Showing papers by "University of Southern Denmark published in 2004"

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

Abstract: We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

The ABC's (and XYZ's) of peptide sequencing

Abstract: Proteomics is an increasingly powerful and indispensable technology in molecular cell biology. It can be used to identify the components of small protein complexes and large organelles, to determine post-translational modifications and in sophisticated functional screens. The key — but little understood — technology in mass-spectrometry-based proteomics is peptide sequencing, which we describe and review here in an easily accessible format.

IntAct: an open source molecular interaction database

Abstract: IntAct provides an open source database and toolkit for the storage, presentation and analysis of protein interactions. The web interface provides both textual and graphical representations of protein interactions, and allows exploring interaction networks in the context of the GO annotations of the interacting proteins. A web service allows direct computational access to retrieve interaction networks in XML format. IntAct currently contains approximately 2200 binary and complex interactions imported from the literature and curated in collaboration with the Swiss-Prot team, making intensive use of controlled vocabularies to ensure data consistency. All IntAct software, data and controlled vocabularies are available at http://www.ebi.ac.uk/intact.

Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.

Abstract: To study the global dynamics of phosphotyrosine-based signaling events in early growth factor stimulation, we developed a mass spectrometric method that converts temporal changes to differences in peptide isotopic abundance. The proteomes of three cell populations were metabolically encoded with different stable isotopic forms of arginine. Each population was stimulated by epidermal growth factor for a different length of time, and tyrosine-phosphorylated proteins and closely associated binders were affinity purified. Arginine-containing peptides occurred in three forms, which were quantified; we then combined two experiments to generate five-point dynamic profiles. We identified 81 signaling proteins, including virtually all known epidermal growth factor receptor substrates, 31 novel effectors and the time course of their activation upon epidermal growth factor stimulation. Global activation profiles provide an informative perspective on cell signaling and will be crucial to modeling signaling networks in a systems biology approach.

LNA (Locked Nucleic Acid): High-Affinity Targeting of Complementary RNA and DNA†

Abstract: Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.

Extending collostructional analysis: A corpus-based perspective on `alternations'

Abstract: This paper introduces an extension of distinctive-collocate analysis that takes into account grammatical structure and is specifically geared to investigating pairs of semantically similar grammatical constructions and the lexemes that occur in them. The method, referred to as `distinctive-collexeme analysis', identifies lexemes that exhibit a strong preference for one member of the pair as opposed to the other, and thus makes it possible to identify subtle distributional differences between the members of such a pair. The method can be applied in the context of what is sometimes referred to as `grammatical alternation' (e.g. the dative alternation), but it can also be applied to other choices provided by the grammar (such as the two future tense constructions in English). The method has two main applications. First, it can reveal subtle differences between seemingly synonymous constructions, many of which are difficult to identify on the basis of more traditional approaches. Second, it can be used to investigate the very notion of `alternation'; we show that many alternations are much more restricted than has hitherto been assumed, and thus confirm the claims of recent, non-derivational views of grammar.

Human protein reference database as a discovery resource for proteomics

Abstract: The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein-protein interactions, post-translational modifications, enzyme-substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease.

Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate.

Abstract: Background Cleft lip or palate (or the two in combination) is a common birth defect that results from a mixture of genetic and environmental factors. We searched for a specific genetic factor contributing to this complex trait by examining large numbers of affected patients and families and evaluating a specific candidate gene. Methods We identified the gene that encodes interferon regulatory factor 6 (IRF6) as a candidate gene on the basis of its involvement in an autosomal dominant form of cleft lip and palate, Van der Woude's syndrome. A single-nucleotide polymorphism in this gene results in either a valine or an isoleucine at amino acid position 274 (V274I). We carried out transmission-disequilibrium testing for V274I in 8003 individual subjects in 1968 families derived from 10 populations with ancestry in Asia, Europe, and South America, haplotype and linkage analyses, and case–control analyses, and determined the risk of cleft lip or palate that is associated with genetic variation in IRF6. Results ...

Features of the Metabolic Syndrome Are Associated With Objectively Measured Physical Activity and Fitness in Danish Children: The European Youth Heart Study (EYHS)

Abstract: OBJECTIVE —Features of the metabolic syndrome are becoming increasingly evident in children. Decreased physical activity is likely to be an important etiological factor, as shown previously for subjective measures of physical activity in selected groups. The purpose of this study was to examine the relationship between the metabolic syndrome and objectively measured physical activity and whether fitness modified this relationship. RESEARCH DESIGN AND METHODS —A total of 589 Danish children (310 girls, 279 boys, mean [±SD] age 9.6 ± 0.44 years, mean weight 33.6 ± 6.4 kg, mean height 1.39 ± 0.06 m) were randomly selected. Physical activity was measured with the uni-axial Computer Science & Applications accelerometer (MTI actigraph) worn at the hip for at least 3 days (≥10 h/day) and fitness with a maximal bike test. As outcomes, we measured sitting systolic and diastolic blood pressure, degree of adiposity (sum of four skinfolds), and, finally, insulin, glucose, triglicerides, and HDL cholesterol in fasting blood samples. The outcome variables were statistically normalized and expressed as the number of SDs from the mean. (i.e., Z scores). A metabolic syndrome risk score was computed as the mean of these Z scores. Multiple linear regression was used to test the association between physical activity and metabolic risk, adjusted primarily for age, sex, sexual maturation, ethnicity, parental smoking, socioeconomic factors, and the Computer Science & Applications unit, as well as for fitness. Robust SEs were computed by clustering on school. RESULTS —All children were in the nondiabetic range of fasting glucose. Metabolic risk was inversely related to physical activity ( P = 0.008). The relationship was weakened after adjustment for fitness, but there was a significantly positive interaction between physical activity and fitness. CONCLUSIONS —Physical activity is inversely associated with metabolic risk, independently of potential confounders. The interaction between physical activity and fitness suggests that the potential beneficial effect of activity may be greatest in children with lower cardiorespiratory fitness.

The bacterial Sm-like protein Hfq: a key player in RNA transactions.

Abstract: The conserved RNA-binding protein Hfq, originally discovered in Escherichia coli as a host factor for Qbeta replicase, has emerged as a pleiotropic regulator that modulates the stability or the translation of an increasing number of mRNAs. During the past 5 years, Hfq-mediated control has been an area of increasing focus because the protein has been linked to the action of many versatile RNA-based regulators that use basepairing interactions to regulate the expression of target mRNAs. The recent findings that Hfq assists in bimolecular RNA-RNA interactions and is similar structurally and functionally to eukaryotic Sm proteins have further fueled interest in this important post-transcriptional regulator. Here, we summarize the history of Hfq and highlight results that have led to an important gain in insight into the physiology, biochemistry and evolution of Hfq and its homologues.

Phosphoproteomics of the Arabidopsis Plasma Membrane and a New Phosphorylation Site Database

Abstract: Functional genomic technologies are generating vast amounts of data describing the presence of transcripts or proteins in plant cells. Together with classical genetics, these approaches broaden our understanding of the gene products required for specific responses. Looking to the future, the focus of research must shift to the dynamic aspects of biology: molecular mechanisms of function and regulation. Phosphorylation is a key regulatory factor in all aspects of plant biology; but it is difficult, if not impossible, for most researchers to identify in vivo phosphorylation sites within their proteins of interest. We have developed a large-scale strategy for the isolation of phosphopeptides and identification by mass spectrometry (Nuhse et al., 2003b). Here, we describe the identification of more than 300 phosphorylation sites from Arabidopsis thaliana plasma membrane proteins. These data will be a valuable resource for many fields of plant biology and overcome a major impediment to the elucidation of signal transduction pathways. We present an analysis of the characteristics of phosphorylation sites, their conservation among orthologs and paralogs, and the existence of putative motifs surrounding the sites. These analyses yield general principles for predicting other phosphorylation sites in plants and provide indications of specificity determinants for responsible kinases. In addition, more than 50 sites were mapped on receptor-like kinases and revealed an unexpected complexity of regulation. Finally, the data also provide empirical evidence on the topology of transmembrane proteins. This information indicates that prediction programs incorrectly identified the cytosolic portion of the protein in 25% of the transmembrane proteins found in this study. All data are deposited in a new searchable database for plant phosphorylation sites maintained by PlantsP (http://plantsp.sdsc.edu) that will be updated as the project expands to encompass additional tissues and organelles.

What is the lowest possible reheating temperature

Abstract: We study models in which the universe exits reheating at temperatures in the MeV regime. By combining light element abundance measurements with cosmic microwave background and large scale structure data we find a fairly robust lower limit on the reheating temperature of ${T}_{\mathrm{RH}}\ensuremath{\gtrsim}4\mathrm{MeV}$ at 95% C.L. However, if the heavy particle whose decay reheats the universe has a direct decay mode to neutrinos, there are some small islands left in parameter space where a reheating temperature as low as 1 MeV is allowed. The derived lower bound on the reheating temperature also leads to very stringent bounds on models with n large extra dimensions. For $n=2$ the bound on the compactification scale is $M\ensuremath{\gtrsim}2000\mathrm{TeV},$ and for $n=3$ it is 100 TeV. These are currently the strongest available bounds on such models.

Branched equation modeling of simultaneous accelerometry and heart rate monitoring improves estimate of directly measured physical activity energy expenditure

Abstract: The combination of heart rate (HR) monitoring and movement registration may improve measurement precision of physical activity energy expenditure (PAEE). Previous attempts have used either regressi...

What's so special about cholesterol?

Abstract: Cholesterol (or other higher sterols such as ergosterol and phytosterols) is universally present in large amounts (20-40 mol%) in eukaryotic plasma membranes, whereas it is universally absent in the membranes of prokaryotes. Cholesterol has a unique ability to increase lipid order in fluid membranes while maintaining fluidity and diffusion rates. Cholesterol imparts low permeability barriers to lipid membranes and provides for large mechanical coherence. A short topical review is given of these special properties of cholesterol in relation to the structure of membranes, with results drawn from a variety of theoretical and experimental studies. Particular focus is put on cholesterol's ability to promote a special membrane phase, the liquid-ordered phase, which is unique for cholesterol (and other higher sterols like ergosterol) and absent in membranes containing the cholesterol precursor lanosterol. Cholesterol's role in the formation of special membrane domains and so-called rafts is discussed.

Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.

Abstract: Protein methylation is a stable post-translational modification (PTM) with important biological functions. It occurs predominantly on arginine and lysine residues with varying numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing methods for identifying methylation sites are laborious, require large amounts of sample and cannot be applied to complex mixtures. We have previously described stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC, cells metabolically convert [(13)CD(3)]methionine to the sole biological methyl donor, [(13)CD(3)]S-adenosyl methionine. Heavy methyl groups are fully incorporated into in vivo methylation sites, directly labeling the PTM. This provides markedly increased confidence in identification and relative quantitation of protein methylation by mass spectrometry. Using antibodies targeted to methylated residues and analysis by liquid chromatography-tandem mass spectrometry, we identified 59 methylation sites, including previously unknown sites, considerably extending the number of in vivo methylation sites described in the literature.

A New Strategy for Identification of N-Glycosylated Proteins and Unambiguous Assignment of Their Glycosylation Sites Using HILIC Enrichment and Partial Deglycosylation

Abstract: Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-β-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by le...

Outcomes in Type 1 Diabetic Pregnancies: A nationwide, population-based study

Abstract: OBJECTIVE —The aim of this study was to compare pregnancy outcomes in type 1 diabetic pregnancies with the background population. RESEARCH DESIGN AND METHODS —This nationwide prospective multicenter study took place in eight Danish centers treating pregnant women with type 1 diabetes during 1993–1999. A total of 990 women with 1,218 pregnancies and delivery after 24 weeks ( n = 1,215) or early termination due to severe congenital malformations ( n = 3) were included. Data were collected prospectively by one to three caregivers in each center and reported to a central registry. RESULTS —The perinatal mortality rate was 3.1% in type 1 diabetic pregnancies compared with 0.75% in the background population (RR 4.1 [95% CI 2.9–5.6]), and the stillbirth rate was 2.1% compared with 0.45 (4.7 [3.2–7.0]). The congenital malformation rate was 5.0% in the study population and 2.8% (1.7 [1.3–2.2]) in the background population. Six of the perinatal deaths (16%) were related to congenital malformations. Only 34% of women performed daily home monitoring of blood glucose at conception, and 58% received preconceptional guidance. Pregnancies with serious adverse outcomes (perinatal death and/or congenital malformations) were characterized by higher HbA1c values before and during pregnancy and a lesser degree of maternal self-care and preconceptional guidance. Women who performed daily self-monitoring of blood glucose at any time during pregnancy had lower HbA1c values than women who did not measure their daily profile. Likewise, daily self-monitoring was associated with a reduction in serious adverse outcomes. The caesarean section rate was 55.9 and 12.6%, respectively, and the risk of preterm delivery was 41.7 and 6.0%, respectively. CONCLUSIONS —Type 1 diabetic pregnancies are still complicated by considerably higher rates of severe perinatal complications compared with the background population, and women with poor self-care are at the highest risk. Adequate glycemic control using daily glucose monitoring before and during pregnancy is a crucial step toward reaching the goals of the St. Vincent declaration.

Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.

Abstract: Protein methylation is a stable post-translational modification (PTM) with important biological functions. It occurs predominantly on arginine and lysine residues with varying numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing methods for identifying methylation sites are laborious, require large amounts of sample and cannot be applied to complex mixtures. We have previously described stable isotope labeling by amino acids in cell culture (SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC, cells metabolically convert [(13)CD(3)]methionine to the sole biological methyl donor, [(13)CD(3)]S-adenosyl methionine. Heavy methyl groups are fully incorporated into in vivo methylation sites, directly labeling the PTM. This provides markedly increased confidence in identification and relative quantitation of protein methylation by mass spectrometry. Using antibodies targeted to methylated residues and analysis by liquid chromatography-tandem mass spectrometry, we identified 59 methylation sites, including previously unknown sites, considerably extending the number of in vivo methylation sites described in the literature.

The transition to a sulphidic ocean ∼ 1.84 billion years ago

Abstract: The Proterozoic aeon (2.5 to 0.54 billion years (Gyr) ago) marks the time between the largely anoxic world of the Archean (> 2.5 Gyr ago)1 and the dominantly oxic world of the Phanerozoic (< 0.54 Gyr ago). The course of ocean chemistry through the Proterozoic has traditionally been explained by progressive oxygenation of the deep ocean in response to an increase in atmospheric oxygen around 2.3 Gyr ago. This postulated rise in the oxygen content of the ocean is in turn thought to have led to the oxidation of dissolved iron, Fe(II), thus ending the deposition of banded iron formations (BIF) around 1.8 Gyr ago1,2. An alternative interpretation suggests that the increasing atmospheric oxygen levels enhanced sulphide weathering on land and the flux of sulphate to the oceans. This increased rates of sulphate reduction, resulting in Fe(II) removal in the form of pyrite as the oceans became sulphidic3. Here we investigate sediments from the ∼1.8-Gyr-old Animikie group, Canada, which were deposited during the final stages of the main global period of BIF deposition. This allows us to evaluate the two competing hypotheses for the termination of BIF deposition. We use iron–sulphur–carbon (Fe–S–C) systematics to demonstrate continued ocean anoxia after the final global deposition of BIF and show that a transition to sulphidic bottom waters was ultimately responsible for the termination of BIF deposition. Sulphidic conditions may have persisted until a second major rise in oxygen between 0.8 to 0.58 Gyr ago4,5, possibly reducing global rates of primary production and arresting the pace of algal evolution6.

An inquiry into born‐global firms in Europe and the USA

Abstract: Companies that internationalise at or near their founding, “born globals,” are emerging in great numbers world‐wide Characterised by a specific Gestalt of marketing‐related competencies, they are playing an increasing role in international trade Born globals are investigated using data from case and survey‐based studies in Denmark and the USA First introduces and describes the born‐global phenomenon Then, hypotheses are developed and tested via a structural model that represents key factors in the international success of this important breed of firm Results suggest that born‐global international performance is enhanced in the wake of managerial emphasis on foreign customer focus and marketing competence Product quality and differentiation strategy also play important roles, particularly in the US firms These and additional findings are discussed in light of their theoretical and practical implications

The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane‐bound complex

Abstract: Summary MreB proteins of Escherichia coli , Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus , the mreB gene is essential. However, in E. coli , mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD -encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Molecule-Independent Electrical Switching in Pt/Organic Monolayer/Ti Devices

Abstract: Electronic devices comprising a Langmuir−Blodgett molecular monolayer sandwiched between planar platinum and titanium metal electrodes functioned as switches and tunable resistors over a 102−105 Ω range under current or voltage control. Reversible hysteretic switching and resistance tuning was qualitatively similar for three very different molecular species, indicating a generic switching mechanism dominated by electrode properties or electrode/molecule interfaces, rather than molecule-specific behavior.

Proteomic Analysis of the Arabidopsis Nucleolus Suggests Novel Nucleolar Functions

Abstract: The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.

The firm as an interactor: firms as vehicles for habits and routines

Abstract: This paper pursues a research agenda inspired by Richard Nelson and Sidney Winter's Evolutionary Theory of Economic Change (1982). This seminal work applied the Darwinian concepts of variation, replication and selection to the evolution of firms. It proposed a level of evolution, replication and selection at a level higher than individuals or genes, involving the replication and selection of routines and institutions. Significantly, the applicability or otherwise of these Darwinian concepts depends on precise definitions of terms such as replication and selection. The present essay builds on previous work where the concepts of replication (Godfrey-Smith, 2000; Aunger, 2002; Hodgson, 2003b) and selection (Price, 1995; Frank, 1998; Knudsen, 2002b, 2003) have been refined. We deploy the key concepts of 'replicator' and 'interactor' from the modern philosophy of biology (Hull, 1981, 1988). It is shown that while habits and routines can be regarded as replicators, there is a case for regarding firms and similarly cohesive organizations as interactors. We explore some of the implications of this result and provide an important component in the construction of a multiple-level evolutionary theory, involving replicating units at several socio-economic levels.