scholarly journals Preparation of mitochondria to measure superoxide flashes in angiosperm flowers

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6708
Author(s):  
Chulan Zhang ◽  
Fengshuo Sun ◽  
Biao Xiong ◽  
Zhixiang Zhang
Keyword(s):  
Reactive Oxygen Species ◽  
Arabidopsis Thaliana ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Complex Method ◽  
Ros Production ◽  
Oxygen Species ◽  
Magnolia Denudata ◽  
Good Coincidence

Background Mitochondria are the center of energy metabolism and the production of reactive oxygen species (ROS). ROS production results in a burst of “superoxide flashes”, which is always accompanied by depolarization of mitochondrial membrane potential. Superoxide flashes have only been studied in the model plant Arabidopsis thaliana using a complex method to isolate mitochondria. In this study, we present an efficient, easier method to isolate functional mitochondria from floral tissues to measure superoxide flashes. Method We used 0.5 g samples to isolate mitochondria within <1.5 h from flowers of two non-transgenic plants (Magnolia denudata and Nelumbo nucifera) to measure superoxide flashes. Superoxide flashes were visualized by the pH-insensitive indicator MitoSOX Red, while the mitochondrial membrane potential (ΔΨ m) was labelled with TMRM. Results Mitochondria isolated using our method showed a high respiration ratio. Our results indicate that the location of ROS and mitochondria was in a good coincidence. Increased ROS together with a higher frequency of superoxide flashes was found in mitochondria isolated from the flower pistil. Furthermore, a higher rate of depolarization of the ΔΨ m was observed in the pistil. Taken together, these results demonstrate that the frequency of superoxide flashes is closely related to depolarization of the ΔΨ m in petals and pistils of flowers.

Effect of Single Layer Centrifugation on reactive oxygen species and sperm mitochondrial membrane potential in cooled stallion semen

2017 ◽  
Vol 29 (5) ◽  
pp. 1039 ◽  
Author(s):  
J. M. Morrell ◽  
A. Lagerqvist ◽  
P. Humblot ◽  
A. Johannisson
Keyword(s):  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Single Layer ◽  
Ros Production ◽  
Oxygen Species ◽  
Species Production

Additional means are needed for evaluating the quality of stallion spermatozoa in semen doses for AI. Mitochondrial membrane potential (ΔΨm) has been linked to fertility in some species, but is rarely used in the evaluation of cooled stallion semen; metabolic activity may be associated with reactive oxygen species production (ROS). In the present study, ΔΨm and ROS production were measured in doses of cooled stallion semen. The effect of colloid centrifugation on these parameters was also investigated. In this case, colloid centrifugation involves centrifuging a sperm sample through a silane-coated silica colloid formulation to retrieve the most robust spermatozoa. High and low ΔΨm in cooled stallion semen varied between stallions and between ejaculates, but was not affected by single-layer centrifugation (SLC). The SLC-selected spermatozoa produced significantly less hydrogen peroxide than controls (P < 0.001), which could explain the increased longevity and retention of fertilising capacity seen in previous studies. For SLC samples, ΔΨm was positively associated with viable spermatozoa that were not producing reactive oxygen species (r = 0.49; P < 0.001) and negatively associated with ROS production (for superoxide: r = –0.4, P < 0.01; for hydrogen peroxide: r = –0.39, P < 0.05). There was no clear association between ΔΨm and ROS production in control samples.

scholarly journals Ultraviolet light-emitting diode irradiation induces reactive oxygen species production and mitochondrial membrane potential reduction in HL-60 cells

2021 ◽  
Vol 49 (5) ◽  
pp. 030006052110166
Author(s):  
Dong Xie ◽  
Yun-Long Li ◽  
Gui-Fen Wang ◽  
Jian Jiang ◽  
Li-Rong Sun
Keyword(s):  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Light Emitting Diode ◽  
Ros Production ◽  
Oxygen Species ◽  
Light Emitting ◽  
Uv Led ◽  
Led Irradiation

Objective Ultraviolet light-emitting diode (UV LED) irradiation at 280 nm has been confirmed to induce apoptosis in cultured HL-60 cells, but the underlying mechanisms remain unclear. This study aimed to investigate the effects of 280 nm UV LED irradiation on reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) in HL-60 cells. Methods HL-60 cells were irradiated with 0, 8, 15, or 30 J/m2 of 280 nm UV LED and incubated for 2 hours. The intracellular ROS levels were assessed using the fluorescent probe 2ʹ-7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA) and a fluorescence plate reader. MMP was determined by flow cytometry using 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazol-carbocyanine iodide (JC-1) staining. The apoptosis-related proteins Bax and Bcl-2 were evaluated by western blot. Results UV LED irradiation at 280 nm induced a dose-dependent increase in ROS production and loss of MMP, and it activated apoptosis at irradiation doses of 8 to 30 J/m2. These results were consistent with a previous apoptosis study from the authors’ group. Conclusion Enhanced ROS production and mitochondrial depolarization are two distinct but interacting events, and both are involved in UV LED-induced apoptosis in HL-60 cells.

Reserpine Induces Apoptosis and Cell Cycle Arrest in Hormone Independent Prostate Cancer Cells through Mitochondrial Membrane Potential Failure

2019 ◽  
Vol 18 (9) ◽  
pp. 1313-1322 ◽  
Author(s):  
Manjula Devi Ramamoorthy ◽  
Ashok Kumar ◽  
Mahesh Ayyavu ◽  
Kannan Narayanan Dhiraviam
Keyword(s):  
Prostate Cancer ◽  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Membrane Potential ◽  
Cancer Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Prostate Cancer Cells

Background: Reserpine, an indole alkaloid commonly used for hypertension, is found in the roots of Rauwolfia serpentina. Although the root extract has been used for the treatment of cancer, the molecular mechanism of its anti-cancer activity on hormonal independent prostate cancer remains elusive. Methods: we evaluated the cytotoxicity of reserpine and other indole alkaloids, yohimbine and ajmaline on Prostate Cancer cells (PC3) using MTT assay. We investigated the mechanism of apoptosis using a combination of techniques including acridine orange/ethidium bromide staining, high content imaging of Annexin V-FITC staining, flow cytometric quantification of the mitochondrial membrane potential and Reactive Oxygen Species (ROS) and cell cycle analysis. Results: Our results indicate that reserpine inhibits DNA synthesis by arresting the cells at the G2 phase and showed all standard sequential features of apoptosis including, destabilization of mitochondrial membrane potential, reduced production of reactive oxygen species and DNA ladder formation. Our in silico analysis further confirmed that indeed reserpine docks to the catalytic cleft of anti-apoptotic proteins substantiating our results. Conclusion: Collectively, our findings suggest that reserpine can be a novel therapeutic agent for the treatment of androgen-independent prostate cancer.

Supplementation of kaempferol to in vitro maturation medium regulates oxidative stress and enhances subsequent embryonic development in vitro

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 59-64
Author(s):  
Yuhan Zhao ◽  
Yongnan Xu ◽  
Yinghua Li ◽  
Qingguo Jin ◽  
Jingyu Sun ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Treated Group ◽  
Control Group ◽  
Oxygen Species

SummaryKaempferol (KAE) is one of the most common dietary flavonols possessing biological activities such as anticancer, anti-inflammatory and antioxidant effects. Although previous studies have reported the biological activity of KAE on a variety of cells, it is not clear whether KAE plays a similar role in oocyte and embryo in vitro culture systems. This study investigated the effect of KAE addition to in vitro maturation on the antioxidant capacity of embryos in porcine oocytes after parthenogenetic activation. The effects of kaempferol on oocyte quality in porcine oocytes were studied based on the expression of related genes, reactive oxygen species, glutathione and mitochondrial membrane potential as criteria. The rate of blastocyst formation was significantly higher in oocytes treated with 0.1 µm KAE than in control oocytes. The mRNA level of the apoptosis-related gene Caspase-3 was significantly lower in the blastocysts derived from KAE-treated oocytes than in the control group and the mRNA expression of the embryo development-related genes COX2 and SOX2 was significantly increased in the KAE-treated group compared with that in the control group. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased after KAE treatment. Mitochondrial membrane potential (ΔΨm) was increased and the activity of Caspase-3 was significantly decreased in the KAE-treated group compared with that in the control group. Taken together, these results suggested that KAE is beneficial for the improvement of embryo development by inhibiting oxidative stress in porcine oocytes.

scholarly journals Effects of Berberine on Cell Cycle, DNA, Reactive Oxygen Species, and Apoptosis in L929 Murine Fibroblast Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Manman Gu ◽  
Jing Xu ◽  
Chunyang Han ◽  
Youxi Kang ◽  
Tengfei Liu ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Cycle ◽  
Dna Damage ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Cell Apoptosis ◽  
Mitochondrial Membrane ◽  
Oxygen Species ◽  
Cycle Arrest ◽  
Murine Fibroblast

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines (TCM), exhibits a strong antimicrobial activity in the treatment of diarrhea. However, it causes human as well as animal toxicity from heavy dosage. The present study was conducted to investigate the cytotoxicity of berberine and its possible trigger mechanisms resulting in cell cycle arrest, DNA damage, ROS (reactive oxygen species) level, mitochondrial membrane potential change, and cell apoptosis in L929 murine fibroblast (L929) cells. The cells were culturedin vitroand treated with different concentrations of berberine for 24 h. The results showed that cell viability was significantly decreased in a subjected dose-dependent state; berberine concentrations were higher than 0.05 mg/mL. Berberine at a concentration above 0.1 mg/mL altered the morphology of L929 cells. Cells at G2/M phase were clear that the level of ROS and cell apoptosis rates increased in 0.1 mg/mL group. Each DNA damage indicator score (DIS) increased in groups where concentration of berberine was above 0.025 mg/mL. The mitochondrial membrane potential counteractive balance mechanics were significantly altered when concentrations of berberine were above 0.005 mg/mL. In all, the present study suggested that berberine at high dosage exhibited cytotoxicity on L929 which was related to resultant: cell cycle arrest; DNA damage; accumulation of intracellular ROS; reduction of mitochondrial membrane potential; and cell apoptosis.

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