Mapping glucose uptake, transport and metabolism in the bovine lens
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ABSTRACT: To spatially correlate the pattern of glucose uptake to glucose transporter distributions in cultured lenses and map glucose metabolism in different lens regions.
Project description:The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that require a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected over 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, over 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic, autophagy, and mitophagy transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. Differentiation-state transcriptional analysis of embryonic chicken lenses was performed following microdissection of 100 embryonic day 13 (E13) chicken lenses into four distinct regions that represent a continuum of lens cell differentiation states: lens central epithelium (EC), equatorial epithelium (EQ), cortical fibers (FP), and central fibers (FC). Further analysis of the transcriptional content of biologically replicate samples was performed by Illumina directional mRNA sequencing and resulting reads mapped by TopHat and assembled with Cufflinks.
Project description:Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (Cx46) results in the formation of lens cataracts. The timing of the onset of this lens opacity is affected by the genetic background, i.e. the mouse strain. To elucidate the mechanism by which cataracts form in the 129Sv/Jae strain earlier than in the C57BL/6J strain, global gene expression was quantitated in the lenses of these strains. Lens cDNAs were analyzed by hybridization to DNA microarrays and with real time-PCR. Theories are proposed based on the observed higher level of expression of the stress-response genes in the C57BL/6J strain and variations in the expression levels of genes involved in protein synthesis, metabolism, catabolism and cell proliferation. How these variations in gene expression might affect the response of lens fiber cells to the increased calcium, caused by lack of alpha3Cx46, is considered. The possibility that the proteins coded by the strain-variable genes might influence the cataract-associated proteolysis of gamma-crystallin is also addressed. Experiment Overall Design: To determine differences in transcript expression between the lenses of two mouse wild type strains (129 SvJae and C57BL/6J), as well as between alpha3Cx46 KO and wild-type mice. The former comparison may lead to identification of potential candidate(s) genes that prevent (or promote) cataract formation, whereas the latter comparison may provide insights into the mechanism by which cataract formation occurs in the alpha3Cx46 KO mice. Total 8 samples were used ( two separate samples for each of the following 4 types of mice: 129SvJae wild type, 129SvJae alpha3Cx46 KO, C57BL/6J wild type and C57BL/6J alpha3Cx46 KO)
Project description:In order to investigate the mechanism for the progressive lens degeneration caused by targeted deletion of Dicer, we compared expression profiles of protein-coding genes in wild type and DicerCN lenses at E13.5, at a time before gross morphological changes had occurred. We identified distinct classes of differentially expressed genes in the conditional knockout lenses. Intact lenses were dissected from five control and five DicerCN mice at E13.5 for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Conditional disruption of Klf4 in the ectoderm-derived tissues of the eye results in defective cornea, conjunctiva and the lens. Expression of Klf4 is first detected in the embryonic day-12 (E12) mouse lens, peaks at E16, and declines thereafter. Lenses from Mice which were conditional null for Klf4 (Klf4CN) were characterized by morphology and transcriptome. Wild type and Klf4-conditional null mouse lens gene expression at embryonic day 16.5 (E16.5) and postnatal day 56 (PN56) was surveyed using Affymetrix mouse whole genome 430 2 arrays.
Project description:The E2F family consists of transcriptional repressors and activators that control cell proliferation. In the classic paradigm of cell cycle regulation, the three activators, E2F1, E2F2 and E2F3, are invariably depicted as the final components of a CDK/Rb signaling cascade that executes the transcriptional program necessary to commit cells to enter S phase. Unexpectedly, we find through analysis of Affymetrix expression array data that mature lens epithelial cells deficient for E2F1-3 fail to repress cell cycle-regulated genes (and other targets of E2F) and that this corresponds with subsequent apoptosis and cellular collapse in the lens. Murine lenses were collected at two stages of development for RNA extraction and hybridization on Affymetrix microarrays. Our aim was to determine key events that lead to cellular collapse of lenses triply deficient for E2F1, E2F2, and E2F3 in neonates.
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.
Project description:This SuperSeries is composed of the following subset Series: GSE22322: Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and their denucleation [lens tissue] GSE25168: Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and their denucleation [eyeball tissue] Refer to individual Series
Project description:Three types of BMs from adult human eyes, the inner limiting membrane, the retinal vascular BMs and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS.
Project description:Prolonged use of glucocorticoids can lead to the formation of a cataract, however the mechanism is not known. We recently reported the presence of the functional glucocorticoid receptor in immortalized cultured mammalian lens epithelial cells (Gupta & Wagner, Invest Ophthalmol Vis Sci 2003), but the short term biological effect of glucocorticoid action in lens epithelial cells is not known. This study seeks to examine glucocorticoid induced changes in global gene expression in LECs. RNA isolated from HLE B-3 cells, treated with Dex or Veh for 4 or 16 hours, was used to analyze global changes in gene expression by microarray hybridization. Microarray studies at 2 time periods demonstrate that glucocorticoids modulate gene expression in immortalized human LECs, reveal novel changes in gene expression, and confirms an endogenous genomic lens glucocorticoid response. Experiment Overall Design: HLE B-3 cells were treated with 1uM dexamethasone or vehicle for 4 or 16 hours in triplicate. Total RNA was converted to cRNA and hybridized to Affymetrix HUM_133A genechips. Images were acquired using the Affymetrix GeneArray scanner. Data was extracted using Affymetrix Microarray Suite 5.0.
Project description:Proteomes of lens nuclei from young (4 years old) and old (15-16 years old) rhesus macaques (Macaca mulatta) were analyzed to determine similarity of the proteomic profile to that of human lenses, age-related differences in protein solubility, and association of various posttranslational modifications with age and protein solubility. Lens core proteins were separated into water-soluble and water-insoluble fractions using aqueous buffer and centrifugation. The water-insoluble fraction was solubilized using SDS. Proteins were processed using S-trap columns and peptide digests were analyzed using high-resolution, label-free DDA proteomics. Open modification searches were performed using MSFragger to identify possible PTMs. The number of modified peptide tandem mass spectra confidently assigned to samples by age or solubility were compared to find PTMs with statistically significant count differences. The overall proteomic profile of rhesus macaque lenses was very similar to human lenses, consisting of 80.2% crystallins, 1.1% beaded filament proteins, and 18.7% other proteins. The crystallin fraction consisted of 27% alpha crystallins, 67.6% beta/gamma crystallins, and 5.4% taxon-specific psi crystallin. Glycolytic enzymes, beta/gamma crystallins, and a few glutathione-related enzymes were found to have age-related shifts to the water-insoluble fraction. There were significant differences in deamidation, dioxidation, carbamylation, carboxymethylation, and trioxidation based on age and/or solubility of proteins.