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- Kotarsky, Knut, et al.
(författare)
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Lysophosphatidic Acid Binds to and Activates GPR92, a G Protein-Coupled Receptor Highly Expressed in Gastrointestinal Lymphocytes
- 2006
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Ingår i: Journal of Pharmacology and Experimental Therapeutics. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 1521-0103 .- 0022-3565. ; 318:2, s. 619-628
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Tidskriftsartikel (refereegranskat)abstract
- Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (K(D) = 6.4 +/- 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA(1-3) receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.
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- Mårtensson, Ulrika, et al.
(författare)
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The mouse chemerin receptor gene, mcmklr1, utilizes alternative promoters for transcription and is regulated by all-trans retinoic acid
- 2005
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Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 350:1, s. 65-77
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Tidskriftsartikel (refereegranskat)abstract
- CMKLR1 (chemoattractant-like receptor 1) is a G-protein-coupled receptor implicated in cartilage and bone development and is expressed in organs like the parathyroid gland, brain, and lung. The receptor is also expressed in dendritic cells and in macrophages where it acts as a co-receptor for entry of HIV/SIV isolates into human CD4(+) cells. Recently, a protein named "chemerin" (also known as TIG2) was isolated from human inflammatory fluids and hemofiltrate and found to be the endogenous ligand for CMKLR1. We have previously described the genomic organization of the cmklr1 gene and characterized its promoter in mouse neuroblastoma NB4 1A3 cells. In the present study we identify a second transcript, cmklr1b, in mouse microglia BV2 cells. Cmklr1b is transcribed from an alternative promoter with a transcription start site located 6780 by downstream of the previously identified exon 1 (cmklr1a). The cmklr1b promoter lacks a TATA box but contains two CCAAT boxes in opposite directions. 5 ' Deletion analysis of the promoter region in BV2 cells using a luciferase reporter gene assay indicates two regions, between 623-755 by and 56-125 by upstream of transcription start site, to be important for promoter function. The proximal promoter region includes both CCAAT boxes, and site-directed mutagenesis separately within these elements revealed that only the forward CCAAT element was important for transcription. Although the forward CCAAT element is essential for transcription electrophoretic mobility shift and super-shift assays demonstrated that both CCAAT elements actually bind nuclear proteins from BV2 cells and identified the binding factor as NFY. Real-time reverse transcriptase-PCR experiments of cmklr1b expression in all-traps retinoic acid (ATRA)- stimulated BV2 cells showed strong up-regulation of receptor transcript. Luciferase reporter gene assay of the promoter in ATRA-stimulated BV2 cells confirmed that transcriptional activity of the cmklr1b promoter is increased by ATRA. However, deletion analysis could not identify an ATRA-responsive element within the promoter region suggesting that gene activation is likely to occur through alternative mechanisms. The results emphasise a possible role of cmklr1 in bone modelling.
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- Sabirsh, Alan, et al.
(författare)
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Exploring the pharmacology of the leukotriene B-4 receptor BLT1, without the confounding effects of BLT2
- 2004
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Ingår i: European Journal of Pharmacology. - : Elsevier BV. - 1879-0712 .- 0014-2999. ; 499:1-2, s. 53-65
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Tidskriftsartikel (refereegranskat)abstract
- Most previous studies of leukotriene B-4 (LTB4) pharmacology using primary leukocyte cultures and myeloid cell lines do not differentiate between leukotriene BLT1 and BLT1 receptor activation because both receptors are often expressed by these cells. Here we show that in HeLa cells expressing BLT1 but not BLT2 receptors, BLT1 receptor activation resulted in IP3 mediated calcium release from intracellular stores initially, followed by calcium influx through cell membrane channels. BLT1 calcium signalling was sensitive to the activity of protein kinase C (PKC). protein kinase A (PKA) and protein-tyrosine kinases (PTKs), as well as changes in membrane cholesterol levels and treatments that are known to disrupt normal membrane physiology and/or lipid rafts. Inhibition of MAP kinases, Rho-associated kinases, or phosphomositol-3-kinases (PI3K) had no effect on BLT1 receptor induced calcium signalling, and the receptor was insensitive to the redox state of the extracellular compartment. (C) 2004 Elsevier B.V. All rights reserved.
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- Sabirsh, A, et al.
(författare)
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Fluorescent leukotriene B-4: potential applications
- 2005
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Ingår i: Journal of Lipid Research. - 1539-7262 .- 0022-2275. ; 46:6, s. 1339-1346
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Tidskriftsartikel (refereegranskat)abstract
- Leukotriene B-4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a K-d of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.
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- Sabirsh, A, et al.
(författare)
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Non-specific effects of leukotriene synthesis inhibitors on HeLa cell physiology
- 2005
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Ingår i: Prostaglandins, Leukotrienes and Essential Fatty Acids. - : Elsevier BV. - 0952-3278. ; 73:6, s. 431-440
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Tidskriftsartikel (refereegranskat)abstract
- We examined the effects of various leukotriene synthesis inhibitors on calcium signalling in HeLa cells, before and after transfection with BLT1. All of the inhibitors studied were found to reduce increases in intracellular calcium concentration induced by BLT1, but also by an ionophore or activation of various G-protein coupled receptors, regardless of BLT1 expression. In order to explore the mechanism of these apparently general effects we examined HeLa cell expression of leukotriene receptors and biosynthetic enzymes and found that the genes for key leukotriene synthesis enzymes and all of the leukotriene receptors were not expressed. Leukotrienes are involved in the pathology of a variety of cancers, and for HeLa cells leukotrienes have been reported to be important for aspects of the carcinogenic phenotype. We find that leukotriene synthesis inhibitors have non-specific effects, so careful controls are necessary to avoid interpreting non-specific effects as evidence for leukotriene involvement.
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- Sabirsh, A, et al.
(författare)
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Residues from transmembrane helices 3 and 5 participate in leukotriene B-4 binding to BLT1
- 2006
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:18, s. 5733-5744
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Tidskriftsartikel (refereegranskat)abstract
- Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B-4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val 105 and Ile 108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.
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| 10. |
- Tilgmann, Carola, et al.
(författare)
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The State of The Research
- 2014
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Konferensbidrag (refereegranskat)abstract
- Introduction. Information overload, special competences need in the area of information analysis, demand of business innovation, a struggle in the grant jungle are strong factors that influence the daily work flow for a researcher. Taking these aspects into consideration, we introduce a concept that we have named The State of The Research. Aim. We aim to broaden the researcher’s view in his/her research area and give insights into new opportunities for innovation and collaboration. The idea here is that the researchers would also be able to use a part or the whole package of the State of The Research in their future grant applications. Method. Our concept is a comprehensive package containing medical information analysis and visualization, news and social media monitoring, patent landscape analysis, and bibliometric analysis – all based on the particular research area of interest for the individual researcher/research group. We are using text mining tools in combination with visualization related to PubMed and Chemical Abstracts to extract research related questions from larger data sets. By using bibliometric mapping techniques, we can also reveal structures in the studied research field, e.g. networks of influential authors, clusters of key concepts and frequently cited articles. We are approaching the researchers face to face in their environment where we present the compiled material describing The State of The Research. For example, we cover a broad disease area that has been selected in advance in collaboration with the researchers. With our analysis tools, we recognize collaboration opportunities and approximate freedom to operate within the research based on patents. We have taken trend analysis in a form of co-word analysis related to time to indicate possible rising or falling research within a disease area. Preliminary results. Personal communication and an understanding in each individual field of The State of The Research get the researchers attention. This in turn has also led to increased requests for deeper analysis. Future prospects and developments will be to offer education in grant application writing Moreover, we would like to use more general text mining approaches to answer research related questions faster and with larger datasets. In sum, allocation of research funds is becoming more competitive and we need to support our researchers not only with bibliometric analysis but with new concepts like The State of The Research to give support to researcher´s grant applications.
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