epithelial surface of the nasopharynx that mostly appears in advanced stages of the disease, leading to a poor prognosis. To date, a number of mRNA profiling investigations on NPC have been reported in order to identify suitable biomarkers for early detection. However, the results may be specific to each study with distinct sample types. In this study, an integrative meta-analysis of NPC transcriptome data was performed to determine dysregulated pathways, potentially leading to identification of molecular markers. Ten independent NPC gene expression profiling microarray datasets, including 135 samples from NPC cell lines, primary cell lines, and tissues were assimilated into a meta-analysis and cross-validation to identify a cohort of genes that were significantly dysregulated in NPC. Bioinformatics analyses of these genes revealed the significant pathways and individual players involving in cellular metabolism, cell cycle regulation, DNA repair, as well as ErbB pathway. Altogether, we propose that dysregulation of these molecular pathways in NPC might play a role in the NPC pathogenesis, providing clues, which could eventually translate into diagnostic and therapeutic approaches. PMID:25724187

  1. Comparative analysis reveals the underlying mechanism of vertebrate seasonal reproduction.

    Science.gov (United States)

    Ikegami, Keisuke; Yoshimura, Takashi

    2016-02-01

    Animals utilize photoperiodic changes as a calendar to regulate seasonal reproduction. Birds have highly sophisticated photoperiodic mechanisms and functional genomics analysis in quail uncovered the signal transduction pathway regulating avian seasonal reproduction. Birds detect light with deep brain photoreceptors. Long day (LD) stimulus induces secretion of thyroid-stimulating hormone (TSH) from the pars tuberalis (PT) of the pituitary gland. PT-derived TSH locally activates thyroid hormone (TH) in the hypothalamus, which induces gonadotropin-releasing hormone (GnRH) and hence gonadotropin secretion. However, during winter, low temperatures increase serum TH for adaptive thermogenesis, which accelerates germ cell apoptosis by activating the genes involved in metamorphosis. Therefore, TH has a dual role in the regulation of seasonal reproduction. Studies using TSH receptor knockout mice confirmed the involvement of PT-derived TSH in mammalian seasonal reproduction. In addition, studies in mice revealed that the tissue-specific glycosylation of TSH diversifies its function in the circulation to avoid crosstalk. In contrast to birds and mammals, one of the molecular machineries necessary for the seasonal reproduction of fish are localized in the saccus vasculosus from the photoreceptor to the neuroendocrine output. Thus, comparative analysis is a powerful tool to uncover the universality and diversity of fundamental properties in various organisms. PMID:26050562

  2. 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.

  3. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

    Science.gov (United States)

    Cuomo, Christina A; Desjardins, Christopher A; Bakowski, Malina A; Goldberg, Jonathan; Ma, Amy T; Becnel, James J; Didier, Elizabeth S; Fan, Lin; Heiman, David I; Levin, Joshua Z; Young, Sarah; Zeng, Qiandong; Troemel, Emily R

    2012-12-01

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites. PMID:22813931

  4. Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV

    Directory of Open Access Journals (Sweden)

    Botvinnik Anna

    2008-07-01

    Full Text Available Abstract Background Regulated protein-protein interactions (PPIs are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. Results In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3ε and ζ in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We

  5. Quantitative analysis of some mechanisms affecting the yield of oxidative phosphorylation: dependence upon both fluxes and forces.

    Science.gov (United States)

    Rigoulet, M; Leverve, X; Fontaine, E; Ouhabi, R; Guérin, B

    1998-07-01

    The purpose of this work was to show how the quantitative definition of the different parameters involved in mitochondrial oxidative phosphorylation makes it possible to characterize the mechanisms by which the yield of ATP synthesis is affected. Three different factors have to be considered: (i) the size of the different forces involved (free energy of redox reactions and ATP synthesis, proton electrochemical difference); (ii) the physical properties of the inner mitochondrial membrane in terms of leaks (H+ and cations); and finally (iii) the properties of the different proton pumps involved in this system (kinetic properties, regulation, modification of intrinsic stoichiometry). The data presented different situations where one or more of these parameters are affected, leading to a different yield of oxidative phosphorylation. (1) By manipulating the actual flux through each of the respiratory chain units at constant protonmotive force in yeast mitochondria, we show that the ATP/O ratio decreases when the flux increases. Moreover, the highest efficiency was obtained when the respiratory rate was low and almost entirely controlled by the electron supply. (2) By using almitrine in different kinds of mitochondria, we show that this drug leads to a decrease in ATP synthesis efficiency by increasing the H+/ATP stoichiometry ofATP synthase (Rigoulet M et al. Biochim Biophys Acta 1018: 91-97, 1990). Since this enzyme is reversible, it was possible to test the effect of this drug on the reverse reaction of the enzyme i.e. extrusion of protons catalyzed by ATP hydrolysis. Hence, we are able to prove that, in this case, the decrease in efficiency of oxidative phosphorylation is due to a change in the mechanistic stoichiometry of this proton pump. To our knowledge, this is the first example of a modification in oxidative phosphorylation yield by a change in mechanistic stoichiometry of one of the proton pumps involved. (3) In a model of polyunsaturated fatty acid deficiency

  6. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

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

  7. 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.

  8. Sequence analysis reveals mosaic genome of Aichi virus

    Directory of Open Access Journals (Sweden)

    Han Xiaohong

    2011-08-01

    Full Text Available Abstract Aichi virus is a positive-sense and single-stranded RNA virus, which demonstrated to be related to diarrhea of Children. In the present study, phylogenetic and recombination analysis based on the Aichi virus complete genomes available in GenBank reveal a mosaic genome sequence [GenBank: FJ890523], of which the nt 261-852 region (the nt position was based on the aligned sequence file shows close relationship with AB010145/Japan with 97.9% sequence identity, while the other genomic regions show close relationship with AY747174/German with 90.1% sequence identity. Our results will provide valuable hints for future research on Aichi virus diversity. Aichi virus is a member of the Kobuvirus genus of the Picornaviridae family 12 and belongs to a positive-sense and single-stranded RNA virus. Its presence in fecal specimens of children suffering from diarrhea has been demonstrated in several Asian countries 3456, in Brazil and German 7, in France 8 and in Tunisia 9. Some reports showed the high level of seroprevalence in adults 710, suggesting the widespread exposure to Aichi virus during childhood. The genome of Aichi virus contains 8,280 nucleotides and a poly(A tail. The single large open reading frame (nt 713-8014 according to the strain AB010145 encodes a polyprotein of 2,432 amino acids that is cleaved into the typical picornavirus structural proteins VP0, VP3, VP1, and nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D 211. Based on the phylogenetic analysis of 519-bp sequences at the 3C-3D (3CD junction, Aichi viruses can be divided into two genotypes A and B with approximately 90% sequence homology 12. Although only six complete genomes of Aichi virus were deposited in GenBank at present, mosaic genomes can be found in strains from different countries.

  9. Lipidome analysis reveals antifungal polyphenol curcumin affects membrane lipid homeostasis.

    Science.gov (United States)

    Sharma, Monika; Dhamgaye, Sanjiveeni; Singh, Ashutosh; Prasad, Rajendra

    2012-01-01

    This study shows that antifungal curcumin (CUR), significantly depletes ergosterol levels in Candida albicans. CUR while displaying synergy with fluconazole (FLC) lowers ergosterol. However, CUR alone at its synergistic concentration (lower than MIC50), could not affect ergosterol contents. For deeper insight of CUR effects on lipids, we performed high throughput mass spectroscopy (MS) based lipid profiling of C. albicans cells. The lipidome analysis revealed that there were no major changes in phosphoglycerides (PGLs) composition following CUR treatment of Candida, however, significant differences in molecular species of PGLs were detected. Among major SPLs, CUR treatment resulted in the reduction of ceramide and accumulation of IPCs levels. The lipidome of CUR treated cells confirmed a dramatic drop in the ergosterol levels with a simultaneous accumulation of its biosynthetic precursors. This was further supported by the fact that the mutants defective in ergosterol biosynthesis (ERG2 and ERG11) and those lacking the transcription factor regulating ergosterol biosynthesis, UPC2, were highly susceptible to CUR. Our study first time shows that CUR, for its antifungal activity, targets and down regulates delta 5, 6 desaturase (ERG3) resulting in depletion of ergosterol. This results in parallel accumulation of ergosterol biosynthetic precursors, generation of reactive oxygen species (ROS) and cell death. PMID:22201946

  10. 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...

  11. Coupling of Phosphate-Imprinted Mesoporous Silica Nanoparticles-Based Selective Enrichment with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry for Highly Efficient Analysis of Protein Phosphorylation.

    Science.gov (United States)

    Chen, Yang; Li, Daojin; Bie, Zijun; He, Xinpei; Liu, Zhen

    2016-01-19

    Protein phosphorylation is a major post-translational modification and represents a ubiquitous mechanism for the cellular signaling of many different biological processes. Selective enrichment of phosphopeptides from the complex biological samples is a key step for the mass spectrometric (MS) analysis of protein phosphorylation. Herein, we present phosphate-imprinted mesoporous silica nanoparticles (MSNs) as an ideal sorbent for selective enrichment of phosphopeptides and an off-line combination with matrix-asisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for highly efficient analysis of protein phosphorylation. The phosphate-imprinted MSNs were prepared according to a newly reported strategy called dual-template docking oriented molecular imprinting (DTD-OMI). The prepared molecularly imprinted mesoporous material exhibited several significant merits, such as excellent selectivity toward phosphopeptides, tolerance to interference, fast binding equilibrium, and large binding capacity, which made the molecularly imprinted mesoporous material an ideal sorbent for selective enrichment of phosphopeptides. Using β-casein as a representative phosphoprotein, highly efficient phosphorylation analysis by the off-line platform was verified. Phosphorylation analysis of a nonfat milk sample was also well demonstrated. Because of their highly desirable properties, the phosphate-imprinted MSNs could find more applications in the analysis of protein phosphorylation. PMID:26684413

  12. 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

  13. 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.

  14. Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes.

    Science.gov (United States)

    Chevet, E; Wong, H N; Gerber, D; Cochet, C; Fazel, A; Cameron, P H; Gushue, J N; Thomas, D Y; Bergeron, J J

    1999-07-01

    Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER-associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two-dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular-signal regulated kinase-1 (ERK-1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK-1 led to increased association of calnexin with membrane-bound ribosomes. In vivo, calnexin-associated ERK-1 activity was identified by co-immunoprecipitation. This activity was abolished in cells expressing a dominant-negative MEK-1. Activation of ERK-1 in cells by addition of serum led to a 4-fold increase in ribosome-associated calnexin over unstimulated cells. Taken together with studies revealing calnexin association with CK2 and ERK-1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein folding close to the translocon. PMID:10393181

  15. Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.).

    Science.gov (United States)

    Dong, Kun; Zhen, Shoumin; Cheng, Zhiwei; Cao, Hui; Ge, Pei; Yan, Yueming

    2015-01-01

    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 Ser(355) 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. PMID:26635843

  16. 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

  17. Detection of phosphorylated subunits by combined LA-ICP-MS and MALDI-FTICR-MS analysis in yeast mitochondrial membrane complexes separated by blue native/SDS-PAGE

    Science.gov (United States)

    Krause-Buchholz, Udo; Becker, J. Susanne; Zoriy, Miroslav; Pickhardt, Carola; Przybylski, Michael; Rödel, Gerhard; Becker, J. Sabine

    2006-01-01

    We report on the identification of phosphorylated subunits of yeast mitochondrial ATPase using a novel screening technique in combination with BN/SDS-PAGE. Protein complexes present in yeast mitochondrial membranes were separated in their native state in the first dimension and their subunit composition was resolved by SDS-PAGE in the second dimension. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to rapidly screen for the presence of phosphorus in the subunits. The detection limits of elements investigated in selected protein spots are in the low [mu]g g-1 concentration range. Sulfur was used as the internal standard element for quantification. Phosphorus was detected in two of the proteins, that were identified by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) as subunits Atp1p and Atp2p of the ATPase. These results were confirmed by Western blot analysis using antibodies directed against phosphorylated amino acids. The combination of LA-ICP-MS and MALDI-FTICR-MS with BN/SDS-PAGE provides a fast and sensitive tool for structure analysis of phosphorus and metal-containing subunits of membrane protein complexes.

  18. 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.

  19. The in vivo phosphorylation sites of rat brain dynamin I

    DEFF Research Database (Denmark)

    Graham, Mark E; Anggono, Victor; Bache, Nicolai; Larsen, Martin R; Craft, George E; Robinson, Phillip J

    2007-01-01

    resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation...

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Pawel; 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

    2015-01-01

    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, Trichin......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 m......RNAs (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 Fd...

  1. 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

  2. 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.

  3. 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 (<2% of row-crop land), the extent of highly unprofitable land increased to 2.5 Mha, or 27% of row-crop land, in the 2015 projection. Aggregation of these areas to the township level revealed ‘hotspots’ for potential management change in Western, Central, and Northeast Iowa. In these least 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.

  4. 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+...

  5. 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

  6. Protein kinase CK2 interacts with Chk2 and phosphorylates Mre11 on serine 649

    International Nuclear Information System (INIS)

    The Mre11-Rad50-Nbs1 protein complex has been known to be involved in a variety of DNA metabolic events that involve DNA double-strand breaks (DSBs). The phosphorylation of Mre11 is increased in response to ionizing radiation, which suggests that phosphorylation of Mre11 may be an important regulatory mechanism of this complex. Mre11-phosphorylating kinase activities were observed in Chk2 immunoprecipitates and HeLa nuclear extracts. Through the tandem affinity tagging system and conventional chromatography, this kinase was purified and identified as protein kinase CK2. CK2 phosphorylates Mre11 in vitro. In vitro kinase assay with a series of truncated Mre11 proteins as substrates for CK2 and site-directed mutagenesis showed that serine 649 of Mre11 is mainly phosphorylated by CK2 in vitro. In vivo labeling and phosphopeptide mapping analysis revealed that this phosphorylation occurs in vivo. These data implicate CK2 as a potential upstream regulator of Mre11 function

  7. 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.

  8. 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

  9. Mining Conditional Phosphorylation Motifs.

    Science.gov (United States)

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/. PMID:26356863

  10. Massive comparative genomic analysis reveals convergent evolution of specialized bacteria

    OpenAIRE

    Raoult Didier; Pontarotti Pierre; Royer-Carenzi Manuela; Merhej Vicky

    2009-01-01

    Abstract Background Genome size and gene content in bacteria are associated with their lifestyles. Obligate intracellular bacteria (i.e., mutualists and parasites) have small genomes that derived from larger free-living bacterial ancestors; however, the different steps of bacterial specialization from free-living to intracellular lifestyle have not been studied comprehensively. The growing number of available sequenced genomes makes it possible to perform a statistical comparative analysis of...

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

    OpenAIRE

    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.

    2008-01-01

    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-o...

  12. 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...

  13. 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

    myosin 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......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...

  14. Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms

    Directory of Open Access Journals (Sweden)

    Paczia Nicole

    2012-09-01

    Full Text Available Abstract Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis.

  15. Mapping of phosphorylation sites in polyomavirus large T antigen.

    OpenAIRE

    Hassauer, M; Scheidtmann, K H; Walter, G.

    1986-01-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T 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. 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; Nefedov, Mikhail; de Jong, Pieter J; Renfree, Marilyn B; Mardis, Elaine R; Wilson, Richard K

    2008-05-01

    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

  18. Bioimage analysis of Shigella infection reveals targeting of colonic crypts.

    Science.gov (United States)

    Arena, Ellen T; Campbell-Valois, Francois-Xavier; Tinevez, Jean-Yves; Nigro, Giulia; Sachse, Martin; Moya-Nilges, Maryse; Nothelfer, Katharina; Marteyn, Benoit; Shorte, Spencer L; Sansonetti, Philippe J

    2015-06-23

    Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent. PMID:26056271

  19. 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

  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. 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.

  2. 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

    2015-09-01

    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 Ca(2+) 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 Ca(2+) 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

  3. Metatranscriptomic Analysis of Groundwater Reveals an Active Anammox Bacterial Population

    Science.gov (United States)

    Jewell, T. N. M.; Karaoz, U.; Thomas, B. C.; Banfield, J. F.; Brodie, E.; Williams, K. H.; Beller, H. R.

    2014-12-01

    Groundwater is a major natural resource, yet little is known about the contribution of microbial anaerobic ammonium oxidation (anammox) activity to subsurface nitrogen cycling. During anammox, energy is generated as ammonium is oxidized under anaerobic conditions to dinitrogen gas, using nitrite as the final electron acceptor. This process is a global sink for fixed nitrogen. Only a narrow range of monophyletic bacteria within the Planctomycetes carries out anammox, and the full extent of their metabolism, and subsequent impact on nitrogen cycling and microbial community structure, is still unknown. Here, we employ a metatranscriptomic analysis on enriched mRNA to identify the abundance and activity of a population of anammox bacteria within an aquifer at Rifle, CO. Planktonic biomass was collected over a two-month period after injection of up to 1.5 mM nitrate. Illumina-generated sequences were mapped to a phylogenetically binned Rifle metagenome database. We identified transcripts for genes with high protein sequence identities (81-98%) to those of anammox strain KSU-1 and to two of the five anammox bacteria genera, Brocadia and Kuenenia, suggesting an active, if not diverse, anammox population. Many of the most abundant anammox transcripts mapped to a single scaffold, indicative of a single dominant anammox species. Transcripts of the genes necessary for the anammox pathway were present, including an ammonium transporter (amtB), nitrite/formate transporter, nitrite reductase (nirK), and hydrazine oxidoreductase (hzoB). The form of nitrite reductase encoded by anammox is species-dependent, and we only identified nirK, with no evidence of anammox nirS. In addition to the anammox pathway we saw evidence of the anammox bacterial dissimilatory nitrate reduction to ammonium pathway (narH, putative nrfA, and nrfB), which provides an alternate means of generating substrates for anammox from nitrate, rather than relying on an external pool. Transcripts for hydroxylamine

  4. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol 32P/mol calcineurin. Analysis by SDS-PAGE revealed that only the α subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn2+ or Ni2+ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn2+ or Ni2+ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni2+ was a more potent activator of phosphorylated calcineurin compared to Mn2+. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni2+ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca2+

  5. Pyrosequencing-Based Transcriptome Analysis of the Asian Rice Gall Midge Reveals Differential Response during Compatible and Incompatible Interaction

    Directory of Open Access Journals (Sweden)

    Jagadish S. Bentur

    2012-10-01

    Full Text Available The Asian rice gall midge (Orseolia oryzae is a major pest responsible for immense loss in rice productivity. Currently, very little knowledge exists with regard to this insect at the molecular level. The present study was initiated with the aim of developing molecular resources as well as identifying alterations at the transcriptome level in the gall midge maggots that are in a compatible (SH or in an incompatible interaction (RH with their rice host. Roche 454 pyrosequencing strategy was used to develop both transcriptomics and genomics resources that led to the identification of 79,028 and 85,395 EST sequences from gall midge biotype 4 (GMB4 maggots feeding on a susceptible and resistant rice variety, TN1 (SH and Suraksha (RH, respectively. Comparative transcriptome analysis of the maggots in SH and RH revealed over-representation of transcripts from proteolysis and protein phosphorylation in maggots from RH. In contrast, over-representation of transcripts for translation, regulation of transcription and transcripts involved in electron transport chain were observed in maggots from SH. This investigation, besides unveiling various mechanisms underlying insect-plant interactions, will also lead to a better understanding of strategies adopted by insects in general, and the Asian rice gall midge in particular, to overcome host defense.

  6. 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

  7. Developmentally regulated phosphorylation-dephosphorylation of ribosomal proteins from maize embryonic axes

    International Nuclear Information System (INIS)

    Previous work in our lab suggests translational control during maize germination. To test this possibility the present research focuses on the phosphorylated status of the ribosomal proteins of maize axes during germination. Ribosomes from embryonic axes incubated for different periods were in vitro or in vivo labeled by 1h-pulse for 32P orthophosphate. Electrophoretic analysis of the ribosomal proteins and autoradiographs revealed: (a) in vitro, several 32P bands and very similar patterns for all stages tested (0 to 24h); in vivo, less number of labeled bands and a changing pattern from 3 to 24h of incubation. A protein of 30,900 MW did not appear phosphorylated until 8h of incubation, while a 17,000 MW protein was strongly labeled at 3h and fastly dephosphorylated toward 24h. Phosphorylated proteins belong to both the small and the large subunits. The implication of this process will be discussed

  8. Mapping of phosphorylation sites in polyomavirus large T antigen

    International Nuclear Information System (INIS)

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed

  9. Mapping of phosphorylation sites in polyomavirus large T antigen

    Energy Technology Data Exchange (ETDEWEB)

    Hassauer, M.; Scheidtmann, K.H.; Walter, G.

    1986-06-01

    The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, /sup 32/P/sub i/-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.

  10. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    Science.gov (United States)

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  11. Phosphorylation of proteoglycans from human articular cartilage

    International Nuclear Information System (INIS)

    Previous studies have shown that sulfated proteoglycans from human articular and epiphyseal cartilage were phosphorylated. These macromolecules contribute to the stiffness and resiliency of this tissue. We demonstrate here that the phosphate moieties are an integral part of proteoglycan subunits. Specifically, evidence is presented which indicates that proteoglycan monomers contain 3 to 4 phosphate moieties per core protein and that these appear to exist as phosphoserine residues. Furthermore, the data illustrate that human articular cartilage also contains more than 20 different phosphoproteins, some of which are closely associated with proteoglycan aggregates. Proteoglycan subunits were purified from extracts of articular cartilage or from media fractions which had been used to label tissue specimens with 32P-orthophosphate. Chemical and radiographic analyses revealed that the phosphate concentration with respect to sulfate and uronic acid content remained constant when purified proteoglycan monomers were subjected to equilibrium ultracentrifugation and size-exclusion chromatography. That the phosphate moieties were bound to proteoglycan monomers via monoester linkages was indicated by the release of 32P-orthophosphate from proteoglycan subunits incubated under mild alkaline conditions or reacted with acid or alkaline phosphatases. Identification of serine residues in the core protein as the sites of phosphorylation was made by autoradiography of thin layer plates on which hydrolyzed samples of purified 32P-proteoglycan subunits had been subjected to 2-dimensional electrophoresis/chromatography. Quantification of 3 to 4 phosphate moieties per core protein of 200,000 daltons was made by chemical analysis of inorganic phosphate released from proteoglycans by acid hydrolysis

  12. The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

    Directory of Open Access Journals (Sweden)

    Tachibana Taro

    2011-10-01

    Full Text Available Abstract Background Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII, which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq. Results We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII. Conclusions We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

  13. 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

  14. Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE

    OpenAIRE

    Yasunori Sugiyama; Syouichi Katayama; Isamu Kameshita; Keiko Morisawa; Takuma Higuchi; Hiroshi Todaka; Eiji Kinoshita; Emiko Kinoshita-Kikuta; Tohru Koike; Taketoshi Taniguchi; Shuji Sakamoto

    2015-01-01

    Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in ce...

  15. 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

  16. 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.

  17. 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...

  18. 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

  19. Phosphorylating DNA with DNA

    OpenAIRE

    Li, Yingfu; Breaker, Ronald R.

    1999-01-01

    Nearly 50 individual DNAs with polynucleotide kinase-like activity were isolated from a random-sequence pool by using in vitro selection. Each self-phosphorylating deoxyribozyme makes use of one or more of the eight standard NTPs or dNTPs as a source of activated phosphate. Although most prototypic deoxyribozymes poorly differentiate between the ribose and deoxyribose moieties, further optimization by in vitro selection produced variants that display up to 100-fold discrimination between rela...

  20. Synaptic plasticity and phosphorylation

    OpenAIRE

    Lee, Hey-Kyoung

    2006-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-syn...

  1. Integrative transcriptome analysis reveals dysregulation of canonical cancer molecular pathways in placenta leading to preeclampsia

    OpenAIRE

    Moslehi, Roxana; Mills, James L; Signore, Caroline; Kumar, Anil; Ambroggio, Xavier; Dzutsev, Amiran

    2013-01-01

    We previously suggested links between specific XPD mutations in the fetal genome and the risk of placental maldevelopment and preeclampsia, possibly due to impairment of Transcription Factor (TF)IIH-mediated functions in placenta. To identify the underlying mechanisms, we conducted the current integrative analysis of several relevant transcriptome data sources. Our meta-analysis revealed downregulation of TFIIH subunits in preeclamptic placentas. Our overall integrative analysis suggested tha...

  2. X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

    Science.gov (United States)

    Yamaguchi, Maki; Kimura, Masako; Li, Zhao-Bo; Ohno, Tetsuo; Takemori, Shigeru; Hoh, Joseph F Y; Yagi, Naoto

    2016-04-15

    The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments. PMID:26911280

  3. 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.

  4. 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......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  5. Evolutionary constraints of phosphorylation in eukaryotes, prokaryotes, and mitochondria.

    Science.gov (United States)

    Gnad, Florian; Forner, Francesca; Zielinska, Dorota F; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias

    2010-12-01

    High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the

  6. 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.

  7. 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.

  8. Hydrazide functionalized monodispersed silica microspheres: a novel probe with tunable selectivity for a versatile enrichment of phosphopeptides with different numbers of phosphorylation sites in MS analysis.

    Science.gov (United States)

    Xu, Linnan; Ma, Wen; Shen, Sensen; Li, Liping; Bai, Yu; Liu, Huwei

    2016-01-21

    Hydrazide functionalized monodispersed silica microspheres (HFMSM) were developed for the enrichment of phosphopeptides for the first time. With the aid of the tunable selectivity of HFMSM, global enrichment or fractionation of phosphopeptides with different numbers of phosphorylation sites could be realized by a simple modulation of the concentrations of formic acid in buffers. PMID:26596900

  9. Using Interaction Analysis to reveal Self-Regulated Learning in Virtual Communities

    OpenAIRE

    Dettori, Giuliana; Persico, Donatella

    2007-01-01

    Aim of this paper is to analyse whether Interaction Analysis can help investigate the practice and development of Self-Regulated Learning (SRL) in Virtual Learning Communities (VLC). Interaction analysis is increasingly used to study learning dynamics within online activities. It proceeds by searching expressions that reveal the aspects under study in the written messages exchanged by the learners. To this end, we devised and classified a number of indicators suggesting the existence of self-...

  10. 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...

  11. Intestinal Origin of Sourdough Lactobacillus reuteri Isolates as Revealed by Phylogenetic, Genetic, and Physiological Analysis

    OpenAIRE

    Su, Marcia Shu-Wei; Oh, Phaik Lyn; Walter, Jens; Gänzle, Michael G.

    2012-01-01

    Lactobacillus reuteri is both a gut symbiont and a stable member of sourdough microbiota. This study employed multilocus sequence analysis and an analysis of host-specific physiological and genetic traits to assign five sourdough isolates to rodent- or human-specific lineages. Comparative genome hybridization revealed that the model sourdough isolate LTH2584 had a genome content very similar to that of the model rodent isolate 100-23. These results demonstrate that sourdough isolates of L. re...

  12. Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions

    Directory of Open Access Journals (Sweden)

    Rivera-Molina Félix E

    2008-01-01

    Full Text Available Abstract Background The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p, a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Δ causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Δ strains. Results Global mRNA expression profiles of myo1Δ strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p ≤ 0.01 were identified with 263 up regulated and 284 down regulated genes in the myo1Δ strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Δslt2Δ double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Δ strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. Conclusion Following this analysis of global mRNA expression in yeast myo1Δ strains, we conclude that 547 genes were differentially regulated in myo1Δ strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Δ strain viability, supporting that the up

  13. Formation and Dissociation of Phosphorylated Peptide Radical Cations

    OpenAIRE

    Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-kuen; Siu, Chi-Kit; Chu, Ivan K.

    2012-01-01

    In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal–ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N,N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radi...

  14. 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

  15. 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....

  16. Mechanism of influence of phosphorylation on serine 124 on a decrease of catalytic activity of human thymidylate synthase.

    Science.gov (United States)

    Jarmuła, Adam; Fraczyk, Tomasz; Cieplak, Piotr; Rode, Wojciech

    2010-05-15

    Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the 'phosphorylation effect' is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125-134) and the patch (residues 189-192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF. PMID:20430630

  17. 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 (pquantitative differences in ground-base ion radiation and spaceflight experiments respectively. The functions of ground-base radiation and spaceflight proteins were both involved in a wide range of biological processes. Gene Ontology enrichment analysis further revealed that ground-base radiation responsive proteins were mainly involved in removal of superoxide radicals, defense response to stimulus and photosynthesis, while spaceflight responsive proteins mainly participate in nucleoside metabolic 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

  18. 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

    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....... Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 + 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase in...

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

    International Nuclear Information System (INIS)

    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: ► We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. ► The mutation enhances the activity to assemble microtubules. ► Mal3 is phosphorylated in a microtubule-dependent manner. ► The phosphorylation negatively regulates the Mal3 activity.

  20. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

    International Nuclear Information System (INIS)

    Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr308 and AKT-Ser473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three

  1. Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Verner, Zdeněk; Čermáková, P.; Škodová, Ingrid; Kováčová, B.; Lukeš, Julius; Horváth, A.

    2014-01-01

    Roč. 193, č. 1 (2014), s. 55-65. ISSN 0166-6851 R&D Projects: GA ČR GAP305/12/2261; GA MŠk(CZ) EE2.3.30.0032; GA MŠk LH12104 Institutional support: RVO:60077344 Keywords : mitochondrion * oxidative phosphorylation * Trypanosoma * Leishmania * Phytomonas * Crithidia Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.787, year: 2014

  2. 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

  3. 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

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

    Directory of Open Access Journals (Sweden)

    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

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

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    Rachel Deplus

    2014-08-01

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

  6. 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 ...

  7. 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

  8. Autotriploid origin of Carassius auratus as revealed by chromosomal locus analysis.

    Science.gov (United States)

    Qin, Qinbo; Wang, Juan; Hu, Min; Huang, Shengnan; Liu, Shaojun

    2016-06-01

    In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploid forms. Chromosomal and karyotypic analyses have suggested that the polyploid C. auratus has a triploid (3n=150, 3nCC) and a tetraploid origin (4n=200), respectively. However, there is a lack of direct genetic evidence to support this conclusion. In this paper, analysis of the 5S rDNA chromosomal locus revealed that the 3nCC is of triploid origin. Analysis of the species-specific chromosomal centromere locus revealed that 3nCC individuals possess three sets of C. auratus-derived chromosomes. Our results provide direct cytogenetic evidence suggesting that individuals with 150 chromosomes are of autotriploid origin within the C. auratus complex. It marks an important contribution to the study of polyploidization and the evolution of vertebrates. PMID:27084707

  9. 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 ...

  10. Analysis of Hepatitis C Virus-Inoculated Chimpanzees Reveals Unexpected Clinical Profiles

    OpenAIRE

    Bassett, Suzanne E.; Brasky, Kathleen M.; Lanford, Robert E.

    1998-01-01

    The clinical course of hepatitis C virus (HCV) infections in a chimpanzee cohort was examined to better characterize the outcome of this valuable animal model. Results of a cross-sectional study revealed that a low percentage (39%) of HCV-inoculated chimpanzees were viremic based on reverse transcription (RT-PCR) analysis. A correlation was observed between viremia and the presence of anti-HCV antibodies. The pattern of antibodies was dissimilar among viremic chimpanzees and chimpanzees that ...

  11. 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

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

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    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.

  13. 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

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

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    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.

  15. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

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    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  16. Formation and Dissociation of Phosphorylated Peptide Radical Cations

    Science.gov (United States)

    Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-Kuen; Siu, Chi-Kit; Chu, Ivan K.

    2012-12-01

    In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N, N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the p M·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model— N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

  17. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

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    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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

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

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    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.

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

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    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.

  5. 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

  6. 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.

  7. High-speed image analysis reveals chaotic vibratory behaviors of pathological vocal folds

    International Nuclear Information System (INIS)

    Research highlights: → Low-dimensional human glottal area data. → Evidence of chaos in human laryngeal activity from high-speed digital imaging. → Traditional perturbation analysis should be cautiously applied to aperiodic high speed image signals. → Nonlinear dynamic analysis may be helpful for understanding disordered behaviors in pathological laryngeal systems. - Abstract: Laryngeal pathology is usually associated with irregular dynamics of laryngeal activity. High-speed imaging facilitates direct observation and measurement of vocal fold vibrations. However, chaotic dynamic characteristics of aperiodic high-speed image data have not yet been investigated in previous studies. In this paper, we will apply nonlinear dynamic analysis and traditional perturbation methods to quantify high-speed image data from normal subjects and patients with various laryngeal pathologies including vocal fold nodules, polyps, bleeding, and polypoid degeneration. The results reveal the low-dimensional dynamic characteristics of human glottal area data. In comparison to periodic glottal area series from a normal subject, aperiodic glottal area series from pathological subjects show complex reconstructed phase space, fractal dimension, and positive Lyapunov exponents. The estimated positive Lyapunov exponents provide the direct evidence of chaos in pathological human vocal folds from high-speed digital imaging. Furthermore, significant differences between the normal and pathological groups are investigated for nonlinear dynamic and perturbation analyses. Jitter in the pathological group is significantly higher than in the normal group, but shimmer does not show such a difference. This finding suggests that the traditional perturbation analysis should be cautiously applied to high speed image signals. However, the correlation dimension and the maximal Lyapunov exponent reveal a statistically significant difference between normal and pathological groups. Nonlinear dynamic

  8. Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

    International Nuclear Information System (INIS)

    DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [γ-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. The authors conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, they speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity

  9. The relationship between periodic dinucleotides and the nucleosomal DNA deformation revealed by normal mode analysis

    International Nuclear Information System (INIS)

    Nucleosomes, which contain DNA and proteins, are the basic unit of eukaryotic chromatins. Polymers such as DNA and proteins are dynamic, and their conformational changes can lead to functional changes. Periodic dinucleotide patterns exist in nucleosomal DNA chains and play an important role in the nucleosome structure. In this paper, we use normal mode analysis to detect significant structural deformations of nucleosomal DNA and investigate the relationship between periodic dinucleotides and DNA motions. We have found that periodic dinucleotides are usually located at the peaks or valleys of DNA and protein motions, revealing that they dominate the nucleosome dynamics. Also, a specific dinucleotide pattern CA/TG appears most frequently

  10. Novel Phosphorylation Assay Based on Multi-Functionalized Soluble Nanopolymer

    OpenAIRE

    Iliuk, Anton; Martinez, Juan S.; Hall, Mark C.; Tao, W. Andy

    2011-01-01

    Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO that is multi-functionalized with titanium (IV)ions for specific binding to phosphoproteins, and with biotin groups that allow for enzyme-linked spectrometr...

  11. Biphasic responses in multi-site phosphorylation systems

    OpenAIRE

    Suwanmajo, Thapanar; Krishnan, J.

    2013-01-01

    Multi-site phosphorylation systems are repeatedly encountered in cellular biology and multi-site modification is a basic building block of post-translational modification. In this paper, we demonstrate how distributive multi-site modification mechanisms by a single kinase/phosphatase pair can lead to biphasic/partial biphasic dose–response characteristics for the maximally phosphorylated substrate at steady state. We use simulations and analysis to uncover a hidden competing effect which is r...

  12. 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.

  13. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica

    Energy Technology Data Exchange (ETDEWEB)

    Pomraning, Kyle R.; Kim, Young-Mo; Nicora, Carrie D.; Chu, Rosalie K.; Bredeweg, Erin L.; Purvine, Samuel O.; Hu, Dehong; Metz, Thomas O.; Baker, Scott E.

    2016-02-25

    Background Yarrowia lipolytica is an oleaginous ascomycete yeast that stores lipids in response to limitation of nitrogen. While the enzymatic pathways responsible for neutral lipid accumulation in Y. lipolytica are well characterized, regulation of these pathways has received little attention. We therefore sought to characterize the response to nitrogen limitation at system-wide levels, including the proteome, phosphoproteome and metabolome, to deepen our understanding of how this organism regulates and controls lipid metabolism and to identify targets that may be manipulated to improve lipid yield. Results Using a variety of -omic techniques, we found that ribosome structural genes are down-regulated under nitrogen limitation, during which nitrogen containing compounds (alanine, putrescine, spermidine and urea) are depleted and sugar alcohols and TCA cycle intermediates accumulate (citrate, fumarate and malate). We identified 1,219 novel phosphorylation sites in Y. lipolytica, 133 of which change in their abundance during nitrogen limitation. Regulatory proteins, including kinases and DNA binding proteins, are particularly enriched for phosphorylation. Within lipid synthesis pathways, we found that ATP-citrate lyase, acetyl-CoA carboxylase and lecithin cholesterol acyl transferase are phosphorylated during nitrogen limitation while many of the proteins involved in β-oxidation are down-regulated, suggesting that storage lipid accumulation is due either to increased lipogenesis from excess citrate and regulated by phosphorylation of key enzymes, decreased capacity for β-oxidation, or both. Further, we identified short DNA elements that associate specific transcription factor families with up- and down-regulated genes. Conclusions Integration of metabolome, proteome and phosphoproteome data identifies lipid accumulation in response to nitrogen limitation as a two-fold result of increased production of acetyl-CoA from excess citrate and decreased capacity for β-oxidation.

  14. 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

  15. Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

    Science.gov (United States)

    Parker, Jane E

    2009-01-01

    The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.1 Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization. PMID:19847120

  16. 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.

  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. 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

  19. 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.

  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. 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

  2. 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.

  3. Pretreatment and integrated analysis of spectral data reveal seaweed similarities based on chemical diversity.

    Science.gov (United States)

    Wei, Feifei; Ito, Kengo; Sakata, Kenji; Date, Yasuhiro; Kikuchi, Jun

    2015-03-01

    Extracting useful information from high dimensionality and large data sets is a major challenge for data-driven approaches. The present study was aimed at developing novel integrated analytical strategies for comprehensively characterizing seaweed similarities based on chemical diversity. The chemical compositions of 107 seaweed and 2 seagrass samples were analyzed using multiple techniques, including Fourier transform infrared (FT-IR) and solid- and solution-state nuclear magnetic resonance (NMR) spectroscopy, thermogravimetry-differential thermal analysis (TG-DTA), inductively coupled plasma-optical emission spectrometry (ICP-OES), CHNS/O total elemental analysis, and isotope ratio mass spectrometry (IR-MS). The spectral data were preprocessed using non-negative matrix factorization (NMF) and NMF combined with multivariate curve resolution-alternating least-squares (MCR-ALS) methods in order to separate individual component information from the overlapping and/or broad spectral peaks. Integrated analysis of the preprocessed chemical data demonstrated distinct discrimination of differential seaweed species. Further network analysis revealed a close correlation between the heavy metal elements and characteristic components of brown algae, such as cellulose, alginic acid, and sulfated mucopolysaccharides, providing a componential basis for its metal-sorbing potential. These results suggest that this integrated analytical strategy is useful for extracting and identifying the chemical characteristics of diverse seaweeds based on large chemical data sets, particularly complicated overlapping spectral data. PMID:25647718

  4. Copy number and targeted mutational analysis reveals novel somatic events in metastatic prostate tumors.

    Science.gov (United States)

    Robbins, Christiane M; Tembe, Waibov A; Baker, Angela; Sinari, Shripad; Moses, Tracy Y; Beckstrom-Sternberg, Stephen; Beckstrom-Sternberg, James; Barrett, Michael; Long, James; Chinnaiyan, Arul; Lowey, James; Suh, Edward; Pearson, John V; Craig, David W; Agus, David B; Pienta, Kenneth J; Carpten, John D

    2011-01-01

    Advanced prostate cancer can progress to systemic metastatic tumors, which are generally androgen insensitive and ultimately lethal. Here, we report a comprehensive genomic survey for somatic events in systemic metastatic prostate tumors using both high-resolution copy number analysis and targeted mutational survey of 3508 exons from 577 cancer-related genes using next generation sequencing. Focal homozygous deletions were detected at 8p22, 10q23.31, 13q13.1, 13q14.11, and 13q14.12. Key genes mapping within these deleted regions include PTEN, BRCA2, C13ORF15, and SIAH3. Focal high-level amplifications were detected at 5p13.2-p12, 14q21.1, 7q22.1, and Xq12. Key amplified genes mapping within these regions include SKP2, FOXA1, and AR. Furthermore, targeted mutational analysis of normal-tumor pairs has identified somatic mutations in genes known to be associated with prostate cancer including AR and TP53, but has also revealed novel somatic point mutations in genes including MTOR, BRCA2, ARHGEF12, and CHD5. Finally, in one patient where multiple independent metastatic tumors were available, we show common and divergent somatic alterations that occur at both the copy number and point mutation level, supporting a model for a common clonal progenitor with metastatic tumor-specific divergence. Our study represents a deep genomic analysis of advanced metastatic prostate tumors and has revealed candidate somatic alterations, possibly contributing to lethal prostate cancer. PMID:21147910

  5. 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

  6. 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.

  7. 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

  8. REVEAL - A tool for rule driven analysis of safety critical software

    International Nuclear Information System (INIS)

    As the determination of ultrahigh reliability figures for safety critical software is hardly possible, national and international guidelines and standards give mainly requirements for the qualitative evaluation of software. An analysis whether all these requirements are fulfilled is time and effort consuming and prone to errors, if performed manually by analysts, and should instead be dedicated to tools as far as possible. There are many ''general-purpose'' software analysis tools, both static and dynamic, which help analyzing the source code. However, they are not designed to assess the adherence to specific requirements of guidelines and standards in the nuclear field. Against the background of the development of I and C systems in the nuclear field which are based on digital techniques and implemented in high level language, it is essential that the assessor or licenser has a tool with which he can automatically and uniformly qualify as many aspects as possible of the high level language software. For this purpose the software analysis tool REVEAL has been developed at ISTec and the Halden Reactor Project. (author)

  9. 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

  10. 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.

  11. 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.

  12. 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.

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

    Science.gov (United States)

    Liang, Lining; Sun, Hao; 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. Transcriptome analysis reveals novel regulatory mechanisms in a genome-reduced bacterium.

    Science.gov (United States)

    Mazin, Pavel V; Fisunov, Gleb Y; Gorbachev, Alexey Y; Kapitskaya, Kristina Y; Altukhov, Ilya A; Semashko, Tatiana A; Alexeev, Dmitry G; Govorun, Vadim M

    2014-12-01

    The avian bacterial pathogen Mycoplasma gallisepticum is a good model for systems studies due to small genome and simplicity of regulatory pathways. In this study, we used RNA-Seq and MS-based proteomics to accurately map coding sequences, transcription start sites (TSSs) and transcript 3'-ends (T3Es). We used obtained data to investigate roles of TSSs and T3Es in stress-induced transcriptional responses. We identified 1061 TSSs at a false discovery rate of 10% and showed that almost all transcription in M. gallisepticum is initiated from classic TATAAT promoters surrounded by A/T-rich sequences. Our analysis revealed the pronounced operon structure complexity: on average, each coding operon has one internal TSS and T3Es in addition to the primary ones. Our transcriptomic approach based on the intervals between the two nearest transcript ends allowed us to identify two classes of T3Es: strong, unregulated, hairpin-containing T3Es and weak, heat shock-regulated, hairpinless T3Es. Comparing gene expression levels under different conditions revealed widespread and divergent transcription regulation in M. gallisepticum. Modeling suggested that the core promoter structure plays an important role in gene expression regulation. We have shown that the heat stress activation of cryptic promoters combined with the hairpinless T3Es suppression leads to widespread, seemingly non-functional transcription. PMID:25361977

  1. Distribution patterns of firearm discharge residues as revealed by neutron activation analysis

    International Nuclear Information System (INIS)

    A systematic investigation using a variety of handguns has revealed the existence of distinguisable distribution patterns of firearm discharge residues on surfaces below the flight path of a bullet. The residues are identificable even at distances of 12 meters from the gun using nondestructive neutron activation analysis. The results of these investigations show that the distribution pattern for a gun is reproducible using similar ammunition and that there exist two distinct regions to the patterns developed between the firearm and the target-one with respect to the position of the gun and the other in the vicinity of the target. The judicious applications of these findings could be of significant value in criminal investigations. (T.G.)

  2. Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid

    Directory of Open Access Journals (Sweden)

    Daniel Fraher

    2016-02-01

    Full Text Available The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

  3. 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

    on 3,527 specimens from 12 cancer types, revealing a unified classification into 11 major subtypes. Five subtypes were nearly identical to their tissue-of-origin counterparts, but several distinct cancer types were found to converge into common subtypes. Lung squamous, head and neck, and a subset......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...... of bladder cancers coalesced into one subtype typified by TP53 alterations, TP63 amplifications, and high expression of immune and proliferation pathway genes. Of note, bladder cancers split into three pan-cancer subtypes. The multiplatform classification, while correlated with tissue-of-origin, provides...

  4. 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

    constitutively expressed protein isoforms, which were associated with 203 ORFs in the A. balhimycina genome. These data, providing insights on the major metabolic pathways/molecular processes operating in this organism, were used to compile 2-DE reference maps covering 3-10, 4-7 and 4.5-5.5 pH gradients...... available over the World Wide Web as interactive web pages (http://www.unipa.it/ampuglia/Abal-proteome-maps). Functional clustering analysis revealed that differentially expressed proteins belong to functional groups involved in central carbon metabolism, amino acid metabolism and protein biosynthesis......, energetic and redox balance, sugar/amino sugar metabolism, balhimycin biosynthesis and transcriptional regulation or with hypothetical and/or unknown function. Interestingly, proteins involved in the biosynthesis of balhimycin precursors, such as amino acids, amino sugars and central carbon metabolism...

  5. Metagenomic Analysis Reveals Unexpected Subgenomic Diversity of Magnetotactic Bacteria within the Phylum Nitrospirae ▿ †

    Science.gov (United States)

    Lin, Wei; Jogler, Christian; Schüler, Dirk; Pan, Yongxin

    2011-01-01

    A targeted metagenomic approach was applied to investigate magnetotactic bacteria (MTB) within the phylum Nitrospirae in Lake Miyun near Beijing, China. Five fosmids containing rRNA operons were identified. Comparative sequence analysis of a total of 172 kb provided new insights into their genome organization and revealed unexpected subgenomic diversity of uncultivated MTB in the phylum Nitrospirae. In addition, affiliation of two novel MTB with the phylum Nitrospirae was verified by fluorescence in situ hybridization. One of them was morphologically similar to “Candidatus Magnetobacterium bavaricum,” but the other differed substantially in cell shape and magnetosome organization from all previously described “Ca. Magnetobacterium bavaricum”-like bacteria. PMID:21057016

  6. 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.

  7. Bifidobacterium asteroides PRL2011 Genome Analysis Reveals Clues for Colonization of the Insect Gut

    Science.gov (United States)

    Bottacini, Francesca; Milani, Christian; Turroni, Francesca; Sánchez, Borja; Foroni, Elena; Duranti, Sabrina; Serafini, Fausta; Viappiani, Alice; Strati, Francesco; Ferrarini, Alberto; Delledonne, Massimo; Henrissat, Bernard; Coutinho, Pedro; Fitzgerald, Gerald F.; Margolles, Abelardo; van Sinderen, Douwe; Ventura, Marco

    2012-01-01

    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. PMID:23028506

  8. Metagenomic analysis reveals significant changes of microbial compositions and protective functions during drinking water treatment

    Science.gov (United States)

    Chao, Yuanqing; Ma, Liping; Yang, Ying; Ju, Feng; Zhang, Xu-Xiang; Wu, Wei-Min; Zhang, Tong

    2013-12-01

    The metagenomic approach was applied to characterize variations of microbial structure and functions in raw (RW) and treated water (TW) in a drinking water treatment plant (DWTP) at Pearl River Delta, China. Microbial structure was significantly influenced by the treatment processes, shifting from Gammaproteobacteria and Betaproteobacteria in RW to Alphaproteobacteria in TW. Further functional analysis indicated the basic metabolic functions of microorganisms in TW did not vary considerably. However, protective functions, i.e. glutathione synthesis genes in `oxidative stress' and `detoxification' subsystems, significantly increased, revealing the surviving bacteria may have higher chlorine resistance. Similar results were also found in glutathione metabolism pathway, which identified the major reaction for glutathione synthesis and supported more genes for glutathione metabolism existed in TW. This metagenomic study largely enhanced our knowledge about the influences of treatment processes, especially chlorination, on bacterial community structure and protective functions (e.g. glutathione metabolism) in ecosystems of DWTPs.

  9. 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

  10. 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...

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

    DEFF Research Database (Denmark)

    Jayanthi, Lankupalle D; Annamalai, Balasubramaniam; Samuvel, Devadoss J;

    2006-01-01

    G alpha(q)-coupled human neurokinin 1 (hNK-1) receptor coexpressed with the WT-hNET produced effects similar to beta-PMA via PKC stimulation. In striking contrast, an hNET double mutant harboring T258A and S259A failed to show NE uptake inhibition and plasma membrane redistribution by beta-PMA or SP...... hypothesis that PKC-mediated phosphorylation of NET is required for transporter internalization. Phosphoamino acid analysis of 32P-labeled native NETs from rat placental trophoblasts and heterologously expressed wild type human NET (WT-hNET) from human placental trophoblast cells revealed that the phorbol...... ester (beta-PMA)-induced phosphorylation of NET occurs on serine and threonine residues. Beta-PMA treatment inhibited NE transport, reduced plasma membrane hNET levels, and stimulated hNET phosphorylation in human placental trophoblast cells expressing the WT-hNET. Substance P-mediated activation of the...

  18. Analysis of wheat SAGE tags reveals evidence for widespread antisense transcription

    Directory of Open Access Journals (Sweden)

    Gibbings J George

    2008-10-01

    role in the regulation of gene expression. Conclusion Our results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Independent component analysis of DTI data reveals white matter covariances in Alzheimer's disease

    Science.gov (United States)

    Ouyang, Xin; Sun, Xiaoyu; Guo, Ting; Sun, Qiaoyue; Chen, Kewei; Yao, Li; Wu, Xia; Guo, Xiaojuan

    2014-03-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease with the clinical symptom of the continuous deterioration of cognitive and memory functions. Multiple diffusion tensor imaging (DTI) indices such as fractional anisotropy (FA) and mean diffusivity (MD) can successfully explain the white matter damages in AD patients. However, most studies focused on the univariate measures (voxel-based analysis) to examine the differences between AD patients and normal controls (NCs). In this investigation, we applied a multivariate independent component analysis (ICA) to investigate the white matter covariances based on FA measurement from DTI data in 35 AD patients and 45 NCs from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. We found that six independent components (ICs) showed significant FA reductions in white matter covariances in AD compared with NC, including the genu and splenium of corpus callosum (IC-1 and IC-2), middle temporal gyral of temporal lobe (IC-3), sub-gyral of frontal lobe (IC-4 and IC-5) and sub-gyral of parietal lobe (IC-6). Our findings revealed covariant white matter loss in AD patients and suggest that the unsupervised data-driven ICA method is effective to explore the changes of FA in AD. This study assists us in understanding the mechanism of white matter covariant reductions in the development of AD.

  5. Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

    Directory of Open Access Journals (Sweden)

    Marangoni Sérgio

    2009-04-01

    Full Text Available Abstract Background Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area – BA22p identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6 and glial fibrillary acidic protein (GFAP were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

  6. 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.

  7. Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability.

    Science.gov (United States)

    Li, Jia; Liu, Fei; Wang, Qi; Ge, Pupu; Woo, Patrick C Y; Yan, Jinghua; Zhao, Yanlin; Gao, George F; Liu, Cui Hua; Liu, Changting

    2014-01-01

    The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens. PMID:25163721

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Pre-2014 mudslides at Oso revealed by InSAR and multi-source DEM analysis

    Science.gov (United States)

    Kim, J. W.; Lu, Z.; QU, F.

    2014-12-01

    The landslide is a process that results in the downward and outward movement of slope-reshaping materials including rocks and soils and annually causes the loss of approximately $3.5 billion and tens of casualties in the United States. The 2014 Oso mudslide was an extreme event costing nearly 40 deaths and damaging civilian properties. Landslides are often unpredictable, but in many cases, catastrophic events are repetitive. Historic record in the Oso mudslide site indicates that there have been serial events in decades, though the extent of sliding events varied from time to time. In our study, the combination of multi-source DEMs, InSAR, and time-series InSAR analysis has enabled to characterize the Oso mudslide. InSAR results from ALOS PALSAR show that there was no significant deformation between mid-2006 and 2011. The combination of time-series InSAR analysis and old-dated DEM indicated revealed topographic changes associated the 2006 sliding event, which is confirmed by the difference of multiple LiDAR DEMs. Precipitation and discharge measurements before the 2006 and 2014 landslide events did not exhibit extremely anomalous records, suggesting the precipitation is not the controlling factor in determining the sliding events at Oso. The lack of surface deformation during 2006-2011 and weak correlation between the precipitation and the sliding event, suggest other factors (such as porosity) might play a critical role on the run-away events at this Oso and other similar landslides.

  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. 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

  15. Comparative transcriptome and proteome analysis to reveal the biosynthesis of gold nanoparticles in Arabidopsis

    Science.gov (United States)

    Tiwari, Manish; Krishnamurthy, Sneha; Shukla, Devesh; Kiiskila, Jeffrey; Jain, Ajay; Datta, Rupali; Sharma, Nilesh; Sahi, Shivendra V.

    2016-01-01

    A large number of plants have been tested and exploited in search of a green chemistry approach for the fabrication of gold or other precious metal nanomaterials. Despite the potential of plant based methods, very little is known about the underlying biochemical reactions and genes involved in the biotransformation mechanism of AuCl4 into gold nanoparticles (AuNPs). In this research, we thus focused on studying the effect of Au on growth and nanoparticles formation by analyses of transcriptome, proteome and ionome shift in Arabidopsis. Au exposure favored the growth of Arabidopsis seedling and induced formation of nanoparticles in root and shoot, as indicated by optical and hyperspectral imaging. Root transcriptome analysis demonstrated the differential expression of the members of WRKY, MYB and BHLH gene families, which are involved in the Fe and other essential metals homeostasis. The proteome analysis revealed that Glutathione S-transferases were induced in the shoot and suggested its potential role in the biosynthesis AuNPs. This study also demonstrated the role of plant hormone auxin in determining the Au induced root system architecture. This is the first study using an integrated approach to understand the in planta biotransformation of KAuCl4 into AuNPs. PMID:26902325

  16. 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.

  17. 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

  18. Proteomic analysis reveals differentially regulated protein acetylation in human amyotrophic lateral sclerosis spinal cord.

    Directory of Open Access Journals (Sweden)

    Dong Liu

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS identifies glial fibrillary acidic protein (GFAP as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS.

  19. 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.

  20. Comparative Transcriptome Analysis Reveals Different Silk Yields of Two Silkworm Strains

    Science.gov (United States)

    Li, Juan; Qin, Sheng; Yu, Huanjun; Zhang, Jing; Liu, Na; Yu, Ye; Hou, Chengxiang; Li, Muwang

    2016-01-01

    Cocoon and silk yields are the most important characteristics of sericulture. However, few studies have examined the genes that modulate these features. Further studies of these genes will be useful for improving the products of sericulture. JingSong (JS) and Lan10 (L10) are two strains having significantly different cocoon and silk yields. In the current study, RNA-Seq and quantitative polymerase chain reaction (qPCR) were performed on both strains in order to determine divergence of the silk gland, which controls silk biosynthesis in silkworms. Compared with L10, JS had 1375 differentially expressed genes (DEGs; 738 up-regulated genes and 673 down-regulated genes). Nine enriched gene ontology (GO) terms were identified by GO enrichment analysis based on these DEGs. KEGG enrichment analysis results showed that the DEGs were enriched in three pathways, which were mainly associated with the processing and biosynthesis of proteins. The representative genes in the enrichment pathways and ten significant DEGs were further verified by qPCR, the results of which were consistent with the RNA-Seq data. Our study has revealed differences in silk glands between the two silkworm strains and provides a perspective for understanding the molecular mechanisms determining silk yield. PMID:27159277

  1. Structure analysis reveals the flexibility of the ADAMTS-5 active site

    Energy Technology Data Exchange (ETDEWEB)

    Shieh, Huey-Sheng; Tomasselli, Alfredo G.; Mathis, Karl J.; Schnute, Mark E.; Woodard, Scott S.; Caspers, Nicole; Williams, Jennifer M.; Kiefer, James R.; Munie, Grace; Wittwer, Arthur; Malfait, Anne-Marie; Tortorella, Micky D. (Pfizer)

    2012-03-02

    A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.

  2. Fission yeast Rad52 phosphorylation restrains error prone recombination pathways.

    Science.gov (United States)

    Bellini, Angela; Girard, Pierre-Marie; Tessier, Ludovic; Sage, Evelyne; Francesconi, Stefania

    2014-01-01

    Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive. PMID:24748152

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

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    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

  4. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

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    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.

  5. 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

  6. 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.

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

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    Hong Hao

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

  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. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    Science.gov (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  11. Genome sequencing and analysis reveals possible determinants of Staphylococcus aureus nasal carriage

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    Cole Alexander M

    2008-09-01

    Full Text Available Abstract Background Nasal carriage of Staphylococcus aureus is a major risk factor in clinical and community settings due to the range of etiologies caused by the organism. We have identified unique immunological and ultrastructural properties associated with nasal carriage isolates denoting a role for bacterial factors in nasal carriage. However, despite extensive molecular level characterizations by several groups suggesting factors necessary for colonization on nasal epithelium, genetic determinants of nasal carriage are unknown. Herein, we have set a genomic foundation for unraveling the bacterial determinants of nasal carriage in S. aureus. Results MLST analysis revealed no lineage specific differences between carrier and non-carrier strains suggesting a role for mobile genetic elements. We completely sequenced a model carrier isolate (D30 and a model non-carrier strain (930918-3 to identify differential gene content. Comparison revealed the presence of 84 genes unique to the carrier strain and strongly suggests a role for Type VII secretion systems in nasal carriage. These genes, along with a putative pathogenicity island (SaPIBov present uniquely in the carrier strains are likely important in affecting carriage. Further, PCR-based genotyping of other clinical isolates for a specific subset of these 84 genes raise the possibility of nasal carriage being caused by multiple gene sets. Conclusion Our data suggest that carriage is likely a heterogeneic phenotypic trait and implies a role for nucleotide level polymorphism in carriage. Complete genome level analyses of multiple carriage strains of S. aureus will be important in clarifying molecular determinants of S. aureus nasal carriage.

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

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    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. Importance of glycolysis and oxidative phosphorylation in advanced melanoma

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    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.

  14. 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

  15. 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

  16. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    International Nuclear Information System (INIS)

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with 32P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with 32P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C

  17. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

  18. An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

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    Diego Forni

    2014-03-01

    Full Text Available The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region. In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin. Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human

  19. An evolutionary analysis of antigen processing and presentation across different timescales reveals pervasive selection.

    Science.gov (United States)

    Forni, Diego; Cagliani, Rachele; Tresoldi, Claudia; Pozzoli, Uberto; De Gioia, Luca; Filippi, Giulia; Riva, Stefania; Menozzi, Giorgia; Colleoni, Marta; Biasin, Mara; Lo Caputo, Sergio; Mazzotta, Francesco; Comi, Giacomo P; Bresolin, Nereo; Clerici, Mario; Sironi, Manuela

    2014-03-01

    The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes

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

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    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 lipids reveals differences between 'Mycobacterium habana' and Mycobacterium simiae.

    Science.gov (United States)

    Mederos, L M; Valdivia, J A; Sempere, M A; Valero-Guillén, P L

    1998-05-01

    Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of 'M. habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II', GPL-II and GPL-III; group C contained GPL-II', GPL-II and GPL-III; group D did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of alpha-, alpha'- and ketomycolates. Partial structural analysis by MS carried out in strains 'M. habana' TMC 5135 and M. simiae ATCC 25275T revealed that alpha- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and alpha'-mycolates from 58 to 67 carbon atoms. The alpha- and ketomycolates belonged to several structural series, and minor variations were found between the two strain examined. The data obtained justified the synonymy between 'M. habana' and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of 'M. habana' can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains. PMID:9611792

  4. 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

  5. LRpath analysis reveals common pathways dysregulated via DNA methylation across cancer types

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    Kim Jung H

    2012-10-01

    Full Text Available Abstract Background The relative contribution of epigenetic mechanisms to carcinogenesis is not well understood, including the extent to which epigenetic dysregulation and somatic mutations target similar genes and pathways. We hypothesize that during carcinogenesis, certain pathways or biological gene sets are commonly dysregulated via DNA methylation across cancer types. The ability of our logistic regression-based gene set enrichment method to implicate important biological pathways in high-throughput data is well established. Results We developed a web-based gene set enrichment application called LRpath with clustering functionality that allows for identification and comparison of pathway signatures across multiple studies. Here, we employed LRpath analysis to unravel the commonly altered pathways and other gene sets across ten cancer studies employing DNA methylation data profiled with the Illumina HumanMethylation27 BeadChip. We observed a surprising level of concordance in differential methylation across multiple cancer types. For example, among commonly hypomethylated groups, we identified immune-related functions, peptidase activity, and epidermis/keratinocyte development and differentiation. Commonly hypermethylated groups included homeobox and other DNA-binding genes, nervous system and embryonic development, and voltage-gated potassium channels. For many gene sets, we observed significant overlap in the specific subset of differentially methylated genes. Interestingly, fewer DNA repair genes were differentially methylated than expected by chance. Conclusions Clustering analysis performed with LRpath revealed tightly clustered concepts enriched for differential methylation. Several well-known cancer-related pathways were significantly affected, while others were depleted in differential methylation. We conclude that DNA methylation changes in cancer tend to target a subset of the known cancer pathways affected by genetic aberrations.

  6. Human processing of short temporal intervals as revealed by an ERP waveform analysis

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    Yoshitaka Nakajima

    2011-12-01

    Full Text Available To clarify the time course over which the human brain processes information about durations up to ~300 ms, we reanalyzed the data that were previously reported by Mitsudo et al. (2009 using a multivariate analysis method. Event-related potentials were recorded from 19 scalp electrodes on 11 (9 original and 2 additional participants while they judged whether two neighboring empty time intervals—called t1 and t2 and marked by three tone bursts—had equal durations. There was also a control condition in which the participants were presented the same temporal patterns but without a judgment task. In the present reanalysis, we sought to visualize how the temporal patterns were represented in the brain over time. A correlation matrix across channels was calculated for each temporal pattern. Geometric separations between the correlation matrices were calculated, and subjected to multidimensional scaling. We performed such analyses for a moving 100-ms time window after the t1 presentations. In the windows centered at < 100 ms after the t2 presentation, the analyses revealed the local maxima of categorical separation between temporal patterns of perceptually equal durations versus perceptually unequal durations, both in the judgment condition and in the control condition. Such categorization of the temporal patterns was prominent only in narrow temporal regions. The analysis indicated that the participants determined whether the two neighboring time intervals were of equal duration mostly within 100 ms after the presentation of the temporal patterns. A very fast brain activity was related to the perception of elementary temporal patterns without explicit judgments. This is consistent with the findings of Mitsudo et al., and it is in line with the processing time hypothesis proposed by Nakajima et al. (2004. The validity of the correlation matrix analyses turned out to be an effective tool to grasp the overall responses of the brain to temporal

  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. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Genome-wide analysis reveals adaptation to high altitudes in Tibetan sheep.

    Science.gov (United States)

    Wei, Caihong; Wang, Huihua; Liu, Gang; Zhao, Fuping; Kijas, James W; Ma, Youji; Lu, Jian; Zhang, Li; Cao, Jiaxue; Wu, Mingming; Wang, Guangkai; Liu, Ruizao; Liu, Zhen; Zhang, Shuzhen; Liu, Chousheng; Du, Lixin

    2016-01-01

    Tibetan sheep have lived on the Tibetan Plateau for thousands of years; however, the process and consequences of adaptation to this extreme environment have not been elucidated for important livestock such as sheep. Here, seven sheep breeds, representing both highland and lowland breeds from different areas of China, were genotyped for a genome-wide collection of single-nucleotide polymorphisms (SNPs). The FST and XP-EHH approaches were used to identify regions harbouring local positive selection between these highland and lowland breeds, and 236 genes were identified. We detected selection events spanning genes involved in angiogenesis, energy production and erythropoiesis. In particular, several candidate genes were associated with high-altitude hypoxia, including EPAS1, CRYAA, LONP1, NF1, DPP4, SOD1, PPARG and SOCS2. EPAS1 plays a crucial role in hypoxia adaption; therefore, we investigated the exon sequences of EPAS1 and identified 12 mutations. Analysis of the relationship between blood-related phenotypes and EPAS1 genotypes in additional highland sheep revealed that a homozygous mutation at a relatively conserved site in the EPAS1 3' untranslated region was associated with increased mean corpuscular haemoglobin concentration and mean corpuscular volume. Taken together, our results provide evidence of the genetic diversity of highland sheep and indicate potential high-altitude hypoxia adaptation mechanisms, including the role of EPAS1 in adaptation. PMID:27230812

  16. A Simple Geotracer Compositional Correlation Analysis Reveals Oil Charge and Migration Pathways

    Science.gov (United States)

    Yang, Yunlai; Arouri, Khaled

    2016-03-01

    A novel approach, based on geotracer compositional correlation analysis is reported, which reveals the oil charge sequence and migration pathways for five oil fields in Saudi Arabia. The geotracers utilised are carbazoles, a family of neutral pyrrolic nitrogen compounds known to occur naturally in crude oils. The approach is based on the concept that closely related fields, with respect to filling sequence, will show a higher carbazole compositional correlation, than those fields that are less related. That is, carbazole compositional correlation coefficients can quantify the charge and filling relationships among different fields. Consequently, oil migration pathways can be defined based on the established filling relationships. The compositional correlation coefficients of isomers of C1 and C2 carbazoles, and benzo[a]carbazole for all different combination pairs of the five fields were found to vary extremely widely (0.28 to 0.94). A wide range of compositional correlation coefficients allows adequate differentiation of separate filling relationships. Based on the established filling relationships, three distinct migration pathways were inferred, with each apparently being charged from a different part of a common source kitchen. The recognition of these charge and migration pathways will greatly aid the search for new accumulations.

  17. 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

  18. 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

  19. Rapid Holocene coastal change revealed by high-resolution micropaleontological analysis, Pamlico Sound, North Carolina, USA

    Science.gov (United States)

    Grand, Pre C.; Culver, S.J.; Mallinson, D.J.; Farrell, K.M.; Corbett, D.R.; Horton, B.P.; Hillier, C.; Riggs, S.R.; Snyder, S.W.; Buzas, M.A.

    2011-01-01

    Foraminiferal analyses of 404 contiguous samples, supported by diatom, lithologic, geochronologic and seismic data, reveal both rapid and gradual Holocene paleoenvironmental changes in an 8.21-m vibracore taken from southern Pamlico Sound, North Carolina. Data record initial flooding of a latest Pleistocene river drainage and the formation of an estuary 9000. yr ago. Estuarine conditions were punctuated by two intervals of marine influence from approximately 4100 to 3700 and 1150 to 500. cal. yr BP. Foraminiferal assemblages in the muddy sand facies that accumulated during these intervals contain many well-preserved benthic foraminiferal species, which occur today in open marine settings as deep as the mid shelf, and significant numbers of well-preserved planktonic foraminifera, some typical of Gulf Stream waters. We postulate that these marine-influenced units resulted from temporary destruction of the southern Outer Banks barrier islands by hurricanes. The second increase in marine influence is coeval with increased rate of sea-level rise and a peak in Atlantic tropical cyclone activity during the Medieval Climate Anomaly. This high-resolution analysis demonstrates the range of environmental variability and the rapidity of coastal change that can result from the interplay of changing climate, sea level and geomorphology in an estuarine setting. ?? 2011 University of Washington.

  20. Proteome analysis of Paenibacillus larvae reveals the existence of a putative S-layer protein.

    Science.gov (United States)

    Fünfhaus, Anne; Genersch, Elke

    2012-04-01

    Honey bee pathology has attracted much interest recently due to the problems with honey bee declines in many regions of the world. American Foulbrood (AFB) caused by Paenibacillus larvae is the most devastating bacterial brood disease of the Western honey bee (Apis mellifera) causing considerable economic losses to beekeepers worldwide. AFB outbreaks are mainly caused by two differentially virulent genotypes of P. larvae, P. larvae ERIC I and ERIC II. To better understand AFB pathogenesis and to complement already existing data from the genetic level we aimed at obtaining expression data from the protein level. We successfully developed a protocol for two-dimensional proteome analysis of P. larvae with subsequent mass-spectrometry based protein sequencing. Based on the obtained master protein maps of P. larvae genotypes ERIC I and II we identified the dominantly expressed cytosolic proteins of both genotypes, some of them presumably linked to pathogenesis and virulence. Comparing the master maps of both genotypes revealed differentially expressed proteins, i.e. a putative S-layer protein which is expressed by P. larvae ERIC II but absent from the proteome of P. larvae ERIC I. The implications of our findings for pathogenesis of AFB and virulence of P. larvae will be discussed. PMID:23757273

  1. 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

  2. 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

  3. Conserved Ser residues in the basic region of the bZIP-type transcription factor HBP-1a(17): importance in DNA binding and possible targets for phosphorylation.

    Science.gov (United States)

    Meshi, T; Moda, I; Minami, M; Okanami, M; Iwabuchi, M

    1998-01-01

    HBP-1a(17) is representative of a group of plant bZIP-type transcription factors which includes HBP-1a proteins and G-box-binding factors. We found kinase activity in wheat nuclear extract that phosphorylated HBP-1a(17). Experiments using recombinant HBP-1a(17) derivatives as substrates revealed that all three of the Ser residues in the basic region, Ser-261, Ser-265, and Ser-269, were phosphorylated in a Ca(2+)-stimulated manner. DNA-binding analysis of mutants with a Ser-to-Glu change, prepared to mimic the phosphorylated proteins, indicated that introduction of a negative charge at position 265 or 269 prevents HBP-1a(17) from binding DNA not only in the homodimer of mutants but also in heterodimers with a wild-type protein. It is therefore suggested that the phosphorylation regulates the function of HBP-1a(17) at least at the level of DNA binding. Since Ser-265 and Ser-269 are highly conserved among the plant bZIP-type factors known to date, a common Ca(2+)-mediated regulatory mechanism may exert an effect on the bZIP-type factors through phosphorylation of these conserved Ser residues. PMID:9484468

  4. Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

    International Nuclear Information System (INIS)

    KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it

  5. Proteomic and bioinformatic analysis of epithelial tight junction reveals an unexpected cluster of synaptic molecules

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    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

  6. 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

  7. 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

  8. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  9. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    International Nuclear Information System (INIS)

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β and δ subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the δ subunit 20-fold over basal phosphorylation and induced phosphorylation of the α subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 μM in the presence of 35 μM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of δ subunit phosphorylation was almost maximal within 5 min, whereas stimulation of α subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the δ subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the δ subunit increases the rate of AcChoR desensitization in rat myotubes

  10. 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.

  11. 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

  12. 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.

  13. Phosphorylation and disassembly of intermediate filaments in mitotic cells

    International Nuclear Information System (INIS)

    As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of Pi per mol of protein in interphase to 1.9 mol of Pi per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of Pi per mol of protein to 1.5 mol of Pi per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins

  14. Phosphorylation and disassembly of intermediate filaments in mitotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Yinghao; Rosevear, E.; Goldman, R.D. (Northwestern Univ. Medical School, Chicago, IL (USA))

    1989-03-01

    As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of P{sub i} per mol of protein in interphase to 1.9 mol of P{sub i} per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of P{sub i} per mol of protein to 1.5 mol of P{sub i} per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional {sup 32}P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins.

  15. Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements

    Science.gov (United States)

    Liu, Shu; Bolger, Joshua K.; Kirkland, Lindsay O.; Premnath, Padmavathy N.; McInnes, Campbell

    2012-01-01

    An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized

  16. 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

  17. 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

  18. 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

  19. DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

    Science.gov (United States)

    Hovey, Raymond; Lentes, Sabine; Ehrenreich, Armin; Salmon, Kirsty; Saba, Karla; Gottschalk, Gerhard; Gunsalus, Robert P; Deppenmeier, Uwe

    2005-05-01

    Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes. PMID:15902489

  20. Analysis of small nucleolar RNAs reveals unique genetic features in malaria parasites

    Directory of Open Access Journals (Sweden)

    Sharma Amit

    2009-02-01

    Full Text Available Abstract Background Ribosome biogenesis is an energy consuming and stringently controlled process that involves hundreds of trans-acting factors. Small nucleolar RNAs (snoRNAs, important components of ribosome biogenesis are non-coding guide RNAs involved in rRNA processing, nucleotide modifications like 2'-O-ribose methylation, pseudouridylation and possibly gene regulation. snoRNAs are ubiquitous and are diverse in their genomic organization, mechanism of transcription and process of maturation. In vertebrates, most snoRNAs are present in introns of protein coding genes and are processed by exonucleolytic cleavage, while in plants they are transcribed as polycistronic transcripts. Results This is a comprehensive analysis of malaria parasite snoRNA genes and proteins that have a role in ribosomal biogenesis. Computational and experimental approaches have been used to identify several box C/D snoRNAs from different species of Plasmodium and confirm their expression. Our analyses reveal that the gene for endoribonuclease Rnt1 is absent from Plasmodium falciparum genome, which indicates the existence of alternative pre-rRNA processing pathways. The structural features of box C/D snoRNAs are highly conserved in Plasmodium genus; however, unlike other organisms most parasite snoRNAs are present in single copy. The genomic localization of parasite snoRNAs shows mixed patterns of those observed in plants, yeast and vertebrates. We have localized parasite snoRNAs in untranslated regions (UTR of mRNAs, and this is an unprecedented and novel genetic feature. Akin to mammalian snoRNAs, those in Plasmodium may also behave as mobile genetic elements. Conclusion This study provides a comprehensive overview on trans-acting genes involved in ribosome biogenesis and also a genetic insight into malaria parasite snoRNA genes.

  1. 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.

  2. 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.

  3. Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1 gene family.

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    Mia T Levine

    Full Text Available Heterochromatin is the gene-poor, satellite-rich eukaryotic genome compartment that supports many essential cellular processes. The functional diversity of proteins that bind and often epigenetically define heterochromatic DNA sequence reflects the diverse functions supported by this enigmatic genome compartment. Moreover, heterogeneous signatures of selection at chromosomal proteins often mirror the heterogeneity of evolutionary forces that act on heterochromatic DNA. To identify new such surrogates for dissecting heterochromatin function and evolution, we conducted a comprehensive phylogenomic analysis of the Heterochromatin Protein 1 gene family across 40 million years of Drosophila evolution. Our study expands this gene family from 5 genes to at least 26 genes, including several uncharacterized genes in Drosophila melanogaster. The 21 newly defined HP1s introduce unprecedented structural diversity, lineage-restriction, and germline-biased expression patterns into the HP1 family. We find little evidence of positive selection at these HP1 genes in both population genetic and molecular evolution analyses. Instead, we find that dynamic evolution occurs via prolific gene gains and losses. Despite this dynamic gene turnover, the number of HP1 genes is relatively constant across species. We propose that karyotype evolution drives at least some HP1 gene turnover. For example, the loss of the male germline-restricted HP1E in the obscura group coincides with one episode of dramatic karyotypic evolution, including the gain of a neo-Y in this lineage. This expanded compendium of ovary- and testis-restricted HP1 genes revealed by our study, together with correlated gain/loss dynamics and chromosome fission/fusion events, will guide functional analyses of novel roles supported by germline chromatin.

  4. 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.

  5. 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.

  6. 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.

  7. Meta-analysis of muscle transcriptome data using the MADMuscle database reveals biologically relevant gene patterns

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    Teusan Raluca

    2011-02-01

    Full Text Available Abstract Background DNA microarray technology has had a great impact on muscle research and microarray gene expression data has been widely used to identify gene signatures characteristic of the studied conditions. With the rapid accumulation of muscle microarray data, it is of great interest to understand how to compare and combine data across multiple studies. Meta-analysis of transcriptome data is a valuable method to achieve it. It enables to highlight conserved gene signatures between multiple independent studies. However, using it is made difficult by the diversity of the available data: different microarray platforms, different gene nomenclature, different species studied, etc. Description We have developed a system tool dedicated to muscle transcriptome data. This system comprises a collection of microarray data as well as a query tool. This latter allows the user to extract similar clusters of co-expressed genes from the database, using an input gene list. Common and relevant gene signatures can thus be searched more easily. The dedicated database consists in a large compendium of public data (more than 500 data sets related to muscle (skeletal and heart. These studies included seven different animal species from invertebrates (Drosophila melanogaster, Caenorhabditis elegans and vertebrates (Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus. After a renormalization step, clusters of co-expressed genes were identified in each dataset. The lists of co-expressed genes were annotated using a unified re-annotation procedure. These gene lists were compared to find significant overlaps between studies. Conclusions Applied to this large compendium of data sets, meta-analyses demonstrated that conserved patterns between species could be identified. Focusing on a specific pathology (Duchenne Muscular Dystrophy we validated results across independent studies and revealed robust biomarkers and new pathways of interest

  8. 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

  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. Star-disk interaction in classical T Tauri stars revealed using wavelet analysis

    Science.gov (United States)

    López-Santiago, J.; Crespo-Chacón, I.; Flaccomio, E.; Sciortino, S.; Micela, G.; Reale, F.

    2016-04-01

    Context. The extension of the corona of classical T Tauri stars (CTTS) is is being widely discussed. The standard model of magnetic configuration of CTTS predicts that coronal magnetic flux tubes connect the stellar atmosphere to the inner region of the disk. However, differential rotation may disrupt these long loops. The results from hydrodynamic modeling of X-ray flares observed in CTTS that confirm the star-disk connection hypothesis are still controversial. Some authors suggest the presence of the accretion disk prevents the stellar corona extending beyond the co-rotation radius, while others are simply not confident with the methods used to derive loop lengths. Aims: We use independent procedures to determine the length of flaring loops in stars of the Orion Nebula Cluster, which has previously been analyzed using hydrodynamic models. Our aim is to disentangle the two scenarios that have been proposed. Methods: We present a different approach for determining the length of flaring loops that is based on the oscillatory nature of the loops after strong flares. We use wavelet tools to reveal oscillations during several flares. The subsequent analysis of these oscillations is based on the physics of coronal seismology. Results: Our results likely confirm the large extension of the corona of CTTS and the hypothesis of star-disk magnetic interaction in at least three CTTS of the Orion Nebula Cluster. Conclusions: Analyzing oscillations in flaring events is a powerful tool to determine the physical characteristics of magnetic loops in coronae in stars other than the Sun. The results presented in this work confirm the star-disk magnetic connection in CTTS.

  11. 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

  12. 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

  13. 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⊙.

  14. 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

  15. 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

  16. 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.

  17. Analysis of global gene expression in Brachypodium distachyon reveals extensive network plasticity in response to abiotic stress.

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    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.

  18. 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

  19. Exploring the intramolecular phosphorylation sites in human Chk2

    DEFF Research Database (Denmark)

    Olsen, Birgitte B; Larsen, Martin R; Boldyreff, Brigitte;

    2008-01-01

    A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time......-fold after DNA damage. Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells...... it was 8. Except for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria....

  20. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.;

    2003-01-01

    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...... extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to...... cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in...

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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...

  6. Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

    Directory of Open Access Journals (Sweden)

    Timmers Marc HTH

    2010-12-01

    Full Text Available Abstract Background The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ. Results Comparing by proteomics WT and nat3Δ strains, using metabolic 15N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation. Conclusions Protein expression levels change only marginally in between WT and nat3Δ. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in nat3Δ revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.

  7. Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei.

    Science.gov (United States)

    Verner, Zdeněk; Cermáková, Petra; Skodová, Ingrid; Kováčová, Bianka; Lukeš, Julius; Horváth, Anton

    2014-01-01

    Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites. PMID:24556248

  8. The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific

    DEFF Research Database (Denmark)

    Raule, Nicola; Sevini, Federica; Li, Shengting;

    2014-01-01

    To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification......, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se...... analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes....

  9. 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...

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  18. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    International Nuclear Information System (INIS)

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  19. 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

  20. 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.

  1. Promiscuity and Selectivity in Phosphoryl Transferases

    OpenAIRE

    Barrozo, Alexandre

    2016-01-01

    Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of ...

  2. 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.

  3. 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

  4. 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; Ruckerbauer, David E; Hannibal-Bach, Hans Kristian; Zanghellini, Jürgen; Jensen, Ole N; Ejsing, Christer S

    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...... of peroxisomal biogenesis, via the heterodimeric Oaf1/Pip2 transcription factor. Our work demonstrates the pivotal role of lipid metabolism in adaptive processes and provides a resource to investigate its regulation at the cellular level....

  5. 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...

  6. Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems

    OpenAIRE

    Tay, Tuan Leng; Ronneberger, Olaf; Ryu, Soojin; Nitschke, Roland; Driever, Wolfgang

    2011-01-01

    Essential components of animal behaviour are modulated by dopaminergic (DA) and noradrenergic circuitry. In this study, we reveal at cellular resolution the complete set of projections ('projectome') of every single type of DA and noradrenergio neurons in the central nervous system of zebrafish larvae. The most extensive DA projections are established by posterior tubercular otp-dependent neurons, with individual somata integrating the ascending DA system, the descending diencephalospinal, as...

  7. 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,...

  8. 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 ...

  9. Do Ethical Consumers Care About Price? A Revealed Preference Analysis of Fair Trade Coffee Purchases

    OpenAIRE

    2006-01-01

    The existing literature on socially responsible purchasing relies heavily on stated preference measures elicited through surveys that utilize hypothetical market choices. This paper explores consumers’ revealed purchasing behavior with regard to fair trade coffee and is apparently the first to do so in an actual market setting. In a series of experiments, we investigated differences in consumer responsiveness to relative price changes in fair trade and non-fair trade brewed coffees. In orde...

  10. 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

  11. 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.

  12. 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.

  13. Increased phosphorylation of histone H3 at serine 10 is involved in Epstein-Barr virus latent membrane protein-1-induced carcinogenesis of nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Increased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC). The expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed. Immunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (χ2=6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1-induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation. EBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of

  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. 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

  16. 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.

  17. 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

  18. Genetic variability of brazilian Alternaria spp. isolates as revealed by RAPD analysis

    Directory of Open Access Journals (Sweden)

    Tigano Myrian Silvana

    2003-01-01

    Full Text Available The genetic variability of 22 isolates of the fungi Alternaria alternata and A. cassiae, obtained from Senna obtusifolia, was studied by RAPD analysis. A total of 491 scorable bands were produced with the use of 28 primers. Cluster analysis based on similarities computed from RAPD markers showed two distinct genetic groups of isolates related to both species. RAPD analysis proved to be an efficient method for detecting genetic variability of A. cassiae and A. alternata isolates occurring in S. obtusifolia, and also for distinguishing Alternaria species.

  19. 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...

  20. 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

    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...... aeruginosa isolated from the airways of cystic fibrosis patients. RESULTS: Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene...... suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. CONCLUSIONS: Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering...

  1. 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.

  2. 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...

  3. 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...

  4. Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

    OpenAIRE

    Ellen, Albert F.; Albers, Sonja-Verena; Huibers, Wim; Pitcher, Angela; Hobel, Cedric F. V.; Schwarz, Heinz; Folea, Mihaela; Schouten, Stefan; Boekema, Egbert J.; Poolman, Bert; Driessen, Arnold J. M.

    2009-01-01

    The crenarchaea Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii, release membrane vesicles into the medium. These membrane vesicles consist of tetraether lipids and are coated with an S-layer. A proteomic analysis reveals the presence of proteins homologous to subunits of the eukaryotic endosomal sorting complex required for transport (ESCRT). Immunodetection of one of these homologs suggest a cell surface localization in intact cells. These data suggest that the membrane vesicles ...

  5. 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.

  6. Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

    Science.gov (United States)

    Hsiao, Felix Shih-Hsiang; Sutandy, Fx Reymond; Syu, Guan-Da; Chen, Yi-Wen; Lin, Jun-Mu; Chen, Chien-Sheng

    2016-01-01

    Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host-GAG binding have been described; however, a systematic study on microbial proteome-mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin. PMID:27323865

  7. 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.

  8. Phosphorylation of the multidrug resistance associated glycoprotein

    International Nuclear Information System (INIS)

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 μM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [γ-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance

  9. Phosphorylation of the multidrug resistance associated glycoprotein.

    Science.gov (United States)

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  10. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening. PMID:22419220

  11. 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.

  12. A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

    DEFF Research Database (Denmark)

    Graham, Mark E; Thaysen-Andersen, Morten; Bache, Nicolai;

    2011-01-01

    Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved...... AP180 purified from rat brain contains a phosphorylated O-GlcNAc (O-GlcNAc-P) within a highly conserved sequence. O-GlcNAc or O-GlcNAc-P, but not phosphorylation alone, was found at Thr-310. Analysis of synthetic GlcNAc-6-P produced identical fragmentation products to GlcNAc-P from AP180. Direct O......-linkage of GlcNAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation...

  13. 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+

  14. 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.

  15. Transcriptome network analysis reveals potential candidate genes for squamous lung cancer.

    Science.gov (United States)

    Bai, Jing; Hu, Sheng

    2012-01-01

    Squamous lung cancer is a common type of lung cancer; however, its mechanism of oncogenesis is still unknown. The aim of this study was to screen candidate genes of squamous lung cancer using a bioinformatics strategy and elucidate the mechanism of squamous lung cancer. Published microarray data of the GSE3268 series was obtained from Gene Expression Omnibus (GEO). Significance analysis of microarrays was performed using the software R, and differentially expressed genes by R analysis were harvested. The relationship between transcription factors and target genes in cancer were collected from the Transcriptional regulatory element database. A transcriptome network analysis method was used to construct gene regulation networks and select the candidate genes for squamous lung cancer. SPI1, FLI1, FOS, ETS2, EGR1 and PPARG were defined as candidate genes for squamous lung cancer by the transcriptome network analysis method. Among them, 5 genes had been reported to be involved in lung cancer, except SPI1 and FLI1. Effective recall on previous knowledge conferred strong confidence in these methods. It is demonstrated that transcriptome network analysis is useful in the identification of candidate genes in disease. PMID:21922129

  16. 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.

  17. 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

  18. Mass Spectrometric-Based Selected Reaction Monitoring of Protein Phosphorylation during Symbiotic Signaling in the Model Legume, Medicago truncatula.

    Directory of Open Access Journals (Sweden)

    Lori K Van Ness

    Full Text Available 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.

  19. 11-cis-retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors.

    Science.gov (United States)

    Ablonczy, Zsolt; Crouch, Rosalie K; Goletz, Patrice W; Redmond, T Michael; Knapp, Daniel R; Ma, Jian-Xing; Rohrer, Barbel

    2002-10-25

    Rpe65(-/-) mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65(-/-) mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65(-/-) mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65(-/-) mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein. PMID:12176991

  20. 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.

  1. 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...... programmed cell death protein 8(AIF) in native and purified samples of complex I analysed. The possible physiological relevance of these findings is discussed....

  2. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

    DEFF Research Database (Denmark)

    Erba, Elisabetta Boeri; Matthiesen, Rune; Bunkenborg, Jakob; Schulze, Waltraud X; Di Stefano, Paola; Cabodi, Sara; Tarone, Guido; Defilippi, Paola; Jensen, Ole N

    2007-01-01

    Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium...

  3. 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.

  4. Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments

    OpenAIRE

    Sobieszek, Apolinary; Sarg, Bettina; Seow, Chun Y.; Lindner, Herbert

    2010-01-01

    Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol Pi per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate f...

  5. Induction of foci of phosphorylated H2AX histones and premature chromosome condensation after DNA damage in Vicia faba root meristem

    International Nuclear Information System (INIS)

    Immunocytochemical analysis using antibody raised against human H2AX histones phosphorylated at serine 139 (gamma-H2AX) demonstrates that root meristem cells of Vicia faba exposed to UV-radiation or incubated with hydroxyurea (HU) reveal discrete foci at the border of the nucleolus and perinucleolar chromatin or scattered over the whole area of cell nucleus. Western blots detected only one protein band at the position expected for the phosphorylated form of H2AX. The dose-effect relationship was demonstrated following treatment with 2.5 and 10 mM HU. Proteins extracted from root meristems incubated for 2 h either with HU and caffeine or with HU and sodium metavanadate showed unchanged amounts of bound gamma-H2AX antibodies as compared to root meristems treated with 2.5 mM HU. Higher quantities of phosphorylated H2AX histones were detected in proteins extracted from roots treated with HU and 2-aminopurine. All treatments were effective in producing evident aberrations of premature mitosis: broken and lagging chromatids, acentric fragments, chromosomal bridges and micronuclei. Our results show that phosphorylation of H2AX at the carboxy-terminal Ser-Gln-Glu sequence is among the earliest responses to double-strand breaks and, presumably, one of the key ATM/ATR-dependent signals indispensable for the repair of spontaneous and induced DNA damage in plant cells

  6. Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: Effects on affinity for IGF-I

    Energy Technology Data Exchange (ETDEWEB)

    Jones, J.I.; D' Ercole, A.J.; Camacho-Hubner, C.; Clemmons, D.R. (Univ. of North Carolina, Chapel Hill (United States))

    1991-09-01

    The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). The authors and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted them to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells and human hepatoma (hepG2) cells secrete {sup 32}P-labeled IGFBP-1 following incubation with ({sup 32}P)orthophosphate. Phospho amino acid analysis of {sup 32}P-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. They conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

  7. Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Camp, David G.; Gritsenko, Marina A.; Luo, Quanzhou; Kelly, Ryan T.; Clauss, Therese RW; Brinkley, William R.; Smith, Richard D.; Stenoien, David L.

    2007-11-16

    The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.

  8. Role of insulin receptor phosphorylation in the insulinomimetic effects of hydrogen peroxide

    International Nuclear Information System (INIS)

    The oxidant H2O2 has many insulin-like effects in rat adipocytes. To determine whether these effects could be mediated by the tyrosine kinase activity of the insulin receptor, the ability of H2O2 to stimulate receptor phosphorylation in intact adipocytes and partially purified insulin receptors has been examined. Phosphorylation of the β subunit of the insulin receptor was increased. Stimulation of receptor phosphorylation was rapid, reaching maximal levels within 5 min, and preceded activation of glucose transport. Phosphoamino acid analysis of insulin receptors from H2O2-treated adipocytes showed that 32P incorporation into phosphotyrosine and phosphoserine residues of the β subunit was enhanced. Furthermore, partially purified receptors from H2O2-treated cells exhibit increased tyrosine kinase activity, as measured by phosphorylation of the peptide Glu80Tyr20. To define the factors involved in H2O2's effect, the authors have examined receptor phosphorylation in fat cell homogenates and purified plasma membranes. Although insulin stimulated receptor phosphorylation in both of these systems, H2O2 was only effective in the cell homogenates. These data demonstrate that, under certain conditions, H2O2 stimulates insulin receptor phosphorylation and tyrosine kinase activity, suggesting that the insulin-like effects of H2O2 may be mediated by stimulation of insulin receptor phosphorylation. This does not appear to be a direct effect of H2O2 on the insulin receptor and requires nonplasma membrane cellular constituents

  9. RNA-seq analysis of Pichia anomala reveals important mechanisms required for survival at low pH

    DEFF Research Database (Denmark)

    Fletcher, Eugene; Feizi, Amir; Kim, SungSoo;

    2015-01-01

    associated with different biological processes including proton efflux, the electron transfer chain and oxidative phosphorylation were highly expressed in P. anomala cells grown in low pH media. We therefore present data supporting the notion that a high expression of proton pumps in the plasma membrane...

  10. 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....

  11. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation

    Science.gov (United States)

    Arosio, Paolo; Michaels, Thomas C. T.; Linse, Sara; Månsson, Cecilia; Emanuelsson, Cecilia; Presto, Jenny; Johansson, Jan; Vendruscolo, Michele; Dobson, Christopher M.; Knowles, Tuomas P. J.

    2016-03-01

    It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.

  12. Heavy metal distribution in Suillus luteus mycorrhizas - as revealed by micro-PIXE analysis

    International Nuclear Information System (INIS)

    Suillus luteus/Pinus sylvestris mycorrhizas, collected from zinc wastes in Southern Poland, were selected as potential biofilters on the basis of earlier studies carried out with energy dispersive spectrometry (EDS) microanalytical system coupled to scanning electron microscope (SEM) and transmission electron microscope (TEM). Using the National Accelerator Centre (NAC) nuclear microprobe, elemental concentrations in the ectomycorrhiza parts were for the first time estimated quantitatively. Micro-proton-induced X-ray emission (PIXE) true elemental maps from freeze-dried and chemically fixed mycorrhizas revealed strong accumulation of Ca, Fe, Zn and Pb within the fungal mantle and in the rhizomorph. Vascular tissue was enriched with P, S and K, while high concentrations of Si and Cl were present in the endodermis. Cu was the only element showing elevated concentrations in the cortex region. Elemental losses and redistributions were found in mycorrhizas prepared by chemical fixation. Some problems related to elemental imaging are discussed

  13. Heavy metal distribution in Suillus luteus mycorrhizas - as revealed by micro-PIXE analysis

    Energy Technology Data Exchange (ETDEWEB)

    Turnau, K. E-mail: ubturnau@cyf-kr.edu.pl; Przybylowicz, W.J.; Mesjasz-Przybylowicz, J

    2001-07-01

    Suillus luteus/Pinus sylvestris mycorrhizas, collected from zinc wastes in Southern Poland, were selected as potential biofilters on the basis of earlier studies carried out with energy dispersive spectrometry (EDS) microanalytical system coupled to scanning electron microscope (SEM) and transmission electron microscope (TEM). Using the National Accelerator Centre (NAC) nuclear microprobe, elemental concentrations in the ectomycorrhiza parts were for the first time estimated quantitatively. Micro-proton-induced X-ray emission (PIXE) true elemental maps from freeze-dried and chemically fixed mycorrhizas revealed strong accumulation of Ca, Fe, Zn and Pb within the fungal mantle and in the rhizomorph. Vascular tissue was enriched with P, S and K, while high concentrations of Si and Cl were present in the endodermis. Cu was the only element showing elevated concentrations in the cortex region. Elemental losses and redistributions were found in mycorrhizas prepared by chemical fixation. Some problems related to elemental imaging are discussed.

  14. 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

  15. 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.

  16. cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, Pinctada fucata martensii.

    Science.gov (United States)

    Shi, Yaohua; Zheng, Xing; Zhan, Xin; Wang, Aimin; Gu, Zhifeng

    2016-06-01

    Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E ≤ -10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster. PMID:27184264

  17. Global multilocus sequence typing analysis of Mycoplasma bovis isolates reveals two main population clusters.

    Science.gov (United States)

    Rosales, R S; Churchward, C P; Schnee, C; Sachse, K; Lysnyansky, I; Catania, S; Iob, L; Ayling, R D; Nicholas, R A J

    2015-03-01

    Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins. PMID:25540400

  18. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

    DEFF Research Database (Denmark)

    Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming;

    2012-01-01

    RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed a...

  19. Proteomic analysis of human metaphase chromosomes reveals Topoisomerase II alpha as an Aurora B substrate

    DEFF Research Database (Denmark)

    Morrison, Ciaran; Henzing, Alexander J; Jensen, Ole Nørregaard; Osheroff, Neil; Dodson, Helen; Kandels-Lewis, Stefanie E; Adams, Richard R; Earnshaw, William C

    2002-01-01

    B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein...

  20. 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

  1. Identification of phosphorylated proteins in erythrocytes infected by the human malaria parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Xia Dong

    2009-05-01

    Full Text Available Abstract Background Previous comparative proteomic analysis on Plasmodium falciparum isolates of different adhesion properties suggested that protein phosphorylation varies between isolates with different cytoadherence properties. But the extent and dynamic changes in phosphorylation have not been systematically studied. As a baseline for these future studies, this paper examined changes in the phosphoproteome of parasitized red blood cells (pRBC. Methods Metabolic labelling with [35S] methionine on pRBC and 2D gel electrophoresis (2-DE has previously been used to show the expression of parasite proteins and changes in protein iso-electric point (PI. 2-DE of different parasite strains was combined with immunoblotting using monoclonal antibodies specifically to phosphorylated serine/threonine and tyrosine, to obtain the phosphorylation profiles throughout the erythrocytic lifecycle. Affinity chromatography was used to purify/enrich phosphorylated proteins and these proteins from mature trophozoite stages which were identified using high-accuracy mass spectrometry and MASCOT search. Results 2D-immunoblots showed that P. falciparum infection greatly increased phosphorylation of a set of proteins in pRBC, the dominant size classes for phosphorylated tyrosine proteins were 95, 60, 50 and 30 kDa and for phosphorylated serine/threonine were 120, 95, 60, 50, 43, 40 and 30 kDa. The most abundant molecules from 2D-gel mapping of phosphorylated proteins in ItG infected RBCs were identified by MALDI-TOF. A proteomic overview of phosphorylated proteins in pRBC was achieved by using complementary phosphorylated protein enrichment techniques combined with nano-flow LC/MS/MS analysis and MASCOT MS/MS ions search with phosphorylation as variable modifications. The definite phosphoproteins of pRBC are reported and discussed. Conclusion Protein phosphorylation is a major process in P. falciparum-parasitized erythrocytes. Preliminary screens identified 170 P

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Maria Jesus Iglesias

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

  4. PhosphoScore: An Open-Source Phosphorylation Site Assignment Tool for MSn Data

    OpenAIRE

    Ruttenberg, Brian E.; Pisitkun, Trairak; Knepper, Mark A.; Jason D. Hoffert

    2008-01-01

    Correct phosphorylation site assignment is a critical aspect of phosphoproteomic analysis. Large-scale phosphopeptide data sets that are generated through liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis often contain hundreds or thousands of phosphorylation sites that require validation. To this end, we have created PhosphoScore, an open-source assignment program that is compatible with phosphopeptide data from multiple MS levels (MSn). The algorithm takes into acco...

  5. 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.

  6. 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.

  7. 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.

  8. Computational Image Analysis Reveals Intrinsic Multigenerational Differences between Anterior and Posterior Cerebral Cortex Neural Progenitor Cells.

    Science.gov (United States)

    Winter, Mark R; Liu, Mo; Monteleone, David; Melunis, Justin; Hershberg, Uri; Goderie, Susan K; Temple, Sally; Cohen, Andrew R

    2015-10-13

    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. PMID:26344906

  9. 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.

  10. 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...

  11. Revealing the X-ray Variability of AGN with Principal Component Analysis

    OpenAIRE

    Parker, M L; Fabian, A.C.; Matt, G.; Koljonen, K. I. I.; Kara, E.; Alston, W.; Walton, D. J.; Marinucci, A.; Brenneman, L.; Risaliti, G.

    2014-01-01

    We analyse a sample of 26 active galactic nuclei (AGN) with deep XMM–Newton observations, using principal component analysis (PCA) to find model-independent spectra of the different variable components. In total, we identify at least 12 qualitatively different patterns of spectral variability, involving several different mechanisms, including five sources which show evidence of variable relativistic reflection (MCG–6-30-15, NGC 4051, 1H 0707−495, NGC 3516 and Mrk 766) and three which show evi...

  12. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    OpenAIRE

    Zhao, Zhongtao; Liu, Huiquan; Wang, Chenfang; Xu, Jin-Rong

    2013-01-01

    Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZyme...

  13. 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...

  14. 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...

  15. Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation

    Directory of Open Access Journals (Sweden)

    Treuner-Lange Anke

    2010-04-01

    Full Text Available Abstract Background Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities. Results Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate

  16. Regionally selective atrophy of subcortical structures in prodromal HD as revealed by statistical shape analysis

    OpenAIRE

    Younes, L.; Ratnanather, JT; Brown, T.; Aylward, E; Nopoulos, P.; Johnson, H.; Magnotta, VA; Paulsen, JS; Margolis, RL; Albin, RL; Miller, MI; Ross, CA; Wassink, T; Cross, S.; Kimble, M

    2012-01-01

    Huntington disease (HD) is a neurodegenerative disorder that involves preferential atrophy in the striatal complex and related subcortical nuclei. In this article, which is based on a dataset extracted from the PREDICT-HD study, we use statistical shape analysis with deformation markers obtained through "Large Deformation Diffeomorphic Metric Mapping" of cortical surfaces to highlight specific atrophy patterns in the caudate, putamen, and globus pallidus, at different prodromal stages of the ...

  17. 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.

  18. 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...

  19. Genome Wide Analysis of Chromatin Regulation by Cocaine Reveals a Novel Role for Sirtuins

    OpenAIRE

    Renthal, William; Kumar, Arvind; Xiao, Guanghua; Wilkinson, Matthew; Covington, Herbert E.; Maze, Ian; Sikder, Devanjan; Robison, Alfred J.; LaPlant, Quincey; Dietz, David M.; Russo, Scott J.; Vialou, Vincent; Chakravarty, Sumana; Kodadek, Thomas J.; Stack, Ashley

    2009-01-01

    Changes in gene expression contribute to the long-lasting regulation of the brain’s reward circuitry seen in drug addiction, however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide chromatin changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings...

  20. Meta-analysis of muscle transcriptome data using the MADMuscle database reveals biologically relevant gene patterns

    OpenAIRE

    Teusan Raluca; Bihouée Audrey; Dubois Emeric; Baron Daniel; Steenman Marja; Jourdon Philippe; Magot Armelle; Péréon Yann; Veitia Reiner; Savagner Frédérique; Ramstein Gérard; Houlgatte Rémi

    2011-01-01

    Abstract Background DNA microarray technology has had a great impact on muscle research and microarray gene expression data has been widely used to identify gene signatures characteristic of the studied conditions. With the rapid accumulation of muscle microarray data, it is of great interest to understand how to compare and combine data across multiple studies. Meta-analysis of transcriptome data is a valuable method to achieve it. It enables to highlight conserved gene signatures between mu...

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

    OpenAIRE

    Barbosa, Catarina; García Martínez, José; Pérez Ortín, José Enrique; Mendes Ferreira, A.

    2015-01-01

    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 (12h, 24h and ...

  2. 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...

  3. 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.

  4. Proteomic Analysis Reveals Key Proteins and Phosphoproteins upon Seed Germination of Wheat (Triticum aestivum L.)

    OpenAIRE

    Dong, Kun; Zhen, Shoumin; Cheng, Zhiwei; Cao, Hui; Ge, Pei; Yan, Yueming

    2015-01-01

    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 e...

  5. Stable Isotope Analysis Reveals That Agricultural Habitat Provides an Important Dietary Component for Nonbreeding Dunlin

    OpenAIRE

    Lesley Joan Evans Ogden; Hobson, Keith A.; Lank, David B.; Shabtai Bittman

    2005-01-01

    Although shorebirds spending the winter in temperate areas frequently use estuarine and supratidal (upland) feeding habitats, the relative contribution of each habitat to individual diets has not been directly quantified. We quantified the proportional use that Calidris alpina pacifica (Dunlin) made of estuarine vs. terrestrial farmland resources on the Fraser River Delta, British Columbia, using stable isotope analysis (δ13C, δ15N) of blood from 268 Dunlin over four winters, 1997 t...

  6. Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

    OpenAIRE

    Meierhofer, D.; Mayr, J. A.; Fink, K.; Schmeller, N.; Kofler, B; Sperl, W.

    2006-01-01

    Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHP...

  7. 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.

  8. Landslide Hazard in Aizawl, India Revealed from Field and Geodetic Observations and Hillslope Stability Analysis

    Science.gov (United States)

    Schaeffer, C.; Huang, M. H.; Smedley, A.; Sitar, N.; Dreger, D. S.

    2014-12-01

    Aizawl, the capital city of Mizoram, India, is highly susceptible to deep-seated landslides. Weak and porous shale and sandstone bedrock, high levels of precipitation during the monsoon season, steep hillslopes, and unregulated development contribute to this problem. M > 7 earthquakes along megathrusts beneath the Burma Fold Belt could trigger over 1,100 landslides and endanger over 300,000 people, increasing the severity of the landslide risk. In this study, we surveyed two slides in the south and east parts of Aizawl and from crack orientations we have inferred ground motion and created vector maps of the two slides. A shallow slope stability analysis (SHALSTAB) for the Aizawl area enabled us to map zones of relative hazard in Aizawl and the vicinity. Our SHALSTAB analysis suggests that 44% of our study area in Aizawl is unconditionally unstable. Similarly, preliminary results from a Newmark Deformation Analysis indicate that nearly 50% of our study area would result in landslide-inducing displacement under seismic loading from a magnitude 7 event. Both results indicate unconditionally unstable conditions in the south and east sides of Aizawl, which agree with our field investigation. We are analyzing Interferometry synthetic aperture radar (InSAR) data collected between 1998 and 2010 that we hope will demonstrate a seasonal time series of slope deformation for the region and determine slide velocities. With data from the Tropical Rainfall Measuring Mission (TRMM) we expect to confirm a correlation between slope movement and landslide occurrence with seasonal and annual precipitation.

  9. 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

  10. 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

  11. In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment

    Science.gov (United States)

    van Dam, Peter A.; van Dam, Pieter-Jan H. H.; Rolfo, Christian; Giallombardo, Marco; van Berckelaer, Christophe; Trinh, Xuan Bich; Altintas, Sevilay; Huizing, Manon; Papadimitriou, Kostas; Tjalma, Wiebren A. A.; van Laere, Steven

    2016-01-01

    An in silico pathway analysis was performed in order to improve current knowledge on the molecular drivers of cervical cancer and detect potential targets for treatment. Three publicly available Affymetrix gene expression data-sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total of 9 cervical cancer cell lines (CCCLs), 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples (CCSs). Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. To select cancer cell-specific gen