排序方式: 共有142条查询结果,搜索用时 15 毫秒
31.
Grønborg M Kristiansen TZ Stensballe A Andersen JS Ohara O Mann M Jensen ON Pandey A 《Molecular & cellular proteomics : MCP》2002,1(7):517-527
Although proteins phosphorylated on tyrosine residues can be enriched by immunoprecipitation with anti-phosphotyrosine antibodies, it has been difficult to identify proteins that are phosphorylated on serine/threonine residues because of lack of immunoprecipitating antibodies. In this report, we describe several antibodies that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting. Importantly, these antibodies can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well. Using these antibodies, we have immunoprecipitated proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine/threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated to be a protein kinase A substrate in vitro. We identified several in vivo phosphorylation sites by tandem mass spectrometry using Frigg protein immunoprecipitated from cells. Our method should be applicable as a generic strategy for enrichment and identification of serine/threonine-phosphorylated substrates in signal transduction pathways. 相似文献
32.
Stukenbrock EH Jørgensen FG Zala M Hansen TT McDonald BA Schierup MH 《PLoS genetics》2010,6(12):e1001189
The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000-12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes. 相似文献
33.
C. A. Stein J. Bo Hansen Johnathan Lai SiJian Wu Anatoliy Voskresenskiy Anja H?g Jesper Worm Maj Hedtj?rn Naira Souleimanian Paul Miller Harris S. Soifer Daniella Castanotto Luba Benimetskaya Henrik ?rum Troels Koch 《Nucleic acids research》2010,38(1):e3
For the past 15–20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called ‘gymnosis’) that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities. 相似文献
34.
Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality 总被引:30,自引:6,他引:24
Elmén J Thonberg H Ljungberg K Frieden M Westergaard M Xu Y Wahren B Liang Z Ørum H Koch T Wahlestedt C 《Nucleic acids research》2005,33(1):439-447
Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform. 相似文献
35.
Thomas H. R. Carlsen Troels K. H. Scheel Santseharay Ramirez Steven K. H. Foung Jens Bukh 《Journal of virology》2013,87(3):1385-1399
The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles. 相似文献
36.
Emilia Horjales-Araujo Ditte Demontis Ellen Kielland Lund Nanna Brix Finnerup Anders D. B?rglum Troels Staehelin Jensen Peter Svensson Lene Vase 《PloS one》2013,8(11)
Pain catastrophizing, a coping style characterized by excessively negative thoughts and emotions in relation to pain, is one of the psychological factors that most markedly predicts variability in the perception of pain; however, only little is known about the underlying neurobiology. The aim of this study was to test for associations between psychological variables, such as pain catastrophizing, anxiety and depression, and selected polymorphisms in genes related to monoaminergic neurotransmission, in particular serotonin pathway genes. Three hundred seventy-nine healthy participants completed a set of psychological questionnaires: the Pain Catastrophizing Scale (PCS), the State-Trait Anxiety Inventory and Beck’s Depression Inventory, and were genotyped for 15 single nucleotide polymorphisms (SNPs) in nine genes. The SNP rs1176744 located in the serotonin receptor 3B gene (5-HTR3B) was found to be associated with pain catastrophizing scores: both the global score and the subscales of magnification and helplessness. This is the first study to show an association between 5-HTR3B and PCS scores, thus suggesting a role of the serotonin pathway in pain catastrophizing. Since 5-HTR3B has previously been associated with descending pain modulation pathways, future studies will be of great interest to elucidate the molecular pathways involved in the relation between serotonin, its receptors and pain catastrophizing. 相似文献
37.
Tolkamp BJ Allcroft DJ Barrio JP Bley TA Howie JA Jacobsen TB Morgan CA Schweitzer DP Wilkinson S Yeates MP Kyriazakis I 《American journal of physiology. Regulatory, integrative and comparative physiology》2011,301(2):R378-R393
Meals have long been considered relevant units of feeding behavior. Large data sets of feeding behavior of cattle, pigs, chickens, ducks, turkeys, dolphins, and rats were analyzed with the aims of 1) describing the temporal structure of feeding behavior and 2) developing appropriate methods for estimating meal criteria. Longer (between-meal) intervals were never distributed as the negative exponential assumed by traditional methods, such as log-survivorship analysis, but as a skewed Gaussian, which can be (almost) normalized by log-transformation of interval lengths. Log-transformation can also normalize frequency distributions of within-meal intervals. Meal criteria, i.e., the longest interval considered to occur within meals, can be estimated after fitting models consisting of Gaussian functions alone or of one Weibull and one or more Gaussian functions to the distribution of log-transformed interval lengths. Nonuniform data sets may require disaggregation before this can be achieved. Observations from all species were in conflict with assumptions of random behavior that underlie traditional methods for criteria estimation. Instead, the observed structure of feeding behavior is consistent with 1) a decrease in satiety associated with an increase in the probability of animals starting a meal with time since the last meal and 2) an increase in satiation associated with an increase in the probability of animals ending a meal with the amount of food already consumed. The novel methodology proposed here will avoid biased conclusions from analyses of feeding behavior associated with previous methods and, as demonstrated, can be applied across a range of species to address questions relevant to the control of food intake. 相似文献
38.
Andreas Zankl Emma L. DuncanGraeme R. Clark Evgeny A. GlazovMarie-Claude Addor Troels HerlinChong Ae Kim Bruno P. LeheupJim McGill Steven McTaggartStephen Mittas Anna L. MitchellGeert R. Mortier Stephen P. RobertsonMarie Schroeder Paulien TerhalMatthew A. Brown 《American journal of human genetics》2014
39.
Troels Ring 《Journal of applied physiology》2006,101(2):692-3; author reply 693-4
40.
Troels T Nielsen Johan Jakobsson Nina Rosenqvist Cecilia Lundberg 《BMC biotechnology》2009,9(1):13-12