Higher Reactive Oxygen Species Content Research Articles

Effects of Long-Term Culture on Human Embryonic Stem Cell Aging

In recent years, human embryonic stem (hES) cells have become a promising cell source for regenerative medicine. Although hES cells have the ability for unlimited self-renewal, potential adverse effects of long-term cell culture upon hES cells must be investigated before therapeutic applications of hES cells can be realized. Here we investigated changes in molecular profiles associated with young (<60 passages) and old (>120 passages) cells of the H9 hES cell line as well as young (<85 passages) and old (>120 passages) cells of the PKU1 hES cell line. Our results show that morphology, stem cell markers, and telomerase activity do not differ significantly between young and old passage cells. Cells from both age groups were also shown to differentiate into derivatives of all 3 germ layers upon spontaneous differentiation in vitro. Interestingly, mitochondrial dysfunction was found to occur with prolonged culture. Old passage cells of both the H9 and PKU1 lines were characterized by higher mitochondrial membrane potential, larger mitochondrial morphology, and higher reactive oxygen species content than their younger counterparts. Teratomas derived from higher passage cells were also found to have an uneven preference for differentiation compared with tumors derived from younger cells. These findings suggest that prolonged culture of hES cells may negatively impact mitochondrial function and possibly affect long-term pluripotency.

Flow cytometric analysis of mitochondrial populations in HL-CMS systems of rice under H2O2 stress

Cytoplasmic male sterility (CMS) has often been associated with mitochondrial dysfunction. In this report, the heterogeneity of mitochondria was analyzed in both Honglian (HL) CMS (YtA) rice seedlings and those of its corresponding maintainers (YtB) by flow cytometry and staining with rhodamine-123 (Rh-123). Both lines revealed two distinct fluorescence populations: high fluorescence populations (HFP) and light fluorescence populations (LFP), and a somewhat lower LFP/HFP ratio was detected in conjunction with the higher reactive oxygen species (ROS) content in YtA. In addition, use of the specific effector hydrogen peroxide (H2O2) demonstrated a correlation between the LFP/HFP ratio and ROS levels in both lines. Higher ROS content caused a more swift decrease of F(0)F(1)-ATPase activity and ATP contents in YtA than those in YtB, which accompanied with an obvious decline of the LFP/HFP ratio in YtA. Furthermore, a mitochondrial genomic DNA smear was detected by pulsed field gel electrophoresis. Taken together, these results implied that HL-CMS line rice seedlings and those of its corresponding maintainer have different proportion of Rh-123 staining mitochondria populations, which may be accounted for by ROS contents on the basis of ATPase activity and ATP contents.

Revealing acupuncture meridian-like system by reactive oxygen species visualization

To investigate the reactive oxygen species (ROS) distribution in living animal tissues, two ROS indicators, dichlorofluorescin diacetate (DCFH-DA) and MitoSOX™ Red were applied to visualize ROS on the frontal interior abdominal wall of living SD-rats by tail vein injection and local smearing respectively. Revealed was an unexpected ROS distribution pattern. ROS were demonstrated in a few vertical fluorescent lines, which related to neither veins nor nerves but could be almost perfectly superimposable on a standard human acupuncture meridian network. The phenomenon that cells with high ROS content should be aligned in a regular manner is interesting as well as its resemblance to meridian system.

Fluorescent Approach to Quantitation of Reactive Oxygen Species in Mainstream Cigarette Smoke

A novel approach to monitoring of mainstream smoke reactive oxygen species (ROS) has been developed and applied to the quantitation of smoke oxidants. Redox-active fluorescent probe dihydrorhodamine 6G (DHR-6G) was selected as the molecular probe because it is sensitive to typical smoke ROS. The experimental system couples an automatic cigarette smoke machine fiber-optic fluorometer for real-time monitoring of the reaction progress between cigarette smoke and DHR-6G. Quantitation was achieved based on the amount of rhodamine 6G, which is the sole product from DHR-6G oxidation. With the optimization of the trapping efficiency, we detected 391 nmol of ROS/cigarette in the mainstream CS for a standard cigarette 2R4F under standard Federal Trade Commission smoking protocol. Applying this method, we quantified the ROS of selected cigarettes and found that the cigarettes made of burley tobacco have much ( approximately 10 times) higher ROS content in the smoke than that in the tobacco made of bright tobacco. The smokeless cigarette, Eclipse, has comparable ROS with cigarettes made of bright tobacco.