Similar Datasets
Project description:To investigate the relationship between histones, chaperone function, and cataracts, we performed RNA-seq, isothermal titration calorimetry (ITC), size-exclusion chromatography, and gel electrophoresis of histones. The RNA-seq of postnatal lenses from 2-day-old cryaa -R49C mice revealed increased histone gene expression, suggesting that a α-crystallin mutation regulates histones via a transcriptional mechanism .
2017-08-20 | GSE98027 | GEO
Project description:The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We have generated transgenic mice introducing the onco-Dbl cDNA sequences linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression affected proliferation, migration and differentiation of lens epithelial cells. We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of transgenic mice and observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT). Moreover, genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses, where vascularization can be readily observed. Thus, onco-Dbl expression in mouse lenses induces disruption of the lens architecture, epithelial cell proliferation, EMT, evasion from cell death, and aberrant angiogenesis. Whole lenses were removed from 2, 14, and 42 day old Dbl transgenic and wild type mice. 4 to 8 individual lenses were pooled . Three independent pools from each time and condition were used as replicates.
2009-06-01 | E-GEOD-15694 | biostudies-arrayexpress
Project description:A large variety of low molecular weight (LMW) peptides, derived from the breakdown of crystallins, have been reported in middle to old age human lenses. The proliferation of these LMW peptides coincides with the earliest stages of cataract formation, suggesting that the protein cleavages involved may contribute to the aggregation and insolubilisation of crystallins – these being hall marks of cataractogenesis. This study reports the identification of 238 endogenous LMW crystallin peptides from the cortical extracts of human lenses aged 16, 44, 75 and 83 years. Analysis of the peptide terminal amino acids showed that Lys and Arg were situated at the C-terminus with significantly higher frequency compared to other residues, suggesting that trypsin-like proteolysis may be active in the lens cortical fibre cells. Selected reaction monitoring (SRM) analysis of a prominent αA-crystallin peptide (αA57-65) showed that the concentration of this peptide in the human lens increased gradually to middle age, after which the rate of αA57-65 formation escalated significantly. Using 2-D gel electrophoresis and nanoLC-ESI-MS/MS, 13 protein complexes of 40-150 kDa consisting of multiple crystallin components were characterised from the water soluble cortical extracts of an adult human lens. The detection of these protein complexes suggested the possibility of crystallin cross-linking, with these complexes potentially acting to stabilise degraded crystallins by sequestration into water soluble complexes.
2016-09-29 | PXD001593 | Pride
Project description:Differential expression of HSF4 in null newborn mouse and wildtype lenses was examined to identify putative downstream targets of HSF4. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific aA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIb, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Keywords: Differential mRNA Expression Three biological replicate experiments were performed with HSF null and wildtype lenses.
2010-12-01 | E-GEOD-22362 | biostudies-arrayexpress
Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants
2015-05-01 | E-GEOD-65500 | biostudies-arrayexpress
Project description:The alpha-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataract, the leading cause of human blindness. There are conflicting data in the literature as to what role that alpha-crystallins may play in early lens development. In this study we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three alpha-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed by mass spectrometry and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of alpha A-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either Alpha Ba- or Alpha Bb-crystallin produced no significant lens defects. Mutation of each Alpha-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for Alpha A-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.
2022-01-20 | PXD030918 | panorama
Project description:The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We have generated transgenic mice introducing the onco-Dbl cDNA sequences linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression affected proliferation, migration and differentiation of lens epithelial cells. We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of transgenic mice and observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT). Moreover, genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses, where vascularization can be readily observed. Thus, onco-Dbl expression in mouse lenses induces disruption of the lens architecture, epithelial cell proliferation, EMT, evasion from cell death, and aberrant angiogenesis.
2009-06-01 | GSE15694 | GEO
Project description:Genome-wide approach to identify the cell-autonomous role of Brg1 in lens fiber cell terminal differentiation. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific alphaA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIbeta, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Wild type and dnBrg1 transgenic lenses, 4 biological replicates each
2011-01-01 | E-GEOD-22322 | biostudies-arrayexpress
Project description:Whole lenses were dissected from E15.5, P1, or P30 wild-type C57BL/6J mice or from K6W-Ub transgenic mice, which develop congenital cataracts. Lens homogenates were subjected to quantitative proteomic analysis using tandem mass tag (TMT) isobaric labeling.
2023-09-14 | PXD045040 | Pride
Project description:Protein post-translational modifications (PTMs) have been associated with aging and age-related diseases. PTMs are particularly impactful in long-lived proteins, such as those found in the ocular lens, because they accumulate with age. Two post-translational modifications that lead to protein-protein crosslinks in aged and cataractous lenses are dehydroalanine (DHA) and dehydrobutyrine (DHB); formed from cysteine/serine and threonine residues, respectively. The purpose of this study was to quantitate DHA and DHB in human lens proteins as a function of age and cataract status. Human lenses of various ages were divided into five donor groups: transparent lenses (18–22-year-old, 48–64-year-old, and 70–93-year-old) and cataractous human lenses of two age groups (48–64-year-old lenses, and 70–93-year-old lenses) and were subjected to proteomic analysis. Relative DHA and DHB peptide levels were quantified and compared to their non-modified peptide counterparts. For most lens proteins containing DHA or DHB, higher amounts of DHA- and DHB-modified peptides were detected in aged and cataractous lenses. DHA-containing peptides were classified into three groups based on abundance changes with age and cataract: those that (1) increased only in age-related nuclear cataract (ARNC), (2) increased in aged and cataractous lenses, and (3) decreased in aged lenses and ARNC. There was no indication that DHA or DHB levels were dependent on lens region. In most donor groups, proteins with DHA and DHB were more likely to be found among urea-insoluble proteins rather than among water- or urea-soluble proteins. DHA and DHB formation may induce structural effects that make proteins less soluble in water that leads to age-related protein insolubility and possibly aggregation and light scattering.
2023-10-12 | PXD045734 | Pride