Membrane Potential Generation Research Articles

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2−1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

A semisynthetic borrelidin analogue BN-3b exerts potent antifungal activity against Candida albicans through ROS-mediated oxidative damage

In the process of investigating the antifungal structure-activity relationships (SAR) of borrelidin and discovering antifungal leads, a semisynthetic borrelidin analogue, BN-3b with antifungal activity against Candida albicans, was achieved. In this study, we found that oxidative damage induced by endogenous reactive oxygen species (ROS) plays an important role in the antifungal activity of BN-3b. Further investigation indicated that BN-3b stimulated ROS accumulation, increased malondialdehyde (MDA) levels, and decreased reduced/oxidized glutathione (GSH/GSSG) ratio. Moreover, BN-3b decreased mitochondrial membrane potential (MMP) and ATP generation. Ultrastructure analysis revealed that BN-3b severely damaged the cell membrane of C. albicans. Quantitative PCR (RT-qPCR) analysis revealed that virulence factors of C. albicans SAPs, PLB1, PLB2, HWP1, ALSs, and LIPs were all down-regulated after BN-3b exposure. We also found that BN-3b markedly inhibited the hyphal formation of C. albicans. In addition, in vivo studies revealed that BN-3b significantly prolonged survival and decreased fungal burden in mouse model of disseminated candidiasis.

Petroselinum sativum protects HepG2 cells from cytotoxicity and oxidative stress induced by hydrogen peroxide.

A number of liver diseases are known to be caused by oxidative stress. Petroselinum sativum (P. sativum; parsley) is popular for its anti-inflammatory, antimicrobial, anticancer, antioxidant and antidiabetic activities. However, till date the hepatoprotective potential of chloroform extract of P. sativum (PSA) on hydrogen peroxide (H2O2) induced cytotoxicity and oxidative stress in human liver (HepG2) cells have not been studied. Therefore, this study was framed to evaluate whether the levels of hydrogen peroxide (H2O2) induced cytotoxicity and oxidative stress in HepG2 cells could be diminished by pretreating the cells with PSA. MTT assay, NRU assay, morphological alterations, glutathione (GSH) depletion, lipid peroxidation (LPO), ROS generation and loss of mitochondrial membrane potential (MMP) were assessed by using non-cytotoxic concentrations (5, 10 and 25μg/mL) of PSA against H2O2 (0.25mM) induced damage in HepG2 cells. The results demonstrated that pretreatment of HepG2 cells with PSA offered protective properties by lowering the LPO and ROS generation and elevating the cell viability, GSH and MMP levels. Together, these results suggest that PSA has the hepatoprotective effect on H2O2 induced cell death in HepG2 cells.

Melatonin triggers the anticancer potential of phenylarsine oxide via induction of apoptosis through ROS generation and JNK activation

Melatonin, a safe endogenous hormone and a natural supplement, has recently been recognized to have antiproliferative effects and the ability to sensitize cells to other anticancer therapies. Phenylarsine oxide (PAO) has anticancer potential but it is considered as a toxic agent. In this study we combined melatonin to reduce the toxicity while securing the anti-cancer effects of PAO. Cell viability was determined by MTT assay, whereas cytotoxic assays were performed using an LDH cytotoxicity assay kit. Cell cycle analysis, Annexin V/PI staining, the mitochondrial membrane potential (MMP), mitochondrial calcium and reactive oxygen species (ROS) generation were analyzed using flow cytometry. Sytox stained cells were visualized by fluorescence microscopy and the expression of proteins was detected by western blotting. Melatonin increased the anticancer potential of PAO by decreasing the cell viability and increasing LDH release in various cancer cells. The mode of cell death was determined to be typical apoptosis, as evidenced by Annexin V/PI-stained cells, PARP cleavage, and caspase-3 activation, and with significant modulations in the expression of proapoptotic, antiapoptotic and cell cycle-related proteins. ROS generation played a critical role in induction of cell death by this combined treatment, which is validated by reversal of cytotoxicity upon cotreatment with NAC. Furthermore, the activation of MAPKs, especially JNK, contributed to the induction of cell death, accompanied by endoplasmic reticulum stress and autophagy, affirmed by the abrogation of cytotoxicity after JNK-IN-8 and TUDCA application. Melatonin showed promising potential as a chemotherapeutic agent in combination with PAO to achieve a better anticancer response.

Anthocyanins from Hibiscus syriacus L. Inhibit Oxidative Stress-Mediated Apoptosis by Activating the Nrf2/HO-1 Signaling Pathway.

Hibiscus syriacus L. is distributed widely throughout Eastern and Southern Asia and considered as the national flower of South Korea. The extraction of several plant parts of H. syriacus L. is currently used as a natural remedy for several diseases, including breast and lung cancer, microbial infection, and chronic inflammation. However, the effect of the anthocyanin extract of H. syriacus L. petals (PS) in oxidative stress conditions has not been studied. In this study, we evaluated the cytoprotective effect of PS against H2O2-induced oxidative stress in HaCaT keratinocytes. In this study, we found that PS significantly inhibited H2O2-induced apoptosis of HaCaT keratinocytes. We also revealed that PS mediated-cytoprotective effect was associated with the increased expression of heme oxygenase-1 (HO-1) arising from the activation of nuclear factor erythroid 2-related factor-2 (Nrf2). PS also decreased H2O2-induced excessive intracellular ROS generation and restored H2O2-induced mitochondrial depolarization through the downregulation of mitochondrial ROS production. Furthermore, H2O2-induced Bax and caspase-3 expression was markedly abolished in the presence of PS. The inhibition of HO-1 by zinc protoporphyrin significantly attenuated the cytoprotective effect of PS in H2O2-treated HaCaT keratinocytes along with ROS generation, indicating that HO-1 crucially affects PS-mediated cytoprotective properties. Collectively, our results suggested that, under H2O2-mediated oxidative stress conditions, PS sustained a normal level of mitochondrial membrane potential and ROS generation in HaCaT keratinocytes by activating the Nrf2/HO-1 axis, exerting cytoprotective effects against oxidative stress.

In vitro Antibacterial Activity of Isopropoxy Benzene Guanidine Against Multidrug-Resistant Enterococci.

BackgroundBacterial infections cause a serious public health crisis due to the emergence of resistance towards multiple conventional antibacterial drugs. In particular, multidrug-resistant (MDR) Enterococcus faecium which belongs to “ESKAPE” organisms is causing significant problems worldwide. Hence, there is an urgent need to find alternative therapies. Recently, substituted benzene guanidine compounds have been used as lead structures to discover new promising drugs in both synthetic and medicinal chemistry.PurposeHere we investigated the antimicrobial activity of a new substituted benzene guanidine analog, isopropoxy benzene guanidine, against Enterococci.Material and methodsThe isopropoxy benzene guanidine was synthesized by Guangzhou Insighter Biotechnology Co., Ltd and tested on both reference bacterial strain and 32 clinical MDR Enterococci strains. The in vitro antibacterial activity was evaluated by microdilution method and kill kinetic assays. The potential antibacterial mechanism was measured by fluorescence spectrometry using fluorescent membrane potential probe 3, 3-diethyloxacarbocyanine iodide (DiOC2 (3)).ResultsIsopropoxy benzene guanidine exhibited potent bactericidal activity against both reference strain and MDR Enterococci isolates. The minimum inhibitory concentration (MIC) range for isopropoxy benzene guanidine was 1–4 μg/mL. Minimum bactericidal concentration (MBC) was about 2-8-fold of its MIC values. Time-kill studies showed that isopropoxy benzene guanidine provided superior bactericidal effect against reference and MDR strains within 12 hrs at 2×MIC. Furthermore, isopropoxy benzene guanidine could cause a large reduction in the magnitude of the generated membrane potential compared to that of the untreated cells.ConclusionThe present study highlights the potent bactericidal activity of isopropoxy benzene guanidine on Enterococci by disrupting the cell membrane potential. These findings demonstrate that isopropoxy benzene guanidine may be a good chemical lead for further medicinal chemistry and pharmaceutical development and could be used as a therapeutic agent for infectious diseases caused by MDR Enterococci.

Erythrocyte plasma membrane potential: past and current methods for its measurement.

The plasma membrane functions both as a natural insulator and a diffusion barrier to the movement of ions. A wide variety of proteins transport and pump ions to generate concentration gradients that result in voltage differences, while ion channels allow ions to move across the membrane down those gradients. Plasma membrane potential is the difference in voltage between the inside and the outside of a biological cell, and it ranges from ~- 3 to ~- 90 mV. Most of the most significant discoveries in this field have been made in excitable cells, such as nerve and muscle cells. Nevertheless, special attention has been paid to some events controlled by changes in membrane potential in non-excitable cells. The origins of several blood disorders, for instance, are related to disturbances at the level of plasma membrane in erythrocytes, the structurally simplest red blood cells. The high simplicity of erythrocytes, in particular, made them perfect candidates for the electrophysiological studies that laid the foundations for understanding the generation, maintenance, and roles of membrane potential. This article summarizes the methodologies that have been used during the past decades to determine Δψ in red blood cells, from seminal microelectrodes, through the use of nuclear magnetic resonance or lipophilic radioactive ions to quantify intra and extracellular ions, to continuously renewed fluorescent potentiometric dyes. We have attempted to highlight the advantages and disadvantages of each methodology, as well as to provide a description of the technical aspects involved.

Cathelicidin Antimicrobial Peptide Expression Is Inversely Associated with Absolute Neutrophil Counts in Pediatric HCT Recipients

BACKGROUND: Neutropenia is a major risk factor for infectious complications in hematopoietic cell transplantation (HCT) recipients. However, much less is understood about neutrophil function in the posttransplant period. Cathelicidins are multifunctional host defense peptides. The sole human homolog, Cathelicidin Antimicrobial Peptide (CAMP, OMIM * 600474) is secreted by invading leukocytes, especially neutrophils and plays an important and pleiotropic role in host defense. It acts both as a direct bactericidal agent, but also affects the activity of many immune cells, including prolonging the lifespan of neutrophils. Another critical step in the microbicidal function of neutrophils is the generation of reactive oxygen species. OBJECTIVE: To characterize neutrophil function and CAMP expression over time in children following HCT. METHODS: A prospective study of children undergoing HCT was conducted. Blood samples were collected at 3 months, 6 mo., and 1 y post-HCT timepoints. White blood cells were purified using a PolymorphPrep gradient (Axis-Shield Diagnostics Ltd., Scotland). CAMP expression was measured using Clone H7 antibody (Santa Cruz Biotechnology Inc., USA) in fixed and permeabilized cells. Neutrophil activation was measured as reduction in membrane potential following stimulation with phorbol 12-myristate 13-acetate (PMA) using fluorescence staining with 3,3'-dipentyloxacarbocyanine iodide (DiOC5). Neutrophil hydrogen peroxide generation was measured by oxidation, and resultant increased staining of the probe 2',7' dichlorohydrofluorescein diacetate (DCFH-DA) following PMA stimulation. STATISTICS: Categorical variables were summarized by frequency and percent, and continuous variables were summarized by median and inter-quartile range (IQR). Distributions of continuous variables were examined and were log-transformed if skewed. Linear mixed-effects model was used to evaluate temporal trends and correlations between pairs of variables across time. points. The Bayesian Information Criterion was used to identify the optimal model that balanced model goodness-of-fit and complexity. RESULTS: Sixty-three patients were enrolled, median age 6.1 y (0.4 - 20.5 y). A majority of transplants (69%) were allogeneic. Patient and transplant characteristics are described in Table 1. CAMP was expressed in 95.8% of white blood cells (IQR 80.3 - 99.0%) (Figure 1). CAMP expression in neutrophils, measured as fold-increase above background staining, showed a median value of 474 (IQR 355 - 623). CAMP expression was inversely corelated with absolute neutrophil counts, with 37 folds increase (95% CI 11 to 63 folds, p = 0.006; Figure 2) for every 106 per milliliter reduction in neutrophil counts. Neutrophil activation measured as reduction in membrane potential was expressed as % decrease in DiOC5 staining following PMA stimulation with a median value of 41% (IQR 31 - 56%). Peroxide generation expressed as % increase in DCFH-DA staining following PMA stimulation showed a median value of 304% (IQR 170 - 630%). The reduction in membrane potential was correlated with peroxide generation. For every 1% reduction in membrane potential, hydrogen peroxide production increased by 2% (95% CI 1 to 3%, Figure 3). CAMP expression in neutrophils was not associated with neutrophil function measured by change in membrane potential or peroxide generation. None of the covariates examined (age, transplant type, and timepoints posttransplant) was found to be associated with CAMP expression or measures of neutrophil function. CONCLUSIONS: We have demonstrated that neutrophils, as characterized by changes in membrane potential and reactive oxygen species generation in response to PMA stimulation, are functional in children as early as 3 months following HCT. Of the parameters assessed in this study the increase in CAMP expression in neutrophils in patients with lower numbers of neutrophils in circulation after HCT stands out and this finding may potentially be of importance. Further studies will be necessary to determine if this is specific to HCT setting or if this can also be observed in normal individuals. It will also be important to study if this is reflected in the serum levels of the CAMP-derived active peptides. Disclosures No relevant conflicts of interest to declare.

Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect against proton leak through complex IV

Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we measured the ΔΨ generated by complex III (CIII) to discriminate between these possibilities. First, we found that the decreased ΔΨ observed in the absence of the HIGD1 proteins cannot be due to decreased proton pumping by CIV because CIII, operating alone, also exhibited a decreased ΔΨ when HIGD1 proteins were absent. Because CIII can neither lower its pumping stoichiometry nor transfer protons completely across the IMM, this result indicates that HIGD1 protein ablation enhances proton leak across the IMM. Second, we demonstrate that this proton leak occurs through CIV because ΔΨ generation by CIII is restored when CIV is removed from the cell. Third, the proton leak appeared to take place through an inactive population of CIV that accumulates when HIGD1 proteins are absent. We conclude that HIGD1 proteins in yeast prevent CIV inactivation, likely by preventing the loss of lipids bound within the Cox3 protein of CIV.

Cationic Oligopeptide-Functionalized Mitochondria Targeting Sequence Show Mitochondria Targeting and Anticancer Activity

Mitochondrial drug delivery systems require development of highly selective mitochondria-targeting carriers. In this study, we report that mitochondria targeting sequence (MTS)-hybrid cationic oligopeptide, MTS-H3R9, shows the dual role of a mitochondria targeting vector along with anticancer effect for cancer therapy. In cytotoxicity assays, MTS-H3R9 was shown to be more effective than MTS. MTS-H3R9 showed significant cell penetration and internalization activity compared to that of MTS along with more efficient escape from lysosome to the cytosol. We showed efficient targeting of MTS-H3R9 to mitochondria in HeLa cell line. Furthermore, we exhibited anticancer agent properties that mitochondrial-accumulated MTS-H3R9 caused cell death by reactive oxygen species generation and loss of mitochondrial membrane potential. MTS-H3R9 exhibited dramatically increased anticancer activity in 3D spheroids as well as in a 2D culture model. We demonstrated that MTS-H3R9 provides dual potentials both as a vehicle for targeted delivery and as a cancer treatment agent for therapeutic applications.

Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep

Genetic modification provides a means to enhancing disease resistance in animals. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. To compare semen characteristics including sperm quality, seminal plasma biochemical index, sperm DNA methylation and pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor 4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic sheep of the same breed. There were no significant differences between transgenic and control rams for all semen quality parameters, including semen volume, sperm concentration, sperm viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and viable sperm with high mitochondrial membrane potential in both F0 and F1 generation. Furthermore, no significant differences were found for seminal plasma concentrations of zinc, neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R methylation in sperm DNA. In addition, pregnancy rate was also similar between these two groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm quality, seminal plasma or sperm DNA of transgenic sheep.

Brain-derived neurotrophic factor mimetic, 7,8-dihydroxyflavone, protects against myocardial ischemia by rebalancing optic atrophy 1 processing

Brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) pathway is associated with ischemic heart diseases (IHD). 7,8-dihydroxyflavone (7,8-DHF), BDNF mimetic, is a potent agonist of TrkB. We aimed to investigate the effects and the underlying mechanisms of 7,8-DHF on cardiac ischemia. Myocardial ischemic mouse model was induced by ligation of left anterior descending coronary artery. 7,8-DHF (5 mg/kg) was administered intraperitoneally two days after ischemia for four weeks. Echocardiography, HE staining and transmission electron microscope were used to examine the function, histology and ultrastructure of the heart. H9c2 cells were treated with hydrogen peroxide (H2O2), 7,8-DHF or TrkB inhibitor ANA-12. The effects of 7,8-DHF on cell viability, mitochondrial membrane potential (MMP) and mitochondrial superoxide generation were examined. Furthermore, mitochondrial fission and protein expression of mitochondrial dynamics (Mfn2 [mitofusin 2], OPA1 [optic atrophy 1], Drp1 [dynamin-related protein 1] and Fis-1 [fission 1]) was detected by mitotracker green staining and western blot, respectively. 7,8-DHF attenuated cardiac dysfunction and cardiomyocyte abnormality of myocardial ischemic mice. Moreover, 7,8-DHF increased cell viability and reduced cell death accompanied by improving MMP, inhibiting mitochondrial superoxide and preventing excessive mitochondrial fission of H2O2-treated H9c2 cells. The cytoprotective effects of 7,8-DHF were antagonized by ANA-12. Mechanistically, 7,8-DHF repressed OMA1-dependent conversion of L-OPA1 into S-OPA1, which was abolished by Akt inhibitor. In conclusion, 7,8-DHF protects against cardiac ischemic injury by inhibiting the proteolytic cleavage of OPA1. These findings provide a novel pharmacological effect of 7,8-DHF on mitochondrial dynamics and a new potential target for IHD.

Neuroprotective and Antiapoptotic Effects of N-acetylcystein and Crocus sativus Aqueous Extract on Arsenic-induced Neurotoxicity in SH-SY5Y Human Dopaminergic Neuroblastoma Cells

Abstract: Background: Parkinson’s disease is mainly specified by progressive and selective death of dopaminergic neurons. Crocus sativus L. (saffron) has been widely used to cure a diverse range of diseases in herbal medicine. Aim: The aim of the present study is to investigate the neuroprotective effects of saffron on arsenic-induced neurotoxicity, which was performed in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease and to confirm whether the neuroprotective effects of saffron on Parkinson’s disease are mainly due to their interactions with antioxidant systems. Moreover, as the antioxidant effect of N-acetylcysteine and its effectiveness as an antioxidant agent are proven, we compared saffron with NAC to control saffron extract. Material and Methods: The induction of cell damage was done by arsenic and the survival of the cells was measured using the MTT assay. In addition, the assessment of the generation of intracellular Reactive Oxygen Species (ROS) and mitochondrial membrane potential was done using fluorescence spectrophotometry method. Furthermore, immunoblotting analysis was performed to precisely determine the biomarkers level for apoptosis in the cells. Results: Our study indicated that arsenic had the ability to decrease cells survival rate, enhance the loss of mitochondrial membrane potential and increase the levels of intracellular ROS, c-Fos ratio and caspease-3. Pretreatment of cells with NAC (5 mM) and saffron aqueous extract (10mg/ml) significantly attenuated the mentioned effects in arsenic-treated cells. Conclusion: The outcome of the study has shown that the protective effects of NAC and saffron aqueous extract are produced by their antioxidant and antiapoptotic properties and their therapeutic potential is demonstrated in the treatment of Parkinson’s disease. Key words: Parkinson Disease, N-acetylcysteine, Saffron, Arsenic, SH-SY5Y cells.

Bnip3 regulates airway smooth muscle cell focal adhesion and proliferation.

Increased airway smooth muscle (ASM) mass is a key contributor to airway narrowing and airway hyperresponsiveness in asthma. Besides conventional pathways and regulators of ASM proliferation, recent studies suggest that changes in mitochondrial morphology and function play a role in airway remodeling in asthma. In this study, we aimed at determining the role of mitochondrial Bcl-2 adenovirus E1B 19 kDa-interacting protein, Bnip3, in the regulation of ASM proliferation. Bnip3 is a member of the Bcl-2 family of proteins critical for mitochondrial health, mitophagy, and cell survival/death. We found that Bnip3 expression is upregulated in ASM cells from asthmatic donors compared with that in ASM cells from healthy donors and transient downregulation of Bnip3 expression in primary human ASM cells using an siRNA approach decreased cell adhesion, migration, and proliferation. Furthermore, Bnip3 downregulation altered the structure (electron density) and function (cellular ATP levels, membrane potential, and reacitve oxygen species generation) of mitochondria and decreased expression of cytoskeleton proteins vinculin, paxillin, and actinin. These findings suggest that Bnip3 via regulation of mitochondria functions and expression of adhesion proteins regulates ASM adhesion, migration, and proliferation. This study reveals a novel role for Bnip3 in ASM functions and establishes Bnip3 as a potential target in mitigating ASM remodeling in asthma.

MpsAB is important for Staphylococcus aureus virulence and growth at atmospheric CO2 levels

The mechanisms behind carbon dioxide (CO2) dependency in non-autotrophic bacterial isolates are unclear. Here we show that the Staphylococcus aureus mpsAB operon, known to play a role in membrane potential generation, is crucial for growth at atmospheric CO2 levels. The genes mpsAB can complement an Escherichia coli carbonic anhydrase (CA) mutant, and CA from E. coli can complement the S. aureus delta-mpsABC mutant. In comparison with the wild type, S. aureus mps mutants produce less hemolytic toxin and are less virulent in animal models of infection. Homologs of mpsA and mpsB are widespread among bacteria and are often found adjacent to each other on the genome. We propose that MpsAB represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, possibly acting as a sodium bicarbonate cotransporter.

Mitochondrial Uncoupling: A Key Controller of Biological Processes in Physiology and Diseases.

Mitochondrial uncoupling can be defined as a dissociation between mitochondrial membrane potential generation and its use for mitochondria-dependent ATP synthesis. Although this process was originally considered a mitochondrial dysfunction, the identification of UCP-1 as an endogenous physiological uncoupling protein suggests that the process could be involved in many other biological processes. In this review, we first compare the mitochondrial uncoupling agents available in term of mechanistic and non-specific effects. Proteins regulating mitochondrial uncoupling, as well as chemical compounds with uncoupling properties are discussed. Second, we summarize the most recent findings linking mitochondrial uncoupling and other cellular or biological processes, such as bulk and specific autophagy, reactive oxygen species production, protein secretion, cell death, physical exercise, metabolic adaptations in adipose tissue, and cell signaling. Finally, we show how mitochondrial uncoupling could be used to treat several human diseases, such as obesity, cardiovascular diseases, or neurological disorders.

Cypermethrin exposure reduces the ovarian reserve by causing mitochondrial dysfunction in granulosa cells

Cypermethrin (CYP) is one of the most highly effective synthetic pyrethroid insecticides and is recommended for insect control because it is considered to be relatively non-toxic to humans in all stages of life. However, recent data have shown that CYP has adverse effects on fertility, immune system, cardiovascular, hepatic metabolism and enzyme activity in vertebrates. Our objective was to investigate the toxicity of CYP to the ovary and to elucidate the underlying molecular mechanisms. Twenty 8-week-old CD-1 female mice were randomly assigned to four groups, that were separately exposed to CYP at doses of 12.5, 25 and 50 mg/kg/day or to corn oil (vehicle) for 28 days by intragastric administration. Moreover, human granulosa KGN cells were treated with CYP for 24 h at concentrations of 100 μM and 200 μM or to anhydrous ethanol (vehicle). This study clearly demonstrated that, compared to vehicle exposure, CYP exposure caused abnormal estrous cyclicity and decreased follicle numbers in all stages of mice with a dose-dependent manner. Interestingly, the apoptosis signals were mainly located in granulosa cells, and the expression levels of Caspase 3, Bax and Bcl-2 were increased in the 25 and 50 mg/kg/d groups. In KGN cells, CYP (100 and 200 μM) clearly induced apoptosis together with mitochondrial membrane potential depolarization and abnormal ROS generation compared to the control group. Altogether, these results suggest that CYP exposure reduced the ovarian reserve in mice by inducing apoptosis in granulosa cells via mitochondrial-related pathways.

Mitofusin 2 Is Essential for IP3-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes.

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes.Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates.Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions.Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP3R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways.

Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

Effects of Cigarette Smoke on TSPO-related Mitochondrial Processes.

The 18 kDa translocator protein (TSPO) is an initiator of the mitochondrial apoptosis cascade. Cigarette smoke (CS) exposure provokes alterations in TSPO expression as well as upregulation of its related functions such as mitochondrial membrane potential (ΔψM) and reactive oxygen species generation, which are associated with cell death. In the current study, H1299 lung cancer cell line exposed to CS for various time periods (30 mins, 60 mins and 120 mins) and TSPO expression and cell death processes were studied. CS exposure for 30 mins resulted in a non-significant increase in TSPO expression by 24% (p > 0.05 vs. control). CS exposure for 60 mins and 120 mins resulted in a significant increase by 43% (p < 0.05 vs. control) and by 47% (p < 0.01 vs. control), respectively. Furthermore, TSPO-related mitochondrial functions were upregulated at the 120 mins time point following CS exposure. TSPO expression is upregulated by CS, suggesting that TSPO plays a role in cell death processes induced by CS exposure. Alterations in TSPO-related cell death processes suggest that TSPO may be involved in the tissue damage caused by CS.