MetaboLights MTBLS120 - GNPS Metabolomic Discrimination of Near Isogenic Low and High Phytate Soybean Seeds
ABSTRACT: This data set is downloaded from MetaboLights (http://www.ebi.ac.uk/metabolights/) accession number MTBLS120 Abstract:Methodologies for untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) were developed and applied to lipid-depleted methanolic extracts of soybeans seeds obtained from four near isogenic soybean lines (NILs) that differed in phytate content. The four lines differed in mutations for genes encoding two highly homologous multi-drug resistant proteins (MRPs). The double mutant exhibited the low phytate/emergence phenotype whereas the other three NILs, namely the two single mutants and the wild type, did not. Lipid-depleted aqueous methanol extracts were analyzed by orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Principal component analysis (PCA) of the LC-MS data clearly separated the low phytate line from the other three, with the major metabolite differences residing in the soyasaponin composition. Group A soyasaponins containing C22-terminated acetylated glucosides were of much lower concentration in the low phytate line, which favored terminally acetylated xyloside-based soyasaponins (soyasaponin A4, A5 and A6). Differing levels of the allergen Gly m 1 were also detected.
Project description:Methodologies for untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) were developed and applied to lipid-depleted methanolic extracts of soybeans seeds obtained from four near isogenic soybean lines (NILs) that differed in phytate content. The four lines differed in mutations for genes encoding two highly homologous multi-drug resistant proteins (MRPs). The double mutant exhibited the low phytate/emergence phenotype whereas the other three NILs, namely the two single mutants and the wild type, did not. Lipid-depleted aqueous methanol extracts were analyzed by orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Principal component analysis (PCA) of the LC-MS data clearly separated the low phytate line from the other three, with the major metabolite differences residing in the soyasaponin composition. Group A soyasaponins containing C22-terminated acetylated glucosides were of much lower concentration in the low phytate line, which favored terminally acetylated xyloside-based soyasaponins (soyasaponin A4, A5 and A6). Differing levels of the allergen Gly m 1 were also detected.
Project description:The pattern of histone H4 acetylation in different genomic regions has been investigated by immunoprecipitating oligonucleosomes from a human lymphoblastoid cell line with antibodies to H4 acetylated at lysines 5, 8, 12 or 16. DNA from antibody-bound or unbound chromatin was assayed by slot blotting. Pol I and pol II transcribed genes located in euchromatin were shown to have levels of H4 acetylation at lysines 5, 8 and 12 equivalent to those in input chromatin, but to be slightly enriched in H4 acetylated at lysine 16. In no case did the acetylation level correlate with actual or potential transcriptional activity. All acetylated histone H4 isoforms were depleted in non-coding, simple repeat DNA in heterochromatin, though the extent of depletion varied with the type of heterochromatin and with the isoform. Two single copy genes that map within or adjacent to blocks of paracentric heterochromatin are depleted in H4 acetylated at lysines 5, 8 and 12, but not 16. Consensus sequences of repetitive elements of the Alu family (SINES, enriched in R bands) were associated with H4 that was more highly acetylated at all four lysines than input chromatin, while H4 associated with Kpn I elements (LINES, enriched in G bands) was significantly underacetylated.
Project description:Transcriptionnal profiling of 4 human cell lines treated during 3 hours with human adiponectin Keywords: transcriptionnal analysis Four human cell lines (Hela, HepG2, Panc-1, Hek293) were grown until confluence. These cell lines were serum depleted during 2 hours and then treated with human adiponectin (2,5 µg/ml) during 3 hours. Each adiponectin stimulated cell line was compared to depleted cell line. Depleted cell line was considered as control.
Project description:Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ~30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity.
Project description:Aberrant activation of FGFR3 via overexpression or mutation is a frequent feature of bladder cancer; however, its molecular and cellular consequences and functional relevance to carcinogenesis are not well understood. In this study with a bladder carcinoma cell line expressing inducible FGFR3 shRNAs, we sought to identiy transcriptional targets of FGFR3 and investigate their contribution to bladder cancer development. Bladder cancer cell line RT112 was transduced with a doxycycline-inducible control EGFP shRNA or three independent FGFR3 shRNAs, designated FGFR3 shRNA 2-4, FGFR3 shRNA 4-1 and FGFR3 shRNA 6-16. These four cell lines were treated with or without doxycycline for 48 hr to deplete FGFR3 protein prior to the isolation of mRNA for microarray analysis. Genes that were differentially expressed after doxycycline induction in all three FGFR3-depleted cell lines but not in the control cell line were considered potential FGFR3-regulated genes. Each treatment group was run in triplcates, and there are 24 samples.
Project description:Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the major miRNAs and proteins extracted from the exosomes were similar among the four cell lines. However, the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.
Project description:Lipids are important cellular components which can be significantly altered in a range of disease states including prostate cancer. Here, a unique systematic approach has been used to define lipid profiles of prostate cancer cell lines, using quantitative mass spectrometry (LC-ESI-MS/MS), FTIR spectroscopy and fluorescent microscopy. All three approaches identified significant difference in the lipid profiles of the three prostate cancer cell lines (DU145, LNCaP and 22RV1) and one non-malignant cell line (PNT1a). Specific lipid classes and species, such as phospholipids (e.g., phosphatidylethanolamine 18:1/16:0 and 18:1/18:1) and cholesteryl esters, detected by LC-ESI-MS/MS, allowed statistical separation of all four prostate cell lines. Lipid mapping by FTIR revealed that variations in these lipid classes could also be detected at a single cell level, however further investigation into this approach would be needed to generate large enough data sets for quantitation. Visualisation by fluorescence microscopy showed striking variations that could be observed in lipid staining patterns between cell lines allowing visual separation of cell lines. In particular, polar lipid staining by a fluorescent marker was observed to increase significantly in prostate cancer lines cells, when compared to PNT1a cells, which was consistent with lipid quantitation by LC-ESI-MS/MS and FTIR spectroscopy. Thus, multiple technologies can be employed to either quantify or visualise changes in lipid composition, and moreover specific lipid profiles could be used to detect and phenotype prostate cancer cells.
Project description:We established chromate transformed cell lines by chronic exposure of normal human bronchial epithelial BEAS-2B cells to low doses of hexavalent chromium followed by anchorage-independent growth. The gene expression profiles were analyzed in the established cell lines. The gene expression profiles from six chromate transformed cell lines were remarkably similar to each other yet differed significantly from that of either control cell line or normal Beas-2B cells. A total of 409 differentially expressed genes were identified in chromate transformed cells compared to control cells. We analyzed gene expression profiles from 10 cell lines ( six chromated transformed cells lines, three control cell lines, and parental BEAS-2B cells) using Affymetrix Human Gene 1.0 ST array. No techinical replicates were performed.
Project description:This research investigates the molecular mechanisms of trait deterioration of two experimental lines of entamopathogenic nematodes, an inbred line (L5M) and its original parental line (OHB), created by sub-culturing different experimental lines of the nematode-bacterium complex over 20 passages in insect hosts. These lines differed in their virulence, heat tolerance and fecundity . Transcriptional profiles of the two experimental lines were determined and select differentially expressed genes were validated by quantitative PCR. Samples from four biological replicates each of the parental strain (OHB) and the laboratory strain (L5M) were hybridized to the custom H. bacteriophora arrays.
Project description:A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.