- Peroxisome Proliferator-Activated Receptor alpha (PPAR alpha) down-regulation in cystic fibrosis lymphocytes (2006)
- Background PPARs exhibit anti-inflammatory capacities and are potential modulators of the inflammatory response. We hypothesized that their expression and/or function may be altered in cystic fibrosis (CF), a disorder characterized by an excessive host inflammatory response. Methods PPARα, β and γ mRNA levels were measured in peripheral blood cells of CF patients and healthy subjects via RT-PCR. PPARα protein expression and subcellular localization was determined via western blot and immunofluorescence, respectively. The activity of PPARα was analyzed by gel shift assay. Results In lymphocytes, the expression of PPARα mRNA, but not of PPARβ, was reduced (-37%; p < 0.002) in CF patients compared with healthy persons and was therefore further analyzed. A similar reduction of PPARα was observed at protein level (-26%; p < 0.05). The transcription factor was mainly expressed in the cytosol of lymphocytes, with low expression in the nucleus. Moreover, DNA binding activity of the transcription factor was 36% less in lymphocytes of patients (p < 0.01). For PPARα and PPARβ mRNA expression in monocytes and neutrophils, no significant differences were observed between CF patients and healthy persons. In all cells, PPARγ mRNA levels were below the detection limit. Conclusion Lymphocytes are important regulators of the inflammatory response by releasing cytokines and antibodies. The diminished lymphocytic expression and activity of PPARα may therefore contribute to the inflammatory processes that are observed in CF.
- Wie sekundäre Pflanzeninhaltsstoffe uns vor Krankheiten schützen : von molekularen Wirkmechanismen zu neuen Medikamenten (2006)
- Wirkungen von Heilpflanzen, Gewürzen, Tees und Lebensmitteln werden in der Naturheilkunde seit der Antike genutzt. Pharmakologisch wirksam sind in der Regel nur die sekundären Pflanzeninhaltsstoffe. Diese in den oft aus vielen Bestandteilen zusammengesetzten Naturstoffen aufzuspüren und ihren molekularbiologischen Wirkungsmechanismus im Körper aufzuklären, ist das Ziel eines Forschungsnetzwerks am Frankfurter ZAFES (Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit). So konnten Pharmazeuten und Kliniker gemeinsam herausfinden, wie ein Bestandteil des Rotweins, das Resveratrol, vor Darmkrebs schützt. Die Inhaltsstoffe von Salbei und Rosmarin bieten vielversprechende Ausgangspunkte für neue Medikamente gegen Altersdiabetes. Weihrauch, Myrte und Johanniskraut enthalten Wirkstoffe, die Schlüsselenzyme für Entzündungsreaktionen – etwa bei rheumatischen Beschwerden – hemmen.
- PhAST : pharmacophore alignment search tool (2009)
- We developed the Pharmacophore Alignment Search Tool (PhAST), a text-based technique for rapid hit and lead structure searching in large compound databases. For each molecule, a two-dimensional graph of potential pharmacophoric points (PPPs) is created, which has an identical topology as the original molecule with implicit hydrogen atoms. Each vertex is coloured by a symbol representing the corresponding PPP. The vertices of the graph are canonically labelled . The symbols associated with the vertices are combined to a so-called PhAST-Sequence beginning with the vertex with the lowest canonical label. Due to the canonical labelling the created PhAST-Sequence is characteristic for each molecule. For similarity assessment, PhAST-Sequences are compared using the sequence identity in their global pairwise alignment . The alignment score lies between 0 (no similarity) and 1 (identical PhAST-Sequences). In order to use global pairwise sequence alignment, a score matrix for pharmacophoric symbols was developed and gap penalties were optimized. PhAST performed comparably and sometimes superior to other similarity search tools (CATS2D , MOE pharmacophore quadruples ) in retrospective virtual screenings using the COBRA  collection of drugs and lead structures. Most importantly, the PhAST alignment technique allows for the computation of significance estimates that help prioritize a virtual hit list.
- CD69 is a TGF-β/1α,25-dihydroxyvitamin D3 target gene in monocytes (2013)
- CD69 is a transmembrane lectin that can be expressed on most hematopoietic cells. In monocytes, it has been functionally linked to the 5-lipoxygenase pathway in which the leukotrienes, a class of highly potent inflammatory mediators, are produced. However, regarding CD69 gene expression and its regulatory mechanisms in monocytes, only scarce data are available. Here, we report that CD69 mRNA expression, analogous to that of 5-lipoxygenase, is induced by the physiologic stimuli transforming growth factor-β (TGF-β) and 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in monocytic cells. Comparison with T- and B-cell lines showed that the effect was specific for monocytes. CD69 expression levels were increased in a concentration-dependent manner, and kinetic analysis revealed a rapid onset of mRNA expression, indicating that CD69 is a primary TGF-β/1α,25(OH)2D3 target gene. PCR analysis of different regions of the CD69 mRNA revealed that de novo transcription was initiated and proximal and distal parts were induced concomitantly. In common with 5-lipoxygenase, no activation of 0.7 kb or ~2.3 kb promoter fragments by TGF-β and 1α,25(OH)2D3 could be observed in transient reporter assays for CD69. Analysis of mRNA stability using a transcription inhibitor and a 3′UTR reporter construct showed that TGF-β and 1α,25(OH)2D3 do not influence CD69 mRNA stability. Functional knockdown of Smad3 clearly demonstrated that upregulation of CD69 mRNA, in contrast to 5-LO, depends on Smad3. Comparative studies with different inhibitors for mitogen activated protein kinases (MAPKs) revealed that MAPK signalling is involved in CD69 gene regulation, whereas 5-lipoxygenase gene expression was only partly affected. Mechanistically, we found evidence that CD69 gene upregulation depends on TAK1-mediated p38 activation. In summary, our data indicate that CD69 gene expression, conforming with 5-lipoxygenase, is regulated monocyte-specifically by the physiologic stimuli TGF-β and 1α,25(OH)2D3 on mRNA level, although different mechanisms account for the upregulation of each gene.
- Design of dual ligands using excessive pharmacophore query alignment : from 7th German Conference on Chemoinformatics: 25 CIC-Workshop Goslar, Germany, 6 - 8 November 2011 (2012)
- Oral presentation Dual- or multi-target ligands have gained increased attention in the past years due to several advantages, including more simple pharmacokinetic and phamarcodynamic properties compared to a combined application of several drugs. Furthermore multi-target ligands often possess improved efficacy . We present a new approach for the discovery of dual-target ligands using aligned pharmacophore models combined with a shape-based scoring. Starting with two sets of known active compounds for each target, a number of different pharmacophore models is generated and subjected to pairwise graph-based alignment using the Kabsch-Algorithm [2,3]. Since a compound may be able to bind to different targets in different conformations, the algorithm aligns pairs of pharmacophore models sharing the same features which are not necessarily at the exactly same spatial distance. Using the aligned models, a pharmacophore search on a multi-conformation-database is performed to find compounds matching both models. The potentially “dual” ligands are scored by a shape-based comparison with the known active molecules using ShaEP . Using this approach, we performed a prospective fragment-based virtual screening for dual 5-LO/sEH inhibitors. Both enzymes play an important role in the arachidonic acid cascade and are involved in inflammatory processes, pain, cardiovascular diseases and allergic reactions [5,6]. Beside several new selective inhibitors we were able to find a compound inhibiting both enzymes in low micromolar concentrations. The results indicate that the idea of aligned pharmacophore models can be successfully employed for the discovery of dual-target ligands.
- Trichostatin A induces 5-lipoxygenase promoter activity and mRNA expression via inhibition of histone deacetylase 2 and 3 (2012)
- The 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes. We have previously shown that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) activates 5-LO transcription via recruitment of Sp1, Sp3 and RNA polymerase II to the proximal promoter. To identify the HDACs involved in the regulation of 5-LO promoter activity isoform-specific HDAC inhibitors were applied. 5-LO promoter activity and mRNA expression were up-regulated by the class I HDAC inhibitors apicidin and MS-275 but not by class II inhibitors. Knockdown of HDAC 1, 2 and 3 revealed that HDAC2 and HDAC3 but not HDAC1 is involved in the up-regulation of 5-LO mRNA expression. To analyse the chromatin modifications at the 5-LO promoter associated with HDAC inhibition, the time course of 5-LO mRNA induction by trichostatin A was investigated and the concomitant changes in histone modifications at the 5-LO promoter in HL-60, U937 and Mono Mac6 cells were determined. Chromatin immunoprecipitation analysis revealed that trichostatin A increases acetylation of histones H3 and H4 at the 5-LO core promoter in HL-60 and U937 cells whereas no significant changes were observed in Mono Mac6 cells. The appearance of H3 and H4 acetylation preceded the 5-LO mRNA induction whereas in all three cell lines, induction of 5-LO mRNA expression correlated with histone H3 lysine 4 trimethylation (H3K4me3), a marker for transcriptional activity of gene promoters.
- 5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells (2012)
- Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
- 5-Lipoxygenase: underappreciated role of a pro-inflammatory enzyme in tumorigenesis (2010)
- Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
- Die Zünglein an der Waage : Eicosanoide und ihre Rolle bei physiologischen und pathophysiologischen Prozessen (2011)
- Wie entsteht Schmerz? Und wie kann man ihn lindern? Um diese Fragen beantworten zu können, untersuchen Forscher eine Gruppe von Schlüsselmolekülen, die Eicosanoide, und ihre Abbauprodukte. Dabei machen sie immer wieder überraschende Entdeckungen: Blockiert man etwa durch Schmerzmittel wie Acetylsalicylsäure gezielt die Entstehung des Abbauprodukts Prostaglandin, schützt dies auch vor Krebs. Verhindert man die Entstehung von Leukotrienen, lassen sich allergische Reaktionen wie Asthma, aber auch Krebs, Osteoporose und Herz-Kreislauf-Erkrankungen beeinflussen.