Vår HoloMonitorfrälste forskare från Frankrike, Alain Géloën, ser ut att ha fått kläm på hur använda HoloMonitor på bästa sätt.Nu levererar han tillsammans med forskaren Emmanuelle Berger ånyo en studie baserad på den teknik HoloMonitor medger.Den tidigare studien handlade om vilken påverkan luftföroreningar har på människan. Läs den. Den nu aktuella studien är enligt mig både djupare som bredare.Djupare så tillvida att Alain och Emmanuelle tagit sig an att studera det kroppsegna ämnet Glutation,närmare bestämt reducerad Glutation (Reduced glutathione (GSH).Ämnet är en blandning av olika aminosyror:glycin,cystein och glutaminsyra.En antioxidant som har till uppgift att medverka till celluppbyggnad som att skydda kroppens celler från ex för tidig apoptos (celldöd) påverkad av div toxiska processer.Tidigare forskning i ämnet är knapp.
Abstract
Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of 7-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to transport of GSH out, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.
INTRODUCTION
Oxidative stress results from an imbalance between reactive oxygen species production (ROS) and the abilities of biological systems to eliminate them. In that context, reduced glutathione (GSH) is the central redox agent in most aerobic organisms and a fundamental player in the fine regulation of oxidative stress. GSH is involved in numerous vital functions such as detoxifying electrophiles, scavenging free radicals, maintaining the thiol status of proteins, providing cysteine, modulating DNA synthesis, microtubular-related processes, nitric oxide homeostasis, the activity of neuromodulators as well as immune functions. As a result, reduced GSH plays important roles in cell metabolism, differentiation, proliferation and apoptosis. Disturbances in GSH homeostasis are implicated in numerous human diseases, including cancer, pathologies associated to aging, cardiovascular, inflammatory, immune degenerative diseases and even viral infection.
Although GSH is present in the extracellular fluid, it is believed that it cannot be directly uptaken by cells. It is degraded by cells expressing the ectoprotein gammaglutamyl transpeptidase to produce dipeptides and free amino-acids uptaken by cells and used for de novo GSH synthesis. GSH is a tripeptide (γ-L-glutamyl-L-cysteinylglycine) synthesized by two cytosolic ATP dependent enzymes: glutamate cysteine ligase (GCL) leading to the production of γ-glutamyl-cysteine from glutamate and cysteine and glutathione synthetase (GS) involved in the ligation of γ-glutamyl-cysteine to glycine to produce γ-glutamylcysteinylglycine. After its synthesis, almost 90 % is retained into the cytoplasm (Meredith and Reed 1982, Hwang et al. 1992 in Lu 2009).
Despite the numerous effects described of reduced glutathione on cells, its influence on cell volume has never been reported so far. The present study relates experimental results pointing out the effect of reduced glutathione on cell volume on several cell lines, using different methods. Implications of such a new effect of reduced glutathione will be discussed in the light of recent data from the literature.
Material and Methods (urval)
3.2 Quantitative phase imaging
Quantitative phase imaging was performed using the Holomonitor M4 digital holographic cytometer (DHC) from Phase Holographic Imaging (PHI, Lund, Sweden). The microscope was housed in a standard 37°C cell culture incubator with 5% CO2. Cell area, optical thickness max and optical volume were measured in real-time every five minutes during ten hours after GSH addition.
Figure 2:
Morphological changes induced by glutathione in A549 cells are independent of toxicity. A: Contrast phase photomicrographs at times 0, 8h, 16h and 24h of a time-lapse in presence of 10 mM glutathione. No image of death cells is visible. Compare to time 0, cells reduced their size. Scale bar represents 100µM. B: Cell permeability to Zombie Red analyzed by fluorescence quantification in cytometry 2.5 hours after treatment, in a dose-dependent response to glutathione (mM). Data are presented as mean +/-fold changes Zombie Red fluorescence intensity in reduced GSH treated cells versus control media (n=4).
To further quantify cellular changes observed in response to glutathione we used the label-free single-cell analysis microscope Holomonitor (Phase Holographic Imaging, PHI, Lund, Sweden, Figure 3). The maximum optical thickness of cells significantly increased (Figure 3). Interestingly most of the increase occurs during the first 3 hours of glutathione exposure. As a consequence, cell volume increased markedly (Figure 3C).
Figure 3:
Real-time analysis of A549 cell size modifications induced by glutathione (10mM). Proliferative 459 cells were monitored on Holomonitor microscope during 10 hr (left panels). Maximum optical thickness (upper graph) and average optical volume (lower graph) were registered every 5 min. Data are presented as mean values +/-SEM (n=20 cells, 4 wells), at 8 h (left panel panels) with significant Student’s t-test p-values (p<0.05) in treated (GSH) versus controls (C).
DISCUSSION
The present results show a marked effect of reduced glutathione on cell volume. Following the addition of reduced GSH in the culture medium, the decrease in cell index is fast and marked reaching almost basal values, occurring within 2-3 hours (Figure 1). That effect is observed on different cell types, with differences in the amplitude of cell index decrease, suggesting that the effect of glutathione is not cell specific but likely widely present in all cell types (and not related to cancer cells). The differences in cell responses suggest that cell equipment and/or sites of glutathione action are slightly different in quantity or in nature from one cell type to another. The cell index decrease is so strong that it suggests that cells are dying or even dead. Several observations attest that cells do not die. First is the visual control by microscopy and maintenance of cell membrane integrity (Figure 2). Second, the decrease in cell index is reversible. Indeed, after longer time exposure, cell index slowly increases despite the presence of glutathione and cell number was not affected. After washing out, the cell index increased markedly without fully reaching the control values (not shown). Third, real-time analysis using the quantitative phase contrast microscope Holomonitor M4, confirmed that there is no cell death (result no shown).In conclusion the present study demonstrates that reduced glutathione has a major impact on cell volume of several cell lines. That rises the hypothesis that reduced glutathione produced by cells can exert a direct control on their glycocalix which affect cell adhesion and cell volume. Such an hypothesis adds a new function of glutathione and most likely will necessitate a re-evaluation of cell adhesion functions according to their redox-state.
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