Showing papers by "University of Southern Denmark published in 2002"
TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
5,653 citations
TL;DR: This extensive proteomic analysis of human nucleoli shows that nucleoli have a surprisingly large protein complexity and supports the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis.
989 citations
TL;DR: It is found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N2 production in marine sediments.
Abstract: In the global nitrogen cycle, bacterial denitrification is recognized as the only quantitatively important process that converts fixed nitrogen to atmospheric nitrogen gas, N 2 , thereby influencing many aspects of ecosystem function and global biogeochemistry. However, we have found that a process novel to the marine nitrogen cycle, anaerobic oxidation of ammonium coupled to nitrate reduction, contributes substantially to N 2 production in marine sediments. Incubations with 15 N-labeled nitrate or ammonium demonstrated that during this process, N 2 is formed through one-to-one pairing of nitrogen from nitrate and ammonium, which clearly separates the process from denitrification. Nitrite, which accumulated transiently, was likely the oxidant for ammonium, and the process is thus similar to the anammox process known from wastewater bioreactors. Anaerobic ammonium oxidation accounted for 24 and 67% of the total N 2 production at two typical continental shelf sites, whereas it was detectable but insignificant relative to denitrification in a eutrophic coastal bay. However, rates of anaerobic ammonium oxidation were higher in the coastal sediment than at the deepest site and the variability in the relative contribution to N 2 production between sites was related to large differences in rates of denitrification. Thus, the relative importance of anaerobic ammonium oxidation and denitrification in N 2 production appears to be regulated by the availability of their reduced substrates. By shunting nitrogen directly from ammonium to N 2 , anaerobic ammonium oxidation promotes the removal of fixed nitrogen in the oceans. The process can explain ammonium deficiencies in anoxic waters and sediments, and it may contribute significantly to oceanic nitrogen budgets.
982 citations
TL;DR: This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.
Abstract: In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.
922 citations
TL;DR: Several methods for enrichment of phosphorylated proteins and peptides are outlined and various options for their identification and quantitation are discussed with special emphasis on mass spectrometry-based techniques.
904 citations
TL;DR: It is proposed that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases activeWAVE1–HSPC300 and leads to actin nucleation.
Abstract: Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. Here we report a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 (p53-inducible messenger RNA with a relative molecular mass (M(r)) of 140,000), Nap125 (NCK-associated protein with an M(r) of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. We therefore propose that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation.
844 citations
TL;DR: Sulfur isotope fractionation experiments on marine and freshwater sulfate reducers, together with the isotope record, imply that oceanic Archean sulfate concentrations were less than one-hundredth of present marine sulfate levels and one-fifth of what was previously thought.
Abstract: The size of the marine sulfate reservoir has grown through Earth9s history, reflecting the accumulation of oxygen into the atmosphere. Sulfur isotope fractionation experiments on marine and freshwater sulfate reducers, together with the isotope record, imply that oceanic Archean sulfate concentrations were
708 citations
TL;DR: A large-scale, high-accuracy mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum revealed 1,289 proteins that contain domains that indicate a role in cell–cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation.
Abstract: The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.
667 citations
TL;DR: In this article, the authors focus on the concept of gradual development and find that export intensity, distribution, market selection, and global orientation are not influenced by the firm's year of establishment or first year of exporting activity.
Abstract: Over the past decade, several studies have questioned the stage models of the internationalization process. Many of these studies concentrate on the exporting versus nonexporting factor, identifying an increasing number of firms that are active in international markets shortly after establishment. Limited empirical evidence exists as to whether this actuality indicates simply a reduced time factor in the preexport phase or an important change in the export behavior of firms. Using small and medium-sized exporting firms from Norway, Denmark, and France, the authors focus on the concept of gradual development. The results suggest that export intensity, distribution, market selection, and global orientation are not influenced by the firm’s year of establishment or first year of exporting activity. One-third of the firms sampled reported that the time period between establishment and export commencement was less than two years. In terms of export intensity, these firms outperform those that waited se...
663 citations
TL;DR: The potencies of the pesticides to react as hormone agonists or antagonists are low compared to the natural ligands, but the integrated response in the organism might be amplified by the ability of the pesticide to act via several mechanism and the frequent simultaneous exposure to several pesticides.
584 citations
TL;DR: The bacterial Hfq protein this article belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins.
TL;DR: It is shown that PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles, that also contains at least two other protein components: PSP2 and p54/nrb.
TL;DR: A phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms finds a new par gene family from enteric bacteria that is consistent with lateral gene transfer between Bacteria and Archaea.
Abstract: Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid-encoded par loci specify three components: a cis-acting centromere-like site and two trans-acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par-encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host-encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter-cell positions). Two types of partitioning ATPases are known: the Walker-type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin-like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid-encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome-encoded ATPases, including members from both Gram-negative and Gram-positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.
TL;DR: The Hfq–RNA structure reveals that the single‐stranded hepta‐oligoribonucleotide binds in a circular conformation around a central basic cleft, whereby Tyr42 residues from adjacent subunits stack with six of the bases, and Gln8, outside the Sm motif, provides key protein–base contacts, which suggests a mechanism for H fq function.
Abstract: In prokaryotes, Hfq regulates translation by modulating the structure of numerous RNA molecules by binding preferentially to A/U-rich sequences To elucidate the mechanisms of target recognition and translation regulation by Hfq, we determined the crystal structures of the Staphylococcus aureus Hfq and an Hfq–RNA complex to 155 and 271 Å resolution, respectively The structures reveal that Hfq possesses the Sm-fold previously observed only in eukaryotes and archaea However, unlike these heptameric Sm proteins, Hfq forms a homo-hexameric ring The Hfq–RNA structure reveals that the single-stranded hepta-oligoribonucleotide binds in a circular conformation around a central basic cleft, whereby Tyr42 residues from adjacent subunits stack with six of the bases, and Gln8, outside the Sm motif, provides key protein–base contacts Such binding suggests a mechanism for Hfq function
TL;DR: CMPA is the most common food allergy in early childhood with an incidence of 2 to 3% in the first year of life and the overall prognosis of CMPA in infancy is good with a remission rate of approximately 85 to 90%.
Abstract: Objective The primary objective of this review is to discuss the clinical features, diagnosis, natural history, and prognosis of cow's milk allergy in early childhood and its relationship to development of inhalant allergies. Data Sources A review of 229 PubMed (National Library of Medicine) articles on cow's milk allergy (CMPA) for the years 1967 through 2001 was performed. In addition, references from other review articles have been included. This review represents a synthesis of these sources and the expert opinion of the author. Study Selection The expert opinion of the author was used to select the relevant data for this review. Results The diagnosis of reproducible adverse reactions to cow's milk protein (CMP), ie, CMPA, has to be confirmed by controlled elimination and challenge procedures. The incidence of CMPA in infancy seems to be approximately 2 to 3% in developed countries. Symptoms suggestive of CMPA may be encountered in approximately 5 to 15% of infants emphasizing the importance of controlled elimination/milk challenge procedures. Reproducible clinical reactions to CMP in human milk have been reported in approximately 0.5% of breastfed infants. Most infants with CMPA develop symptoms before 1 month of age, often within 1 week after introduction of CMP-based formula. The majority has two or more symptoms from two or more organ systems. Approximately 50 to 60% have cutaneous symptoms, 50 to 60% have gastrointestinal symptoms, and approximately 20 to 30% respiratory symptoms. Symptoms may occur within 1 hour after milk intake (immediate reactions) or after 1 hour (late reactions). The prognosis of CMPA is good with a remission rate of approximately 45 to 50% at 1 year, 60 to 75% at 2 years, and 85 to 90% at 3 years. Associated adverse reactions to other foods develop in up to 50% and allergy against inhalants in 50 to 80% before puberty. Conclusions CMPA is the most common food allergy in early childhood with an incidence of 2 to 3% in the first year of life. The overall prognosis of CMPA in infancy is good with a remission rate of approximately 85 to 90%. In particular, gastrointestinal symptoms show a good prognosis. An early increased immunoglobulin E-response to CMP is associated with an increased risk of persistent allergy to CMP, development of adverse reactions to other foods, and development of asthma and rhinoconjunctivitis later in childhood.
TL;DR: Several antibodies are described that recognize phosphoserine/phosphothreonine-containing proteins by Western blotting and can be used to enrich for proteins phosphorylated on serine/threonine residues by immunoprecipitation, as well.
TL;DR: It is suggested that low phosphorus availability should have significantly reduced rates of photosynthesis and carbon burial, thereby reducing the long-term oxygen production on the early Earth and contributing to the low concentrations of atmospheric oxygen during the late Archaean and early Proterozoic eras.
Abstract: After the evolution of oxygen-producing cyanobacteria at some time before 2.7 billion years ago, oxygen production on Earth is thought to have depended on the availability of nutrients in the oceans, such as phosphorus (in the form of orthophosphate). In the modern oceans, a significant removal pathway for phosphorus occurs by way of its adsorption onto iron oxide deposits. Such deposits were thought to be more abundant in the past when, under low sulphate conditions, the formation of large amounts of iron oxides resulted in the deposition of banded iron formations. Under these circumstances, phosphorus removal by iron oxide adsorption could have been enhanced. Here we analyse the phosphorus and iron content of banded iron formations to show that ocean orthophosphate concentrations from 3.2 to 1.9 billion years ago (during the Archaean and early Proterozoic eras) were probably only approximately 10-25% of present-day concentrations. We suggest therefore that low phosphorus availability should have significantly reduced rates of photosynthesis and carbon burial, thereby reducing the long-term oxygen production on the early Earth--as previously speculated--and contributing to the low concentrations of atmospheric oxygen during the late Archaean and early Proterozoic.
TL;DR: It is shown here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate.
Abstract: RelE and ChpAK (MazF) toxins of Escherichia coli have previously been described as proteins that mediate efficient cell killing. We show here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate. Later induction of transcription of the antitoxin genes relB or chpAI fully reversed the static condition induced by RelE and ChpAK respectively. We also provide a mechanistic explanation for these findings. Thus, induction of relE transcription severely inhibited translation, whereas induction of chpAK transcription inhibited both translation and replication. Hence, most likely, lack of colony formation is due to inhibition of translation in the case of relE and inhibition of translation and/or replication in the case of chpAK. Consistent with this proposal, later induction of transcription of the cognate antitoxin genes simultaneously reversed cell stasis and the inhibitory effects of RelE and ChpAK on macromolecular syntheses. These results preclude that RelE and ChpAK mediate cell killing during the conditions used here. In vivo and in vitro analyses of a mutant RelE protein supported that inhibition of colony formation was due to inhibition of translation.
TL;DR: Recent contributions to the field are described and factors that probably play a role in vivo are described, including Wnt, transforming growth factor beta, inflammatory cytokines and prostaglandin F(2alpha).
Abstract: The balance of contradictory signals experienced by preadipocytes influences whether these cells undergo adipogenesis. In addition to the endocrine system, these signals originate from the preadipocytes themselves or operate as part of a feedback loop involving mature adipocytes. The factors that regulate adipogenesis either promote or block the cascade of transcription factors that coordinate the differentiation process. Some of the positive factors reviewed include insulin-like growth factor I, macrophage colony-stimulating factor, fatty acids, prostaglandins and glucocorticoids, and negative factors reviewed include Wnt, transforming growth factor β, inflammatory cytokines and prostaglandin F 2α . Tipping the scales towards or away from adipogenesis has profound implications for human health. In this review, we describe recent contributions to the field and will focus on factors that probably play a role in vivo .
TL;DR: Several DQ2-restricted T-cell epitopes exist in gliadin that are located in regions rich in proline, which likely reflects epitope selection at the levels of digestive and antigen-presenting cell processing, transglutaminase-mediated deamidation, and/or peptide binding to D Q2.
TL;DR: Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition to show the biological nature of the process and indicate that the transformations might be attributed to the anammox process.
Abstract: Factors controlling the anaerobic oxidation of ammonium with nitrate and nitrite were explored in a marine sediment from the Skagerrak in the Baltic-North Sea transition. In anoxic incubations with the addition of nitrite, approximately 65% of the nitrogen gas formation was due to anaerobic ammonium oxidation with nitrite, with the remainder being produced by denitrification. Anaerobic ammonium oxidation with nitrite exhibited a biological temperature response, with a rate optimum at 15°C and a maximum temperature of 37°C. The biological nature of the process and a 1:1 stoichiometry for the reaction between nitrite and ammonium indicated that the transformations might be attributed to the anammox process. Attempts to find other anaerobic ammonium-oxidizing processes in this sediment failed. The apparent Km of nitrite consumption was less than 3 μM, and the relative importance of ammonium oxidation with nitrite and denitrification for the production of nitrogen gas was independent of nitrite concentration. Thus, the quantitative importance of ammonium oxidation with nitrite in the jar incubations at elevated nitrite concentrations probably represents the in situ situation. With the addition of nitrate, the production of nitrite from nitrate was four times faster than its consumption and therefore did not limit the rate of ammonium oxidation. Accordingly, the rate of this process was the same whether nitrate or nitrite was added as electron acceptor. The addition of organic matter did not stimulate denitrification, possibly because it was outcompeted by manganese reduction or because transport limitation was removed due to homogenization of the sediment.
TL;DR: Preliminary estimates of phytoremediation potential suggest that P. calomelanos might remove approximately 2% of the soil arsenic load per year, and the option of disposing high arsenic ferns at sea is raised for discussion.
TL;DR: Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.
Abstract: Axons rely on guidance cues to reach remote targets during nervous system development1. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum2,3, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.
TL;DR: This chapter provides an overview of the recent understanding of intracellular transport of cholesterol, aimed to create a link between the well characterized biophysical properties of cholesterol in model membranes and its behavior in living cells.
Abstract: Cholesterol is the single most abundant lipid species in mammalian cells. More than 2×109 years of evolution designed this molecule to perfectly fit into phospholipid bilayers regulating the fluidity, permeability and bending stiffness of biological membranes. Cholesterol also serves as a precursor of steroid hormones, bile acids and oxysterols, and its cellular synthesis is regulated by a complex machinery. While the molecular mechanisms underlying cholesterol synthesis are known in great detail, knowledge is rather sparse about the inter-compartment transport of cholesterol, including trafficking modes and kinetics, as well as control of endomembrane cholesterol content. This chapter provides an overview of our recent understanding of intracellular transport of cholesterol. It is aimed to create a link between the well characterized biophysical properties of cholesterol in model membranes and its behavior in living cells.
TL;DR: The ferns were by far the most proficient plants at accumulating arsenic from soil, attaining concentrations of up to 8350 microg g(-1) (dry mass) in the frond.
TL;DR: Packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet are developed, with a five to one ratio of outlet size to particle diameter appearing to be the upper maximum.
TL;DR: It is concluded that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit and underline the importance of antisense control in bacterial gene regulation.
Abstract: The physiological role of Escherichia coli Spot 42 RNA has remained obscure, even though the 109-nucleotide RNA was discovered almost three decades ago. Structural features of Spot 42 RNA and previous work suggested to us that the RNA might be a regulator of discoordinate gene expression of the galactose operon, a control that is only understood at the phenomenological level. The effects of controlled expression of Spot 42 RNA or deleting the gene (spf) encoding the RNA supported this hypothesis. Down-regulation of galK expression, the third gene in the gal operon, was only observed in the presence of Spot 42 RNA and required growth conditions that caused derepression of the spf gene. Subsequent biochemical studies showed that Spot 42 RNA specifically bound at the galK Shine-Dalgarno region of the galETKM mRNA, thereby blocking ribosome binding. We conclude that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit. Our results reveal an interesting mechanism by which the expression of a promoter distal gene in an operon can be modulated and underline the importance of antisense control in bacterial gene regulation.
TL;DR: This review addresses the question of the origin of life, with emphasis on plausible boundary structures that may have initially provided cellular compartmentation, by investigating nutrient uptake across simple membranes and encapsulated catalyzed reactions in protocellular life forms.
Abstract: This review addresses the question of the origin of life, with emphasis on plausible boundary structures that may have initially provided cellular compartmentation. Some form of compartmentation is a necessary prerequisite for maintaining the integrity of interdependent molecular systems that are associated with metabolism, and for permitting variations required for speciation. The fact that lipid-bilayer membranes define boundaries of all contemporary living cells suggests that protocellular compartments were likely to have required similar, self-assembled boundaries. Amphiphiles such as short-chain fatty acids, which were presumably available on the early Earth, can self-assemble into stable vesicles that encapsulate hydrophilic solutes with catalytic activity. Their suspensions in aqueous media have therefore been used to investigate nutrient uptake across simple membranes and encapsulated catalyzed reactions, both of which would be essential processes in protocellular life forms.
TL;DR: This is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.
TL;DR: In this article, the authors consider a setting in which a firm uses residual income to motivate a manager's investment decision, and they consider a case where the residual income capital charge is adjusted for market risk.
Abstract: We consider a setting in which a firm uses residual income to motivate a manager's investment decision. Textbooks often recommend adjusting the residual income capital charge for market risk, but n...