Homo sapiens isolate:Arachidonate 12 Lipoxygenase Gene
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PROVIDER: PRJNA379762 | ENA |
REPOSITORIES: ENA
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Project description:Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes capable of converting polyunsaturated fatty acids to their hydroperoxy derivatives. They have been implicated in basic cellular processes such as proliferation and differentiation but are also involved in the pathogenesis of various diseases such as inflammatory disorders and atherosclerosis. The catalytic activity of LOXs increases the intracellular peroxide tone, which is an important parameter for expression regulation of redox-sensitive genes. Previously, we showed that induction of 12/15-expression by interleukin-4 profoundly alters the gene expression pattern of human peripheral monocytes, which led to a profound switch of the cellular phenotype. To explore, which of these changes might be a direct consequence of 12/15-LOX expression we stably transfected a permanent human monocytic cell line (U937) with a 12/15-LOX containing eukaryotic expression plasmid and an empty control vector (mock-control). After a multi-step selection procedure a viable clones (12/15-LOX transfectants and mock-transfectants) were isolated and further cultured. We found that 12/15-LOX expression did not significantly alter basic cellular functions and that the cells, which express the 12/15-LOX at similar levels as IL4-treated human monocytes, did not show major functional deficits when compared with the mock-transfectants. However, more detailed comparison of the gene expression profiles of 12/15-LOX transfected cells and that of corresponding mock-transfectants revealed subtle differences. Among the 22,300 genes present on the chip about one half was not expressed regardless whether 12/15-LOX was present in the cells or not. In 12/15-LOX expressing cells 11,000 genes (50 % of the genes present on the chip) and in the corresponding mock-transfectants 10,600 genes (47 % of the genes present on the chip) were expressed. Among the expressed genes only 900 (7 %) were upregulated more than 2-fold and 1,400 (12 %) were down regulated to a similar extent. Among the regulated gene products we detected several connective tissue matrix proteins (collagen isoforms, lamins, fibulins, fibronectins, aggrecans, claudins), extracellular proteinases (serpins, matrix metalloproteinases), tissue inhibitors of metalloproteinases, growth factors and their receptors, and adhesion molecules. In most cases, we did not see simultaneous up- or down-regulation of all family members but rather selective regulatory response of individual proteins (for instance 5-fold upregulation of collagen XV, alpha â??1, but 20-fold downregulation of collagen VII, alpha-1 after 12/15-LOX transfection). These data suggest that induction of 12/15-LOX specifically modifies the gene expression pattern of U937 cells but did not lead to comprehensive alterations. Experiment Overall Design: Cell transfection Experiment Overall Design: The eukaryotic expression vector pcDNA 3 (Invitrogen Life Technologies, Experiment Overall Design: Leek, The Netherlands) bearing the complete coding region of the rabbit Experiment Overall Design: reticulocyte type 15-LOX1 and a corresponding empty vector (mock) were Experiment Overall Design: transfected into U937 cells using FuGENE 6, according to the manufacturerâ??s instructions. Experiment Overall Design: Briefly, 2.5 x 10E5 cells/ml were plated on 35-mm plates and transfected with 2 µg of plasmid using 6 µl of FuGENE 6 reagent. After 72h exposure, the precipitate was removed and the cells were cultured for 4 weeks in selection medium supplemented with 0.5 mg/ml geneticin. Experiment Overall Design: Incubation: at 37 °C with 5% CO2
2008-06-18 | E-GEOD-8173 | biostudies-arrayexpress
Project description:The diploid isolate EM93 is the main ancestor to the widely used Saccharomyces cerevisiae haploid laboratory strain, S288C. In this study, we first hybridise DNA from eight EM93 segregants from two tetrads to Affymetrix genechip S. cerevisiae Tiling 1.0R Arrays to generate a high-resolution overview of the genetic differences between EM93 and S288C. We show that EM93 is heterozygous for many polymorphisms, including large sequence polymorphisms, such as deletions and a Saccharomyces paradoxus introgression. We also find that many of the large sequence polymorphisms are associated with Ty-elements and sub-telomeric regions. We next utilize the hybridization profiles of the 3.2 million Tiling Array probes to identify 2,965 genetic markers, which we then used to design an EM93 genotyping array. By genotyping 120 EM93 tetrads, we deduced the structures of all EM93 chromosomes and found that the average EM93 meiosis produces 144 detectable recombination events, consisting of 87 crossover and 57 non-crossover events; we also identified 203 recombination hot-spots and provide evidence for crossover interference. We find that the effect of heterozygous large sequence polymorphisms on recombination is chromosome position-dependent, with sub-telomeric large sequence polymorphisms having no discernable effect on recombination but non-sub-telomeric large sequence polymorphisms reducing recombination. Finally, we find that recombination hotspots show limited conservation, with some but not all strain-specific hotspots being due to heterozygous non-sub-telomeric large sequence polymorphisms.
2012-06-08 | E-MEXP-3246 | biostudies-arrayexpress
Project description:Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells and involves an interplay between β cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in β cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic (NOD) mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecules PD-L1 and PD-1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets from inhibitor-treated mice revealed significant alteration of cytokine-responsive pathways. RNA sequencing of polarized proinflammatory macrophages showed that VLX-1005 significantly reduced the interferon response. This study highlights a functional equivalence of human ALOX12 and mouse Alox15 in T1D pathogenesis. Our studies provide a platform for the preclinical evaluation of LOX inhibitors and supports the development of 12-LOX inhibitors as a therapeutic strategy for T1D and other inflammatory diseases.
2024-08-23 | GSE272687 | GEO
Project description:Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing β cells and involves an interplay between β cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in β cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic (NOD) mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecules PD-L1 and PD-1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets from inhibitor-treated mice revealed significant alteration of cytokine-responsive pathways. RNA sequencing of polarized proinflammatory macrophages showed that VLX-1005 significantly reduced the interferon response. This study highlights a functional equivalence of human ALOX12 and mouse Alox15 in T1D pathogenesis. Our studies provide a platform for the preclinical evaluation of LOX inhibitors and supports the development of 12-LOX inhibitors as a therapeutic strategy for T1D and other inflammatory diseases.
2024-08-23 | GSE272668 | GEO
Project description:Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-?-linolenic acid (DGLA), eicosapentaenoic acid (EPA), ?-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, ?IIb?3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.
| S-EPMC3494251 | biostudies-literature
Project description:In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12 -/- mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15 -/- mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12 -/- mice and reduced oxidative stress in Alox15 -/- mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12 -/- mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress.
| S-EPMC6484126 | biostudies-literature
Project description:Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes capable of converting polyunsaturated fatty acids to their hydroperoxy derivatives. They have been implicated in basic cellular processes such as proliferation and differentiation but are also involved in the pathogenesis of various diseases such as inflammatory disorders and atherosclerosis. The catalytic activity of LOXs increases the intracellular peroxide tone, which is an important parameter for expression regulation of redox-sensitive genes. Previously, we showed that induction of 12/15-expression by interleukin-4 profoundly alters the gene expression pattern of human peripheral monocytes, which led to a profound switch of the cellular phenotype. To explore, which of these changes might be a direct consequence of 12/15-LOX expression we stably transfected a permanent human monocytic cell line (U937) with a 12/15-LOX containing eukaryotic expression plasmid and an empty control vector (mock-control). After a multi-step selection procedure a viable clones (12/15-LOX transfectants and mock-transfectants) were isolated and further cultured. We found that 12/15-LOX expression did not significantly alter basic cellular functions and that the cells, which express the 12/15-LOX at similar levels as IL4-treated human monocytes, did not show major functional deficits when compared with the mock-transfectants. However, more detailed comparison of the gene expression profiles of 12/15-LOX transfected cells and that of corresponding mock-transfectants revealed subtle differences. Among the 22,300 genes present on the chip about one half was not expressed regardless whether 12/15-LOX was present in the cells or not. In 12/15-LOX expressing cells 11,000 genes (50 % of the genes present on the chip) and in the corresponding mock-transfectants 10,600 genes (47 % of the genes present on the chip) were expressed. Among the expressed genes only 900 (7 %) were upregulated more than 2-fold and 1,400 (12 %) were down regulated to a similar extent. Among the regulated gene products we detected several connective tissue matrix proteins (collagen isoforms, lamins, fibulins, fibronectins, aggrecans, claudins), extracellular proteinases (serpins, matrix metalloproteinases), tissue inhibitors of metalloproteinases, growth factors and their receptors, and adhesion molecules. In most cases, we did not see simultaneous up- or down-regulation of all family members but rather selective regulatory response of individual proteins (for instance 5-fold upregulation of collagen XV, alpha –1, but 20-fold downregulation of collagen VII, alpha-1 after 12/15-LOX transfection). These data suggest that induction of 12/15-LOX specifically modifies the gene expression pattern of U937 cells but did not lead to comprehensive alterations. Keywords: other
2008-05-30 | GSE8173 | GEO
Project description:SNEP: Simultaneous detection of nucleotide and expression polymorphisms using Affymetrix GeneChip
| PRJNA115105 | ENA
Project description:Detection of single feature polymorphisms comparing five barley genotypes. Gene expression under unstressed and drought stressed conditions. Tissue from five entire five day old seedlings from drought stress or unstressed growth conditions was used for RNA extraction. For Barke, Morex and Stepoe the two types of RNA were pooled. For Oregon Wolfe Barley Dominant and Recessive (OWBs), the two types of RNA were handled separately. Targets from three biological replicates of each genotype-treatment were generated and transcript levels were determined using Affymetrix Barley1 GeneChip arrays. Probe set, followed by single probe, comparisons between genotypes allows the identification of single feature polymorphisms in comparisons between genotypes. For the OWBs, comparisons between stressed and unstressed conditions defines stress-regulated genes. Experiment Overall Design: Three genotypes, three replicates each. Two additional genotypes, two sets of three replicates each.
2008-06-12 | E-GEOD-3170 | biostudies-arrayexpress
Project description:Two human 12-lipoxygenase enzyme (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31)-related genes were characterized from 13 distinct clones isolated from three genomic bacteriophage and cosmid libraries. A complete gene (12-lipoxygenase gene 1) spanning approximately 17 kilobases and consisting of 14 exons with sequence matching the cloned platelet/human erythroleukemia (HEL) cell cDNA sequence was identified. Several consensus sites for transcription factors and two potential transcription initiation sites within the 5' flanking region, encompassing the putative promoter region, were identified. A segment of a second, probable pseudogene (12-lipoxygenase gene 2), which displays approximately 85% identity to gene 1 within exon sequences, was also characterized. The presence of two 12-lipoxygenase genes was also substantiated by Southern blot analysis of total human genomic DNA. Exon-intron boundaries for the 12-lipoxygenase genes were located in the identical corresponding positions to the previously cloned human 5-lipoxygenase and rabbit 15-lipoxygenase genes, indicating a highly related gene family. Three lipoxygenase genes (12-lipoxygenase genes 1 and 2, 15-lipoxygenase) were localized to human chromosome 17, whereas the most unrelated lipoxygenase (5-lipoxygenase) was mapped to chromosome 10 by PCR analysis of a human-hamster somatic hybrid DNA panel. 12-Lipoxygenase gene 1 expression could be detected in human erythroleukemia cells, platelets, and human umbilical vein endothelial cells with certainty by reverse transcription-PCR analysis. There was no detectable 12-lipoxygenase gene 2 expression in several tissues and cell lines.
| S-EPMC525611 | biostudies-other