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Uppsala, Sweden

Snir O.,Karolinska University Hospital | Widhe M.,Karolinska University Hospital | Hermansson M.,Karolinska University Hospital | Von Spee C.,Karolinska University Hospital | And 10 more authors.
Arthritis and Rheumatism

Objective. High titers of specific anti-citrullinated protein antibodies (ACPAs) are frequently present in the serum of rheumatoid arthritis (RA) patients, but their presence in synovial fluid is less well characterized. The purpose of this study was to compare the levels of antibody to 4 well-defined citrullinated candidate RA autoantigens in serum and synovial fluid and to determine whether antibodies to one citrullinated antigen are dominant over another. Furthermore, we studied their relationships with mutated citrullinated vimentin (MCV), a newly identified RA-specific serum assay, and the classic cyclic citrullinated peptide (CCP) in the synovial fluid of well-defined HLA-DR groups. Methods. Paired serum and synovial fluid samples from 290 RA patients and serum samples from 100 age- and sex-matched healthy controls were analyzed for the presence of anti-MCV and anti-CCP antibodies and for reactivity to citrullinated fibrinogen, α-enolase, type II collagen, and vimentin. A total of 219 of the 290 patients were genotyped for the HLA-DR shared epitope alleles. Results. Significantly higher proportions of antibodies against all RA-associated citrullinated antigens were found in synovial fluid as compared with serum. This was also true for the MCV and CCP responses but not for non-RA-associated anti-tetanus toxoid antibodies. As expected, we found a high correlation between citrullinated vimentin and MCV responses. All synovial fluid ACPAs were predominantly associated with HLA-DRB1*04 alleles and were confined to the CCP+/ MCV+ subset of patients. Conclusion. MCV and CCP positivity represent a similar subset of RA patients, whereas ACPAs with different fine specificities fall into subgroups of anti-CCP+/anti-MCV+ patients. The levels of all specific ACPAs were elevated in synovial fluid, suggesting that there is local antibody production and/or retention of ACPAs at the site of inflammation governed by RA-predisposing genes. © 2010, American College of Rheumatology. Source

Rask-Andersen M.,Uppsala University | Almen M.S.,Uppsala University | Schioth H.B.,Uppsala University | Schioth H.B.,Uppsala Biomedical Center
Nature Reviews Drug Discovery

The discovery and exploitation of new drug targets is a key focus for both the pharmaceutical industry and academic biomedical research. To provide an insight into trends in the exploitation of new drug targets, we have analysed the drugs that were approved by the US Food and Drug Administration during the past three decades and examined the interactions of these drugs with therapeutic targets that are encoded by the human genome, using the DrugBank database and extensive manual curation. We have identified 435 effect-mediating drug targets in the human genome, which are modulated by 989 unique drugs, through 2,242 drug-target interactions. We also analyse trends in the introduction of drugs that modulate previously unexploited targets, and discuss the network pharmacology of the drugs in our data set. © 2011 Macmillan Publishers Limited. All rights reserved. Source

Xu N.,Uppsala Biomedical Center | Akusjarvi G.,Uppsala Biomedical Center
Methods in Molecular Biology

RNA interference (RNAi) plays novel roles in both host antiviral defense and viral replication. It has been shown that some viruses can exploit the RNAi machinery for their own benefit by encoding for their own viral small RNAs. Here we present a collection of methods to study adenoviral small RNAs, specifically a method for immunopurification of RNA-induced silencing complex (RISC) and a biochemical assay for the activity of purified RISC associated with adenoviral small RNAs. © Springer Science+Business Media, LLC 2011. Source

Martinez E.,Uppsala Biomedical Center | Martinez E.,University of Barcelona | Hamberg M.,Karolinska Institutet | Busquets M.,University of Barcelona | And 3 more authors.
Journal of Biological Chemistry

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy- (8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid.Aseries of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S- 2H]18:1n-9, [7R-2H]18:2n-6, and [8R-2H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)- octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Nilsson T.,Uppsala Biomedical Center | Martinez E.,University of Barcelona | Manresa A.,University of Barcelona | Oliw E.H.,Uppsala Biomedical Center
Rapid Communications in Mass Spectrometry

Pseudomonas aeruginosa is an opportunistic pathogen, which oxidizes oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid (7,10-(OH)2-18:1) of biological and industrial interest. Electrospray tandem mass spectrometric (MS/MS) analysis of hydroxylated fatty acids usually generates characteristic fragments containing the carboxylate anion and formed by a-cleavage at the oxidized carbon. These fragments indicate the positions of the hydroxyl group. In contrast, liquid chromatography (LC)/MS/MS analysis of 7,10-(OH)2-18:1 yielded a series of other ions with structural information. To study the fragmentation mechanism, we prepared 2H-and 18O-labeled isotopomers. We also performed MS3 analysis of the major ions, and for comparison we generated the corresponding 7,10-dihydroxy metabolites of 16:1n-7, 18:2n-6, and 20:1n-11 with a protein extract of P. aeruginosa. The MS/MS spectra of 7,10-(OH)2-18:1 and its isotopomers, 7,10-(OH)2-16:1, and 7,10-(OH)2-20:1, contained a series of prominent fragments that all hold the omega end. The 8,9-double bond was not essential for this fragmentation, as 7,10-(OH)2-18:0, and its isotopomers, formed essentially the same fragments in the lower mass range. In contrast, 7,10-dihydroxy-8(E),12(Z)-octadecadienoic acid (7,10-(OH)2-18:2) fragmented by a-cleavage at the oxidized carbons with formation of carboxylate anions. Our results demonstrate that C16-C20 fatty acids with a 7,10-dihydroxy-8(E) functionality undergo charge-driven fragmentation after charge migration to the v-end, whereas the main ions of 7,10-(HO)2-18:2 retain charge at the carboxyl group. © 2010 John Wiley & Sons, Ltd. Source

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