4 research outputs found
Validation of proteome data.
<p>Based on the proteome data, two differentially regulated mitochondrial proteins (hsp60 and prohibitin 2) were selected for validation in an independent cohort of controls and patients. Raw proteome data reads (a) and quantitation (b) are shown together with (c) western blot analysis of the independent cohort. Samples were pooled from 5 individuals and run as duplicate lanes.</p
The mitochondrial protein enriched proteome of erythroid cells.
<p>Mitochondrial protein enriched preparations from day 10 erythroid cells from normal controls and β°-thalassemia/Hb E patients (mild and severe) were subjected to GelC-MS/MS analysis. Venn diagrams of (a) total proteins identified and (b) mitochondrial proteins as identified by the Mitoproteome database and GoCat ontological analysis of (c) cellular processes and (d) functional categorization of the identified mitochondrial proteins and GoCat ontological analysis of (e) cellular processes and (f) functional categorization of the identified differentially regulated proteins.</p
Mitochondrial Changes in β<sup>0</sup>-Thalassemia/Hb E Disease
<div><p>The compound β°-thalassemia/Hb E hemoglobinopathy is characterized by an unusually large range of presentation from essentially asymptomatic to a severe transfusion dependent state. While a number of factors are known that moderate presentation, these factors do not account for the full spectrum of presentation. Mitochondria are subcellular organelles that are pivotal in a number of cellular processes including oxidative phosphorylation and apoptosis. A mitochondrial protein enriched proteome was determined and validated from erythroblasts from normal controls and β°-thalassemia/Hb E patients of different severities. Mitochondria were evaluated through the use of mitotracker staining, analysis of relative mitochondrial genome number and evaluation of mitochondrial gene expression in addition to assay of overall cellular redox status through the use of alamarBlue assays. Fifty differentially regulated mitochondrial proteins were identified. Mitotracker staining revealed significant differences in staining between normal control erythroblasts and those from β°-thalassemia/Hb E patients. Differences in relative mitochondria number and gene expression were seen primarily in day 10 cells. Significant differences were seen in redox status as evaluated by alamarBlue staining in newly isolated CD34+ cells. Mitochondria mediate oxidative phosphorylation and apoptosis, both of which are known to be dysregulated in differentiating erythrocytes from β°-thalassemia/Hb E patients. The evidence presented here suggest that there are inherent differences in these cells as early as the erythroid progenitor cell stage, and that maximum deficit is seen coincident with high levels of globin gene expression.</p></div
Mitochondria in erythroid precursor cells: activity.
<p>Day 7, 10 and 14 erythroid precursor cells from normal controls (white bars) and from mild (grey bars) and severe (black bars) β°-thalassemia/Hb E patients were examined for expression of (a) ATP6, (b) ATP8 or (c) CYTB by real time PCR on days 7, 10 and 14 of culture. (d) A total of 1 x10<sup>4</sup> erythroid precursor cells from normal controls (white bars) and from mild (grey bars) and severe (black bars) β°-thalassemia/Hb E patients were used in alamarBlue assays. Error bars show ± S.E.M. * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001.</p