Showing papers by "University of Southern Denmark published in 2003"
TL;DR: The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
Abstract: Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.
6,597 citations
TL;DR: In this article, a four-volume set brings together seminal articles on the subject from varied sources, creating an invaluable roadmap for scholars seeking to consolidate their knowledge of CDA, and of its continued development.
Abstract: Since the late 1980s, Critical Discourse Analysis (CDA) has become a well-established field in the social sciences. However, in contrast with some branches of linguistics, CDA is not a discrete academic discipline in the traditional sense, with a fixed set of research methods. The manifold roots of CDA lie in a myriad of disciplines including rhetoric, anthropology, philosophy and cognitive science, to name a few. This four-volume set brings together seminal articles on the subject from varied sources, creating an invaluable roadmap for scholars seeking to consolidate their knowledge of CDA, and of its continued development. Sculpted and edited by a leading voice in the field, this work covers the interdisciplinary roots, the most important approaches and methodologies of CDA, as well as applications in other disciplines in an updated and comprehensive way. Structured thematically, the four volumes cover a wide range of aspects and considerations: Volume One: Histories, Concepts and Interdisciplinarity Volume Two: Theoretical Approaches and Methodologies Volume Three: 'Doing CDA' - Case Studies Volume Four: Applications and Perspectives - New Trends in CDA
4,972 citations
TL;DR: A novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips, finding that the Stage system is well-suited as a universal sample preparation system for proteomics.
Abstract: Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding 300 μL/...
2,358 citations
TL;DR: Stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications and hold tremendous potential to map modification sites in molecular detail.
Abstract: Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.
1,933 citations
TL;DR: A mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins.
Abstract: The centrosome is the major microtubule-organizing centre of animal cells and through its influence on the cytoskeleton is involved in cell shape, polarity and motility. It also has a crucial function in cell division because it determines the poles of the mitotic spindle that segregates duplicated chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity.
1,312 citations
TL;DR: Proteomics is the study of the function of all expressed proteins and further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
Abstract: Proteomics is the study of the function of all expressed proteins Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential
1,092 citations
TL;DR: The Human Protein Reference Database (HPRD) as mentioned in this paper is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease, including protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization.
Abstract: Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.
1,088 citations
TL;DR: This work uses quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components, providing the first large-scale and unbiased evidence for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.
Abstract: Membrane lipids were once thought to be homogenously distributed in the 2D surface of a membrane, but the lipid raft theory suggests that cholesterol and sphingolipids partition away from other membrane lipids. Lipid raft theory further implicates these cholesterol-rich domains in many processes such as signaling and vesicle traffic. However, direct characterization of rafts has been difficult, because they cannot be isolated in pure form. In the first functional proteomic analysis of rafts, we use quantitative high-resolution MS to specifically detect proteins depleted from rafts by cholesterol-disrupting drugs, resulting in a set of 241 authentic lipid raft components. We detect a large proportion of signaling molecules, highly enriched versus total membranes and detergent-resistant fractions, which thus far biochemically defined rafts. Our results provide the first large-scale and unbiased evidence, to our knowledge, for the connection of rafts with signaling and place limits on the fraction of plasma membrane composed by rafts.
858 citations
TL;DR: A proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations produces a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle, which offers new insights into the biogenesis and ancestry of mammalian mitochondria.
856 citations
TL;DR: This article developed a phenomenological account of desire and found that desire is regarded as a powerful cyclic emotion that is both discomforting and pleasurable, and that self-seduction, longing, desire for desire, fear of being without desire, hopefulness, and tensions between seduction and morality underlie and drive the pursuit of desire.
Abstract: Desire is the motivating force behind much of contemporary consumption. Yet consumer research has devoted little specific attention to passionate and fanciful consumer desire. This article is grounded in consumers' everyday experiences of longing for and fantasizing about particular goods. Based on journals, interviews, projective data, and inquiries into daily discourses in three cultures (the United States, Turkey, and Denmark), we develop a phenomenological account of desire. We find that desire is regarded as a powerful cyclic emotion that is both discomforting and pleasurable. Desire is an embodied passion involving a quest for otherness, sociality, danger, and inaccessibility. Underlying and driving the pursuit of desire, we find self-seduction, longing, desire for desire, fear of being without desire, hopefulness, and tensions between seduction and morality. We discuss theoretical implications of these processes for consumer research.
789 citations
TL;DR: Upon recognition of the infectious agent, MBL and the ficolins initiate the lectin pathway of complement activation through attached serine proteases (MASPs), whereas SP-A and SP-D rely on other effector mechanisms: direct opsonization, neutralization, and agglutination to limit the infection and concurrently orchestrates the subsequent adaptive immune response.
Abstract: Collectins and ficolins, present in plasma and on mucosal surfaces, are humoral molecules of the innate immune systems, which recognize pathogen-associated molecular patterns. The human collectins, mannan-binding lectin (MBL) and surfactant protein A and D (SP-A and SP-D), are oligomeric proteins composed of carbohydrate-recognition domains (CRDs) attached to collagenous regions and are thus structurally similar to the ficolins, L-ficolin, M-ficolin, and H-ficolin. However, they make use of different CRD structures: C-type lectin domains for the collectins and fibrinogen-like domains for the ficolins. Upon recognition of the infectious agent, MBL and the ficolins initiate the lectin pathway of complement activation through attached serine proteases (MASPs), whereas SP-A and SP-D rely on other effector mechanisms: direct opsonization, neutralization, and agglutination. This limits the infection and concurrently orchestrates the subsequent adaptive immune response. Deficiencies of the proteins may predispose to infections or other complications, e.g., reperfusion injuries or autoimmune diseases. Structure, function, clinical implications, and phylogeny are reviewed.
TL;DR: Stable isotopic amino acids in cell culture is employed to differentially label proteins in EGF-stimulated versus unstimulated cells and SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
Abstract: Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
TL;DR: Several reports have revealed LNA as a most promising molecule for the development of oligonucleotide-based therapeutics, including high capturing efficiencies and unambiguous scoring of single-nucleotide polymorphisms.
TL;DR: The results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men.
Abstract: A major component of variation in body height is due to genetic differences, but environmental factors have a substantial contributory effect. In this study we aimed to analyse whether the genetic architecture of body height varies between affluent western societies. We analysed twin data from eight countries comprising 30,111 complete twin pairs by using the univariate genetic model of the Mx statistical package. Body height and zygosity were self-reported in seven populations and measured directly in one population. We found that there was substantial variation in mean body height between countries; body height was least in Italy (177 cm in men and 163 cm in women) and greatest in the Netherlands (184 cm and 171 cm, respectively). In men there was no corresponding variation in heritability of body height, heritability estimates ranging from 0.87 to 0.93 in populations under an additive genes/unique environment (AE) model. Among women the heritability estimates were generally lower than among men with greater variation between countries, ranging from 0.68 to 0.84 when an additive genes/shared environment/unique environment (ACE) model was used. In four populations where an AE model fit equally well or better, heritability ranged from 0.89 to 0.93. This difference between the sexes was mainly due to the effect of the shared environmental component of variance, which appears to be more important among women than among men in our study populations. Our results indicate that, in general, there are only minor differences in the genetic architecture of height between affluent Caucasian populations, especially among men.
TL;DR: The anammox reaction is shown to be a globally significant sink for oceanic nitrogen in the anoxic waters of Golfo Dulce, a 200-m-deep coastal bay in Costa Rica, where it accounts for 19–35% of the total N2 formation in the water column.
Abstract: In oxygen-depleted zones of the open ocean, and in anoxic basins and fjords, denitrification (the bacterial reduction of nitrate to give N2) is recognized as the only significant process converting fixed nitrogen to gaseous N2. Primary production in the oceans is often limited by the availability of fixed nitrogen such as ammonium or nitrate1, and nitrogen-removal processes consequently affect both ecosystem function and global biogeochemical cycles. It was recently discovered that the anaerobic oxidation of ammonium with nitrite—the ‘anammox’ reaction, performed by bacteria—was responsible for a significant fraction of N2 production in some marine sediments2. Here we show that this reaction is also important in the anoxic waters of Golfo Dulce, a 200-m-deep coastal bay in Costa Rica, where it accounts for 19–35% of the total N2 formation in the water column. The water-column chemistry in Golfo Dulce is very similar to that in oxygen-depleted zones of the oceans—in which one-half to one-third of the global nitrogen removal is believed to occur3,4. We therefore expect the anammox reaction to be a globally significant sink for oceanic nitrogen.
TL;DR: A scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides is presented.
TL;DR: It is suggested that inhibition of protein synthesis by RelE is reversed with the help of tmRNA, and that RelE plays a regulatory role in bacteria during adaptation to poor growth conditions.
TL;DR: BlueJ, an integrated development environment designed for teaching object-orientation, is introduced and it is discussed how the use of this tool can change the approach to teaching.
Abstract: Many teachers experience serious problems when teaching object orientation to beginners or professionals. Many of these problems could be overcome or reduced through the use of more appropriate tools. In this paper, we introduce BlueJ, an integrated development environment designed for teaching object orientation, and discuss how the use of this tool can change the approach to teaching.
TL;DR: The benefits and pitfalls of some of the most commonly used protocols, focusing on stable isotope labeling with amino acids in cell culture (SILAC), are reviewed and it is speculated that SILAC will soon be a key tool of quantitative proteomics.
TL;DR: The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS-PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions.
Abstract: We have recently described a method, stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of relative protein abundances. Cells were metabolically labeled with deuterated leucine, leading to complete incorporation within about five cell doublings. Here, we investigate fully substituted 13C-labeled arginine in the SILAC method. After tryptic digestion, there is a single label at the C-terminal position in half of the peptides. Labeled and unlabeled peptides coelute in liquid chromatography−mass spectrometric analysis, eliminating quantitation error due to unequal sampling of ion profiles. Tandem mass spectrum interpretation and database identification are aided by the predictable shift of the y-ions in the labeled form. The quantitation of mixtures of total cell lysates in known ratios resolved on a one-dimensional SDS−PAGE gel produced consistent and reproducible results with relative standard deviations better than five percent under optimal conditions. Keywords: q...
TL;DR: Heart rate increases rapidly, before any significant elevations in metHb or extracellular potassium occur, suggesting nitrite-induced vasodilation (possibly via nitric oxide generated from nitrite) that is countered by increased cardiac pumping to re-establish blood pressure.
Abstract: Nitrite is a potential problem in aquatic environments. Freshwater fish actively take up nitrite across the gills, leading to high internal concentrations. Seawater fish are less susceptible but do take up nitrite across intestine and gills. Nitrite has multiple physiological effects. Its uptake is at the expense of chloride, leading to chloride depletion. Nitrite also activates efflux of potassium from skeletal muscle and erythrocytes, disturbing intracellular and extracellular K(+) levels. Nitrite transfer across the erythrocytic membrane leads to oxidation of haemoglobin to methaemoglobin (metHb), compromising blood O(2) transport. Other haem proteins are also oxidised. Hyperventilation is observed, and eventually tissue O(2) shortage becomes reflected in elevated lactate concentrations. Heart rate increases rapidly, before any significant elevations in metHb or extracellular potassium occur. This suggests nitrite-induced vasodilation (possibly via nitric oxide generated from nitrite) that is countered by increased cardiac pumping to re-establish blood pressure. Nitrite can form and/or mimic nitric oxide and thereby interfere with processes regulated by this local hormone. Steroid hormone synthesis may be inhibited, while changes in ammonia and urea levels and excretion rates reflect an influence of nitrite on nitrogen metabolism. Detoxification of nitrite occurs via endogenous oxidation to nitrate, and elimination of nitrite takes place both via gills and urine. The susceptibility to nitrite varies between species and in some cases also within species. Rainbow trout fall into two groups with regard to susceptibility and physiological response. These two groups are not related to sex but show significant different nitrite uptake rates.
TL;DR: Several statistical models for quantitative genetic analyses are described, such as models for the classical twin design, multivariate and longitudinal genetic analyses, extended twin analyses, and linkage and association analyses.
Abstract: With the rapid advances in molecular biology, the near completion of the human genome, the development of appropriate statistical genetic methods and the availability of the necessary computing power, the identification of quantitative trait loci has now become a realistic prospect for quantitative geneticists. We briefly describe the theoretical biometrical foundations underlying quantitative genetics. These theoretical underpinnings are translated into mathematical equations that allow the assessment of the contribution of observed (using DNA samples) and unobserved (using known genetic relationships) genetic variation to population variance in quantitative traits. Several statistical models for quantitative genetic analyses are described, such as models for the classical twin design, multivariate and longitudinal genetic analyses, extended twin analyses, and linkage and association analyses. For each, we show how the theoretical biometrical model can be translated into algebraic equations that may be used to generate scripts for statistical genetic software packages, such as Mx, Lisrel, SOLAR, or MERLIN. For using the former program a web-library (available from http://www.psy.vu.nl/mxbib) has been developed of freely available scripts that can be used to conduct all genetic analyses described in this paper.
TL;DR: An unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity is revealed and it is established that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC 1/ XPF.
01 Jan 2003
TL;DR: In this paper, a simulation model of international tourism is presented that fills the gap of analysis of the global changes in tourism demand, and the current pattern of international tourist flows is modelled using 1995 data on departures and arrivals for 207 countries.
Abstract: The literature on tourism and climate change lacks an analysis of the global changes in tourism demand. Here, a simulation model of international tourism is presented that fills that gap. The current pattern of international tourist flows is modelled using 1995 data on departures and arrivals for 207 countries. Using this basic model the impact on arrivals and departures through changes in population, per capita income and climate change are analysed. In the medium to long term, tourism will grow, however, the change from climate change is smaller than from population and income changes.
TL;DR: A mechanistic explanation for the detrimental effect on cell growth exerted by ChpAK and the homologous ChpBK protein of E.coli RelE is yield and a model that integrates TA loci into general prokaryotic stress physiology is proposed.
TL;DR: Evaluating the reliability and validity of the CSA in a wide walking-running speed range in laboratory and field found between-subject reliability was related to step frequency, presumably due to relatively constant vertical acceleration in running.
Abstract: BRAGE, S., N. WEDDERKOPP, P. W. FRANKS, L. B. ANDERSEN, and K. FROBERG. Reexamination of Validity and Reliability of the CSA Monitor in Walking and Running. Med. Sci. Sports Exerc., Vol. 35, No. 8, pp. 1447–1454, 2003.PurposeTo evaluate the reliability and validity of the CSA (model 7164) ac
TL;DR: Results from analyses of opposite sex pairs showed evidence of sex-specific genetic effects suggesting there may be some differences between men and women in the genetic factors that influence variation in BMI, and encourage the continued search for genes of importance to the body composition and the development of obesity.
Abstract: Body mass index (BMI), a simple anthropometric measure, is the most frequently used measure of adiposity and has been instrumental in documenting the worldwide increase in the prevalence of obesity witnessed during the last decades. Although this increase in overweight and obesity is thought to be mainly due to environmental changes, i.e., sedentary lifestyles and high caloric diets, consistent evidence from twin studies demonstrates high heritability and the importance of genetic differences for normal variation in BMI. We analysed self-reported data on BMI from approximately 37,000 complete twin pairs (including opposite sex pairs) aged 20-29 and 30-39 from eight different twin registries participating in the GenomEUtwin project. Quantitative genetic analyses were conducted and sex differences were explored. Variation in BMI was greater for women than for men, and in both sexes was primarily explained by additive genetic variance in all countries. Sex differences in the variance components were consistently significant. Results from analyses of opposite sex pairs also showed evidence of sex-specific genetic effects suggesting there may be some differences between men and women in the genetic factors that influence variation in BMI. These results encourage the continued search for genes of importance to the body composition and the development of obesity. Furthermore, they suggest that strategies to identify predisposing genes may benefit from taking into account potential sex specific effects.
TL;DR: It is shown here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions, suggesting that the function and target ofrelE may be conserved across the prokaryotic domains.
Abstract: RelE of Escherichia coli is a global inhibitor of translation that is activated by nutritional stress. Activation of RelE depends on Lon-mediated degradation of RelB, the antagonist that neutralizes RelE. In vitro, RelE cleaves synthetic mRNAs positioned at the ribosomal A-site. We show here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions. RelE-mediated cleavage depended on translation of the RNAs and occurred at both sense and stop codons. RelE cleavage of mRNA and tmRNA was also induced by amino acid starvation. An ssrA deletion strain was hypersensitive to RelE, whereas overproduction of tmRNA counteracted RelE toxicity. After neutralization of RelE by RelB, rapid recovery of translation required tmRNA, indicating that tmRNA alleviated RelE toxicity by rescuing ribosomes stalled on damaged mRNAs. RelE proteins from Gram-positive Bacteria and Archaea cleaved tmRNA with a pattern similar to that of E. coli RelE, suggesting that the function and target of RelE may be conserved across the prokaryotic domains.
TL;DR: In this article, the authors argue that there are only two necessary conditions, namely uncertainty and immobility, for the expression of SCA, and that all other conditions are additional.
Abstract: To advance the RBV we propose separation of necessary and additional conditions for the expression of SCA. We argue that there are only two necessary conditions, namely uncertainty and immobility and that all other conditions are additional. Copyright © 2003 John Wiley & Sons, Ltd.
TL;DR: In this article, a single molecular monolayer of bistable rotaxanes sandwiched between two 40-nm metal electrodes was fabricated using imprint lithography, and it was observed that it has high on-off ratios and reversible switching properties.
Abstract: Nanoscale molecular-electronic devices comprising a single molecular monolayer of bistable [2]rotaxanes sandwiched between two 40-nm metal electrodes were fabricated using imprint lithography. Bistable current–voltage characteristics with high on–off ratios and reversible switching properties were observed. Such devices may function as basic elements for future ultradense electronic circuitry.