M. Rohan Fernando

Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependent and thioredoxin reductase-dependent peroxidase activities in vitro and in lens epithelial cells

Thioltransferase (or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. Mitochondrial Grx2 is a recent discovery, but its function is largely unknown. In this study we investigate Grx2 function by examining its potential peroxidase activity using lens epithelial cells (LEC). cDNA for human and mouse Grx2 was cloned into pET21d(+) vector and used to produce respective recombinant Grx2 for kinetic studies. cDNA for human Grx2 was transfected into human LEC and used for in vivo studies. Both human and mouse Grx2 showed glutathione (GSH)‐dependent and thioredoxin reductase (TR)‐dependent peroxidase activity. The catalytic efficiency of human and mouse Grx2 was lower than that of glutathione peroxidases (2.5 and 0.8×104 s−1M−1, respectively), but comparable with TR‐dependent peroxiredoxins (16.5 and 2.7×104 s−1M−1, respectively). TR‐dependent peroxidase activity increased 2‐fold in the transfected cells and was completely abolished by addition of anti‐Grx2 antibody (Ab). Flow cytometry (FACS) analysis and confocal microscopy revealed that cells preloaded with pure Grx2 detoxified peroxides more efficiently. Grx2 over‐expression protected cells against H2O2‐mediated disruption of mitochondrial transmembrane potential. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play a role in protecting the mitochondria from oxidative damage.—Fernando, M. R., Lechner, J. M., Löfgren, S., Gladyshev, V. N., Lou, M. F. Mitochondrial thioltransferase (glutaredoxin 2) has GSH‐dependent and thioredoxin reductase‐dependent peroxidase activities in vitro and in lens epithelial cells. FASEB J. 20, E2240–E2248 (2006)

A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device.

Cell‐free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K3EDTA tubes, when subjected to conditions that may occur during sample storage and shipping.

Effect of thioltransferase (glutaredoxin) deletion on cellular sensitivity to oxidative stress and cell proliferation in lens epithelial cells of thioltransferase knockout mouse.

PURPOSE To examine the physiological function of the thioltransferase (TTase)/glutathione (GSH) system in the lens using TTase knockout mouse (TTase(-/-)) lens epithelial cells (LECs) as a model. METHODS Primary LEC cultures were obtained from wild-type (TTase(+/+)) and TTase(-/-) mice. Characterization and validation of the cells were determined by immunoblotting for TTase and alpha-crystallin proteins and by immunohistochemistry for glutathionylated proteins. Cell proliferation was examined by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and BrdU analysis, and cell apoptosis after H(2)O(2) stress was assessed by fluorescence-activated cell sorter analysis. Reloading of TTase protein into the TTase(-/-) cells was achieved with reagent. RESULTS Primary LEC cultures obtained from wild-type (TTase(+/+)) and TTase(-/-) mice were characterized and found to contain lens-specific alpha-crystallin protein. Western blot analysis confirmed the absence of TTase protein in the TTase(-/-) cells and its presence in the wild-type cells. TTase(-/-) LECs had significantly lower levels of glutathione (GSH) and protein thiols with extensive elevation of glutathionylated proteins, and they exhibited less resistance to oxidative stress than did TTase(+/+) cells. These cells were less viable and more apoptotic, and they had a reduced ability to remove H(2)O(2) after challenge with low levels of H(2)O(2). Reloading of purified TTase into the TTase(-/-) cells restored the antioxidant function in TTase(-/-) cells to a near normal state. CONCLUSIONS These findings confirm the importance of TTase in regulating redox homeostasis and suggest a new physiological function in controlling cell proliferation in the lens epithelial cells.

Stabilization of cell-free RNA in blood samples using a new collection device

OBJECTIVE To investigate whether a new blood collection device stabilizes cell-free RNA (cfRNA) in blood post-phlebotomy when compared to collection using K(3)EDTA tubes. DESIGN AND METHODS Blood samples were drawn from healthy donors into K(3)EDTA tubes and Cell-Free RNA BCTs (BCTs) and stored at room temperature (20-25 °C). At specified time points (days 0-3), plasma was separated and cfRNA was extracted. Reverse transcription real-time PCR was used to quantify mRNA for c-fos, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for 18S rRNA. RESULTS Blood drawn into K(3)EDTA tubes showed a steady increase in RNA concentration over 3 days of ex vivo incubation. Blood drawn into BCTs showed no statistically significant change in RNA copy number except for GAPDH on day 3. CONCLUSIONS The novel chemical cocktail contained in the new device allows for the stabilization of cfRNA in blood samples at room temperature, which potentially enhances the clinical utility of cfRNA.

New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.

WITHDRAWN: Compatibility of a blood collection tube that stabilizes cell-free DNA with a rapid fluorescence assay.

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