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Oxidative stresss in the microenvironment surrounding lesions induces apoptosis of transplanted bone-marrow-derived mesenchymal stem cells (BMSCs). Hence, there is an urgent need for improving antioxidative-stress processes of transplanted BMSCs to further promote their survival. The present study reports the role and mechanism of Parkinson's disease protein 7 (PARK7) in enhancing antioxidative activity in BMSCs. We used a PARK7 lentivirus to transfect BMSCs to up- or downregulate PARK7, and then used H 2 O 2 to simulate oxidative stress in BMSCs in vitro. Overexpression of PARK7 effectively reduced reactive oxygen species and malondialdehyde, protected mitochondrial membrane potential, and resisted oxidative-stress-induced apoptosis of BMSCs, but the expression of PARK7 was downregulated, these results were reversed. At the same time, we also found that overexpression of PARK7 increased extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation and nuclear translocation, as well as upregulated Elk1 phosphorylation and superoxide dismutase (SOD) expression. In contrast, when U0126 was used to block the ERK1/2 pathway, ERK1/2 and Elk1 phosphorylation levels were downregulated, ERK1/2 nuclear translocation and SOD content were significantly reduced, and PARK7-overexperssion-induced antioxidative activity was completely blocked. Collectively, our results suggest that PARK7 overexpression increased antioxidative-stress processes and survival of BMSCs subjected to H 2 O 2 via activating the ERK1/2 signaling pathway. Our findings may guide the development of a PARK7-specific strategy for improving the transplantation efficacy of BMSCs., (© 2020 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals LLC.)

Cerebral ischemia-reperfusion (IR) injury is associated with mitochondrial damage. Macrophage-stimulating 1 (MST1) reportedly stimulates mitochondrial apoptosis by suppressing BCL-2. We investigated whether MST1 promotes the progression of cerebral IR injury by inducing mitochondrial dysfunction in vivo and in vitro. Western blot analysis, quantitative polymerase chain reaction, immunofluorescence, and mitochondrial function assays were conducted in cells from wild-type and Mst1-knockout mice subjected to cerebral IR injury. MST1 expression in wild-type glial cells increased following cerebral IR injury. Cerebral IR injury reduced the mitochondrial membrane potential and mitochondrial metabolism in glial cells, while it enhanced mitochondrial reactive oxygen species generation and mitochondrial calcium levels in these cells. The deletion of Mst1 attenuated cerebral IR injury by improving mitochondrial function and reducing mitochondrial damage. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was suppressed in wild-type glial cell upon cerebral IR injury but was reactivated in Mst1-knockout glial cell. Accordingly, blocking the MAPK/ERK pathway abolished the beneficial effects of Mst1 deletion during cerebral IR injury by inducing mitochondrial damage in glial cells. Our results suggest that cerebral IR injury is associated with MST1 upregulation in the brain, while the genetic ablation of Mst1 can attenuate mitochondrial damage and sustain brain function following cerebral IR injury., (© 2020 Wiley Periodicals, Inc.)

Oxidative stress is a major pathogenesis of some ocular surface diseases. Our previous study demonstrated that epidermal growth factor (EGF)-activated reactive oxygen species (ROS) could protect against human corneal epithelial cell (HCE) injury. In the present study, we aimed to explore the role and mechanisms of oxidative stress and mitochondrial autophagy in HCE cells subjected to scratch injury. CCK-8 assays, EdU assays, Western blot analysis, wound-healing assays, and flow cytometry were conducted to determine cell viability, proliferation, protein expression, cell apoptosis, and intracellular ROS levels, respectively. The results showed that EGF could promote damage repair and inhibit cell apoptosis in scratch injured HCE cells by upregulating ROS (**p < .01, ***p < .001). EGF also induced mitochondrial autophagy and alleviated mitochondrial damage. Interestingly, the combination of the mitochondrial autophagy inhibitor and mitochondrial division inhibitor 1 (MDIVI-1) with EGF could reduce cell proliferation, viability, and the ROS level (*p < .05, **p < .01, ***p < .001). Treatment using the ROS inhibitor N-acetyl- l-cysteine abrogated the increase in mitochondrial membrane potential after EGF treatment. (*p < .05). Taken together, these findings indicated that EGF plays an important role in HCE damage repair and could activate ROS to protect against HCE injury by inducing mitochondrial autophagy via activation of TRPM2., (© 2020 Wiley Periodicals, Inc.)

Increasing evidence suggests that mitochondrial dysfunction plays a critical role in the development of diabetic kidney disease (DKD), however, its specific pathomechanism remains unclear. A-kinase anchoring protein (AKAP) 1 is a scaffold protein in the AKAP family that is involved in mitochondrial fission and fusion. Here, we show that rats with streptozotocin (STZ)-induced diabetes developed podocyte damage accompanied by AKAP1 overexpression and that AKAP1 closely interacted with the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). At the molecular level, high glucose (HG) promoted podocyte injury and Drp1 phosphorylation at Ser637 as proven by decreased mitochondrial membrane potential, elevated reactive oxygen species generation, reduced adenosine triphosphate synthesis, and increased podocyte apoptosis. Furthermore, the AKAP1 knockdown protected HG-induced podocyte injury and suppressed HG-induced Drp1 phosphorylation at Ser637. AKAP1 overexpression aggravated HG-induced mitochondrial fragmentation and podocyte apoptosis. The coimmunoprecipitation assay showed that HG-induced Drp1 interacted with AKAP1, revealing that AKAP1 could recruit Drp1 from the cytoplasm under HG stimulation. Subsequently, we detected the effect of drp1 phosphorylation on Ser637 by transferring several different Drp1 mutants. We demonstrated that activated AKAP1 promoted Drp1 phosphorylation at Ser637, which promoted the transposition of Drp1 to the surface of the mitochondria and accounts for mitochondrial dysfunction events. These findings indicate that AKAP1 is the main pathogenic factor in the development and progression of HG-induced podocyte injury through the destruction of mitochondrial dynamic homeostasis by regulating Drp1 phosphorylation in human podocytes., (© 2020 Wiley Periodicals, Inc.)

Bone marrow mesenchymal stem cells (BMSC) can ameliorate ischemic injury of various tissues. However, the molecular mechanisms involved remain to be clarified. In this study, we intend to investigate the effects of BMSC-derived conditioned medium (BMSC-CM) on hypoxia/reoxygenation (H/R)-induced injury of H9c2 myocardial cells, and the potential mechanisms. Cell injury was determined through level of cell viability, lactate dehydrogenase (LDH) release, total intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and cell apoptosis. Autophagic activity of cells was detected through levels of the autophagy-associated proteins and autophagic flux. Results showed that BMSC-CM alleviated H/R-induced injury in H9c2 cells, as demonstrated by increased cell viability and Δψm, decreased ROS production, LDH release, and cell apoptosis. Furthermore, the H/R treatment induced a decrease in autophagic activity and an increase in Notch2 signaling activation in H9c2 cells. In the presence of BMSC-CM, the autophagic activity impaired by the H/R treatment was upregulated with decreased phosphorylation of mTOR, and the activation of Notch2 signaling was downregulated. These effects of BMSC-CM could be replicated by Notch signaling inhibitor. In contrast, inhibitors of cell autophagy including chloroquine (CQ) and 3-methyladenine, diminished the protective effects of BMSC-CM. Taken together results, our study showed that BMSC-CM could protect H9c2 cells from H/R-induced injury potentially through regulating Notch2/mTOR/autophagy signaling. These findings may provide a novel insight into the mechanisms of BMSC-CM in therapy of myocardial ischemia/reperfusion injury as well as other ischemic diseases., (© 2019 Wiley Periodicals, Inc.)

HIG2A promotes cell survival under hypoxia and mediates the assembly of complex III and complex IV into respiratory chain supercomplexes. In the present study, we show that human HIGD2A and mouse Higd2a gene expressions are regulated by hypoxia, glucose, and the cell cycle-related transcription factor E2F1. The latter was found to bind the promoter region of HIGD2A. Differential expression of the HIGD2A gene was found in C57BL/6 mice in relation to tissue and age. Besides, the silencing of HIGD2A evidenced the modulation of mitochondrial dynamics proteins namely, OPA1 as a fusion protein increases, while FIS1, a fission protein, decreases. Besides, the mitochondrial membrane potential (ΔΨm) increased. The protein HIG2A is localized in the mitochondria and nucleus. Moreover, we observed that the HIG2A protein interacts with OPA1. Changes in oxygen concentration, glucose availability, and cell cycle regulate HIGD2A expression. Alterations in HIGD2A expression are associated with changes in mitochondrial physiology., (© 2019 Wiley Periodicals, Inc.)

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d-aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment., (© 2018 Wiley Periodicals, Inc.)

Pre-diabetes is characterized by impaired glucose tolerance (IGT) and/or impaired fasting glucose. Impairment of skeletal muscle function is closely associated with the progression of diabetes. However, the entire pathological characteristics and mechanisms of pre-diabetes in skeletal muscle remain fully unknown. Here, we established a mouse model of pre-diabetes, in which 6-week-old male C57BL6/J mice were fed either normal diet or high-fat diet (HFD) for 8 or 16 weeks. Both non-fasting and fasting glucose levels and the results of glucose and insulin tolerance tests showed that mice fed an 8-week HFD developed pre-diabetes with IGT; whereas mice fed a 16-week HFD presented with impaired fasting glucose and impaired glucose tolerance (IFG-IGT). Mice at both stages of pre-diabetes displayed decreased numbers of mitochondria in skeletal muscle. Moreover, IFG-IGT mice exhibited decreased mitochondrial membrane potential and ATP production in skeletal muscle and muscle degeneration characterized by a shift in muscle fibers from predominantly oxidative type I to glycolytic type II. Western blotting and histological analysis confirmed that myoblast differentiation was only inhibited in IFG-IGT mice. For primary skeletal muscle satellite cells, inhibition of differentiation was observed in palmitic acid-induced insulin resistance model. Moreover, enhanced myoblast differentiation increased glucose uptake and insulin sensitivity. These findings indicate that pre-diabetes result in mitochondrial dysfunction and inhibition of myoblast differentiation in skeletal muscle. Therefore, interventions that enhance myoblast differentiation may improve insulin resistance of diabetes at the earlier stage., (© 2018 Wiley Periodicals, Inc.)

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC-1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC-1 and MitoTracker. Platelets from 22 canine PCs were stained with JC-1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC-1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis in the evaluation of stored canine PCs. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Fibroblast growth factor 2 (FGF2) is a multifunctional cell growth factor that regulates cell proliferation, differentiation, adhesion, migration, and apoptosis. FGF2 has multiple isoforms, including an 18-kDa low molecular weight isoform (lo-FGF2) and 22-, 23-, 24-, and 34-kDa high molecular weight isoforms (hi-FGF2). Hi-FGF2 overexpression induces chromatin compaction, which requires the mitochondria and leads to apoptosis. Complement component 1 Q subcomponent-binding protein (C1QBP) plays an important role in mitochondria-dependent apoptosis by regulating the opening of the mitochondrial permeability transition pore. However, the interaction between C1QBP and hi-FGF2 and its role in hi-FGF2-mediated apoptosis remain unclear. Here, we found that hi-FGF2 overexpression induced depolarization of the mitochondrial membrane, cytochrome c release into the cytosol, and a considerable increase in C1QBP messenger RNA and protein expression. Furthermore, coimmunoprecipitation results showed that the mitochondrial protein, C1QBP, interacts with hi-FGF2. C1QBP knockdown using small interfering RNA significantly decreased the localization of hi-FGF2 to the mitochondria and increased the rate of apoptosis. Our results highlight a novel mechanism underlying hi-FGF2-induced, mitochondria-driven cell death involving the direct interaction between hi-FGF2 and C1QBP and the upregulation of C1QBP expression., (© 2018 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.)

We have previously shown that protein kinase Cε (PKCε) is involved in mitochondrial dysfunction in renal proximal tubular cells (RPTC). This study examined mitochondrial targets of active PKCε in RPTC injured by the model oxidant tert-butyl hydroperoxide (TBHP). TBHP exposure augmented the levels of phosphorylated (active) PKCε in mitochondria, which suggested translocation of PKCε to mitochondria after oxidant exposure. Oxidant injury decreased state 3 respiration, adenosine triphosphate (ATP) production, ATP content, and complex I activity. Further, TBHP exposure increased ΔΨ m and production of reactive oxygen species (ROS), and induced mitochondrial fragmentation and RPTC death. PKCε activation by overexpressing constitutively active PKCε exacerbated decreases in state 3 respiration, complex I activity, ATP content, and augmented RPTC death. In contrast, inhibition of PKCε by overexpressing dnPKCε mutant restored state 3 respiration, respiratory control ratio, complex I activity, ΔΨ m , and ATP production and content, but did not prevent decreases in F 0 F 1 -ATPase activity. Inhibition of PKCε prevented oxidant-induced production of ROS and mitochondrial fragmentation, and reduced RPTC death. We conclude that activation of PKCε mediates: (a) oxidant-induced changes in ΔΨ m , decreases in mitochondrial respiration, complex I activity, and ATP content; (b) mitochondrial fragmentation; and (c) RPTC death. In contrast, oxidant-induced inhibition of F 0 F 1 -ATPase activity is not mediated by PKCε. These results show that, in contrast to the protective effects of PKCε in the heart, PKCε activation is detrimental to mitochondrial function and viability in RPTC and mediates oxidant-induced injury., (© 2018 Wiley Periodicals, Inc.)

Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro-RNA (miRNA)-based mechanisms. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n = 7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome-c oxidase (COX)-IV activity. Oxidative stress was assessed by Mito-SOX and dihydroethidium staining. Next-generation sequencing (RNA-seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). Compared to Lean-MSCs, mitochondria from Obese-MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX-IV activity decreased in Obese-MSCs, associated with increased mitochondrial oxidative stress. RNA-seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese-MSCs (fold change >2, p < 0.05) and found to target 43 specific mitochondrial genes. Obesity impairs MSC mitochondrial structure and function, possibly mediated partly through miRNA-induced mitochondrial gene regulation, leading to increased oxidative stress. Importantly, these alterations may limit the therapeutic use of autologous MSCs in subjects with obesity., (© 2017 Wiley Periodicals, Inc.)

Mitochondrion is the major cellular energy producing organelle that is at the boundary between chemical reactions and physical processes. Although mitochondria have been shown to be affected by physical methods such as nonthermal plasma, whether static magnetic field (SMF) could also affect them is still unclear. Here we used rat adrenal PC12 cells to compare SMFs of different intensities for their effects on ATP (adenosine-5'-triphosphate), the major energy source produced by mitochondria, which is essential for various cellular processes. Our results show that although 0.26 or 0.50 T SMFs did not affect ATP, 1 T and 9 T SMFs affected ATP level differently and time-dependently. Moreover, SMF-induced ATP level fluctuations are correlated with mitochondrial membrane potential changes. Our study provides insights not only into understanding various cellular effects of SMFs, but also the potential clinical applications of SMFs. Bioelectromagnetics. 39:352-360, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Objective: Defective mitochondrial function attributed to optic atrophy 1 (OPA1) mutations causes primarily optic atrophy and, less commonly, neurodegenerative syndromes. The pathomechanism by which OPA1 mutations trigger diffuse loss of neurons in some, but not all, patients is unknown. Here, we used a tractable induced pluripotent stem cell (iPSC)-based model to capture the biology of OPA1 haploinsufficiency in cases presenting with classic eye disease versus syndromic parkinsonism., Methods: iPSCs were generated from 2 patients with OPA1 haploinsufficiency and 2 controls and differentiated into dopaminergic neurons. Metabolic profile was determined by extracellular flux analysis, respiratory complex levels using immunoblotting, and complex I activity by a colorimetric assay. Mitochondria were examined by transmission electron microscopy. Mitochondrial DNA copy number and deletions were assayed using long-range PCR. Mitochondrial membrane potential was measured by tetramethylrhodamine methyl ester uptake, and mitochondrial fragmentation was assessed by confocal microscopy. Exome sequencing was used to screen for pathogenic variants., Results: OPA1 haploinsufficient iPSCs differentiated into dopaminergic neurons and exhibited marked reduction in OPA1 protein levels. Loss of OPA1 caused a late defect in oxidative phosphorylation, reduced complex I levels, and activity without a significant change in the ultrastructure of mitochondria. Loss of neurons in culture recapitulated dopaminergic degeneration in syndromic disease and correlated with mitochondrial fragmentation., Interpretation: OPA1 levels maintain oxidative phosphorylation in iPSC-derived neurons, at least in part, by regulating the stability of complex I. Severity of OPA1 disease associates primarily with the extent of OPA1-mediated fusion, suggesting that activation of this mechanism or identification of its genetic modifiers may have therapeutic or prognostic value. Ann Neurol 2018;83:915-925., (© 2018 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green ® (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red ® (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future. © 2017 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

B cell leukemia/lymphoma-2 (Bcl-2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl-2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)-induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration-dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ-H2AX, and production of the DNA damage-responsive enzyme poly(ADP-ribose)polymerase-1 (PARP-1). Exposure of TK6 cells to HQ leads to an increase in Bcl-2 and co-localization with PARP-1 in the cytoplasm. Inhibition of Bcl-2 using the BH3 mimetic, ABT-737, suppressed the PARP-1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ-induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT-737 combined with HQ increased the levels of cleaved PARP and γ-H2AX, but significantly decreased the level of P53. Thus, ABT-737 can influence PARP-1 translocation and induce apoptosis via mitochondria-mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP-1 attenuated the HQ-induced production of cleaved PARP and P53. These results identify Bcl-2 as a protective mediator of HQ-induced apoptosis and show that upregulation of Bcl-2 helps to localize PARP-1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49-59, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Background: The limited treatment option for recurrent prostate cancer and eventual resistant to conventional chemotherapy drugs has fueled continued interest in finding new anti-neoplastic agents. WZ35, a chemical analog of curcumin, had been demonstrated to have high chemical stability and potential anticancer effects in gastric cancer cells. The present study aimed to investigate the anti-prostate cancer effects of WZ35 in vitro and in vivo as well as the underlying mechanism., Methods: Two prostate cancer cell lines RM-1 and DU145 were utilized to test the anti-cancer effects of WZ35 and the underlying mechanism. MTT assay was used to assess the cytotoxic effect of WZ35. Cell cycle distribution, apoptosis, alteration of ROS, and [Ca 2+ ] i level were evaluated using flow cytometry. Western blotting assay was applied to measure the levels of proteins associated with apoptosis and cell cycle. Immunofluorescence staining and Electron micrographs were used to evaluate activation of mitochondrial apoptosis pathway. Tumor models in nude mice were induced by injection of RM-1 prostate cancer cells to test the in vivo anticancer action of WZ35., Results: Our results showed that WZ35 treatment induced loss of cell viability, cell apoptosis, and G2/M cycle arrest in both RM-1 and DU145 cells, coupled with ROS overproduction, intracellular calcium surge, and activation of mitochondrial apoptosis pathway in RM-1 cells. Interestingly, all above changes induced by WZ35 were completely reversed by ROS blockage. In addition, prevention of [Ca 2+ ] i elevation by BAPTA/AM also inhibited activation of mitochondrial apoptosis pathway induced by WZ35. In vivo studies, WZ35 treatment significantly inhibited RM-1 homograft tumor growth along with increased ROS accumulation, mitochondrial disruption, and cell apoptosis in tumor tissues., Conclusions: In conclusion, this work provides a novel anticancer candidate for the treatment of prostate cancer and demonstrated that increased ROS mediate the anti-cancer effects of WZ35 via activating mitochondrial apoptosis pathway. Importantly, this work also reveals that targeting ROS generation might be an effective strategy in human androgen-resistant prostate cancer treatment. Prostate 77:489-504, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Energy metabolism could influence amyotrophic lateral sclerosis (ALS) and progressive lateral sclerosis (PLS) pathogenesis and the response to therapy. We developed a novel assay to simultaneously assess mitochondrial content and membrane potential in patients' skin fibroblasts. In ALS and PLS fibroblasts, membrane potential was increased and mitochondrial content decreased, relative to healthy controls. In ALS higher mitochondrial membrane potential correlated with age at diagnosis, and in PLS it correlated with disease severity. These unprecedented findings in ALS and PLS fibroblasts could shed new light onto disease pathogenesis and help in developing biomarkers to predict disease evolution and the individual response to therapy in motor neuron diseases., (© 2014 American Neurological Association.)

Inactivated Sendai virus (HVJ-E) directly kills cancer cells by inducing apoptosis through a mechanism mediated by Janus kinases/signal transducers and activators of transcription (JAK/STAT) signaling pathways. However, whether other signaling pathways are involved remain largely unknown. This study aimed to investigate the mechanism underlying HVJ-E-induced apoptosis in murine B16F10 melanoma cells. We found that HVJ-E induced B16F10 cell apoptosis via the caspase pathway, particularly caspase-9, which mediates the intrinsic apoptotic pathway. Mitogen-activated protein kinase (MAPK) pathway activation also contributed to HVJ-E-induced apoptosis. Whereas caspase pathway involvement depended on both IFN-β promoter stimulator-1 (IPS-1) and type I interferon (IFN), MAPK pathway activation was independent of type I IFN but involved IPS-1. In addition, intratumoral HVJ-E treatment displayed a direct oncolytic effect in an in vivo BALB/c nude mouse melanoma model. Collectively, our data provides new insights into the mechanism underlying HVJ-E-induced apoptosis in tumor cells., (© 2013 UICC.)

We earlier documented that lovastatin (LOV)-mediated inhibition of small Rho GTPases activity protects vulnerable oligodendrocytes (OLs) in mixed glial cell cultures stimulated with Th1 cytokines and in a murine model of multiple sclerosis (MS). However, the precise mechanism of OL protection remains unclear. We here employed genetic and biochemical approaches to elucidate the underlying mechanism that protects LOV treated OLs from Th1 (tumor necrosis factor-α) and Th17 (interleukin-17) cytokines toxicity in in vitro. Cytokines enhanced the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization with corresponding lowering of glutathione (reduced) level in OLs and that were reverted by LOV. In addition, the expression of ROS detoxifying enzymes (catalase and superoxide-dismutase 2) and the transactivation of peroxisome proliferators-activated receptor (PPAR)-α/-β/-γ including PPAR-γ coactivator-1α were enhanced by LOV in similarly treated OLs. Interestingly, LOV-mediated inhibition of small Rho GTPases, i.e., RhoA and cdc42, and Rho-associated kinase (ROCK) activity enhanced the levels of PPAR ligands in OLs via extracellular signal regulated kinase (1/2)/p38 mitogen-activated protein kinase/cytoplasmic phospholipase 2/cyclooxygenase-2 signaling cascade activation. Small hairpin RNA transfection-based studies established that LOV mainly enhances PPAR-α and less so of PPAR-β and PPAR-γ transactivation that enhances ROS detoxifying defense in OLs. In support of this, the observed LOV-mediated protection was lacking in PPAR-α-deficient OLs exposed to cytokines. Collectively, these data provide unprecedented evidence that LOV-mediated inhibition of the Rho-ROCK signaling pathway boosts ROS detoxifying defense in OLs via PPAR-α-dependent mechanism that has implication in neurodegenerative disorders including MS., (Copyright © 2013 Wiley Periodicals, Inc.)

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca(2+)](i), loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia-like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia-induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O(2) (reperfusion-like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high-affinity Zn(2+) chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high-affinity Zn(2+) probe, we showed that ischemia induced an intracellular Zn(2+) rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia-induced oligodendrocyte death. Moreover, UO126 blocked the ischemia-induced increase in poly-[ADP]-ribosylation of proteins, and the poly[ADP]-ribose polymerase 1 (PARP-1) inhibitor DPQ significantly inhibited ischemia-induced oligodendroglial cell death-demonstrating that PARP-1 was required downstream in the Zn(2+)-ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn(2+), blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia-reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Increasing evidence suggests that mitochondria play a central role in regulating cell apoptosis. Survivin, an inhibitor of apoptosis protein (IAP) family member, mediates resistance to cancer chemotherapy particularly in prostate cancers. Therefore, development of anticancer agents targeting mitochondria and survivin is a potential strategy., Method: Cell proliferation was examined by sulforhodamine B, CFSE staining, and clonogenic assays. Mitochondrial membrane potential (ΔΨ(m) ) and reactive oxygen species (ROS) were detected by flow cytometric analysis. Protein expression was detected by Western blot. RNA levels were examined by reverse transcription polymerase chain reaction assay. Overexpression of constitutively active Akt was also used in this study., Results: Ardisianone, a natural benzoquinone derivative, displayed anti-proliferative and apoptotic activities against human hormone-refractory prostate cancer cells (HRPC), PC-3, and DU-145. Ardisianone dramatically induced mitochondrial damage, identified by downregulation of Bcl-2 family proteins, ROS production, and loss of ΔΨ(m) . Ardisianone also inhibited Akt and mTOR/p70S6K pathways and induced a fast downregulation of survivin, leading to activation of mitochondria-involved caspase cascades. Overexpression of constitutively active Akt partly rescued ardisianone-mediated apoptotic signaling cascades. Furthermore, a long-term treatment of ardisianone caused an increase of endoplasmic reticulum (ER) stress, upregulation of cIAP1 and cIAP2, and apoptosis-inducing factor (AIF)-mediated caspase-independent apoptosis., Conclusions: The data suggest that the ardisianone induces apoptosis in human prostate cancers through mitochondrial damage stress, leading to the inhibition of mTOR/p70S6K pathway, downregulation of Bcl-2 family members, degradation of survivin, and activation of caspase cascades. The data provide evidence supporting that ardisianone is a potential anticancer agent against HRPCs., (Copyright © 2012 Wiley Periodicals, Inc.)

In the present study, we explored the possible links between Junctophilin 2 (Jp2) and the mitochondrium-sarcoplasmic reticulum (SR) interaction in embryonic stem cell-derived cardiomyocytes (ESC-CMs), as well as the role of Jp2 in cardiogenesis of ES cells. We found that Ca(2+) transient was abnormal and mitochondria were de-energized within siJp2 ESC-CMs. The essential juxtaposition structure of mitochondrium with SR was destroyed accompanied by selectively downregulation of Pgc-1α, Nrf-1, and Mfn-2. Impaired co-localization of the JP2 and sarcomeres (α-Actinin or Troponin-T) appeared in embryoid bodies (EBs) after Jp2 knockdown. Calsequestrin2 and ryanodine receptor 2 within SR were expressed as early as the initiation of differentiation, while triadin and caveolin3 within t-tubules (TTs) did not appear until the terminal, indicating that JP2 probably did not contribute to anchoring the SR to TTs at the early cardiogenesis stage as usual. In addition, Jp2 knockdown selectively decreased gene transcription toward cardiogenesis (Brachyury, Isl1, and Nkx2.5), subsequently weaken EB beating activity by 60%. Taken together, reducing JP2 expression in ESC-CMs resulted in impaired mitochondrial status due to either abnormal cellular Ca(2+) homeostasis or disturbing of juxtaposition. A sensitive time window of JP2 necessary in cardiac differentiation was found at early stage via an extra non-TTs/SR anchor-dependent role., (Copyright © 2012 Wiley Periodicals, Inc.)

Nanosecond, high-voltage electric pulses (nsEP) induce permeabilization of the plasma membrane and the membranes of cell organelles, leading to various responses in cells including cytochrome c release from mitochondria and caspase activation associated with apoptosis. We report here evidence for nsEP-induced permeabilization of mitochondrial membranes in living cells. Using three different methods with fluorescence indicators-rhodamine 123 (R123), tetramethyl rhodamine ethyl ester (TMRE), and cobalt-quenched calcein-we have shown that multiple nsEP (five pulses or more, 4 ns duration, 10 MV/m, 1 kHz repetition rate) cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. These effects could be a consequence of nsEP permeabilization of the inner mitochondrial membrane or the activation of mitochondrial membrane permeability transition pores. Plasma membrane permeabilization (YO-PRO-1 influx) was detected in addition to mitochondrial membrane permeabilization., (© 2011 Wiley Periodicals, Inc.)

Cell phenotyping and cell cycle analysis are two commonly used assays in both clinical diagnosis and biomedical research. Cell phenotyping by identifying different biomarkers is essential for the diagnosis of hematologic malignancy, sub-classifying diseases, monitoring response to treatment, predicting prognosis, detecting rare cell populations and residual malignant cells. Cell cycle analysis distinguishes cells in different phases of cell cycle and is often used to determine the cellular response to drugs and biological stimulations. These assays have been traditionally carried out by sensitive fluorescence detection methods such as flow cytometry and laser scanning cytometry for fluorescence-based cell population analysis. However, these instruments remain relatively expensive, large in size, and require a considerable amount of maintenance, which may not be feasible for smaller research groups that do not have access to these equipments or field clinics that require quick diagnostic results on site. Recently, a small portable imaging cytometry system (Cellometer Vision) has been developed by Nexcelom Bioscience LLC (Lawrence, MA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Here we report new applications of the Cellometer imaging cytometry for fluorescence-based cell population analysis and compared them with conventional flow cytometry. Cell population analysis assays such as immunophenotyping, cell cycle, and mitochondrial membrane potential detection methods have not yet been reported for the Cellometer Vision system. Using this imaging cytometry method for fluorescence-based assays that are typically done by flow cytometry offers a quick, simple, and inexpensive alternative method for biomedical research, which may be beneficial for smaller research laboratories and clinics., (Copyright © 2011 International Society for Advancement of Cytometry.)

Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders., (© 2010 Wiley-Liss, Inc.)

Endotoxemia by bacterial lipopolysaccharide (LPS) has been reported to affect gut motility specifically depending on Toll-like receptor 4 activation (TLR4). However, the direct impact of LPS ligation to TLR4 on human smooth muscle cells (HSMC) activity still remains to be elucidated. The present study shows that TLR4, its associated molecule MD2, and TLR2 are constitutively expressed on cultured HSMC and that, once activated, they impair HSMC function. The stimulation of TLR4 by LPS induced a time- and dose-dependent contractile dysfunction, which was associated with a decrease of TLR2 messenger, a rearrangement of microfilament cytoskeleton and an oxidative imbalance, i.e., the formation of reactive oxygen species (ROS) together with the depletion of GSH content. An alteration of mitochondria, namely a hyperpolarization of their membrane potential, was also detected. Most of these effects were partially prevented by the NADPH oxidase inhibitor apocynin or the NFkappaB inhibitor MG132. Finally, a 24 h washout in LPS-free medium almost completely restored morphofunctional and biochemical HSMC resting parameters, even if GSH levels remained significantly lower and no recovery was observed in TLR2 expression. Thus, the exposure to bacterial endotoxin directly and persistently impaired gastrointestinal smooth muscle activity indicating that HSMC actively participate to dysmotility during infective burst. The knowledge of these interactions might provide novel information on the pathogenesis of infection-associated gut dysmotility and further clues for the development of new therapeutic strategies.

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4-butanediyl-bismethanethiosulfonate (BMTS)-induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase-9 and caspase-3, and downregulation of Bcl-2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti-oxidant N-acetyl cysteine (NAC) inhibited BMTS-mediated caspase-9 and -3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox-linked apoptosis induced by sulfhydryl-reactive chemicals.

Nontoxic concentrations of peroxynitrite (ONOO(-)) nevertheless commit rat astrocytes to mitochondrial permeability transition-dependent toxicity, however prevented by a signaling response driven by arachidonic acid (ARA). The lipid messenger was released upon ONOO(-)-dependent activation of cytosolic phospholipase A(2) and its pharmacological inhibition, or knock-down, was invariably associated with a prompt apoptotic response sensitive to exogenous ARA, but insensitive to other polyunsaturated fatty acids, as eicosapentaenoic or linoleic acid. Interestingly, while microglia also used ARA to cope with ONOO(-), cerebellar granule cells were killed by the same concentrations of ONOO(-) employed in astrocyte/microglia experiments via a mechanism sensitive to inhibition of ARA release. These results collectively support the notion that resistance of glial cells to ONOO(-), a species extensively produced under neuroinflammatory conditions, is largely based on a critical survival signaling triggered by the inflammatory product ARA. In remarkable contrast with these results, the lipid messenger appears to mediate toxicity in neuronal cells., ((c) 2009 Wiley-Liss, Inc.)

The present study was designed to investigate the effect of vitrification on mitochondrial distribution, membrane potential (Deltapsi) and microtubule distribution in mouse 2-PN embryos, as well as to document the relationship between mitochondrial distribution and developmental ability of those embryos. Mitochondrial distribution was examined by fluorescence microscopy technology. Results indicated that: (1) The rate of mitochondrial ring formation around pronuclei in vitrified 2-PN embryos was significantly lower than in fresh ones (67.3 +/- 3.0% vs. 84.9 +/- 3.1%) (P < 0.05). (2) Blastocyst development rate of vitrified 2-PN embryos without mitochondrial rings (61.7 +/- 4.5%) was significantly lower than that of vitrified embryos with mitochondrial rings (82.1 +/- 2.8%). (3) Following staining by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbo-cyanine iodide (JC-1), most red-colored mitochondria (high Deltapsi) were distributed peripherally around pronuclei and along cell membranes of fresh 2-PN embryos. Conversely, red-colored mitochondria were greatly diminished in vitrified embryos, with green mitochondria (low Deltapsi) evenly distributed throughout the cytoplasm. The proportion of fresh 2-PN embryos with obvious aggregation of high Deltapsi mitochondria (84.2 +/- 2.2%) was significantly higher than that of vitrified embryos (26.7 +/- 3.0%) (P < 0.05). (4) The proportion of fresh embryos with microtubules distributed around pronuclei (83.5 +/- 3.4%) was similar to that of vitrified embryos (74.7 +/- 2.5%). In conclusion, vitrification affected mitochondrial distribution and decreased the mitochondrial membrane potential in mouse 2-PN embryos, events which may affect subsequent developmental viability of such embryos.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease affecting vascular smooth muscle cells of nearly all tissues. Clinical manifestations mainly concern the central nervous system with repeated TIA/stroke, migraine, psychiatric disturbances, and cognitive decline. Minor findings have been reported in muscle, nerve, and skin. CADASIL is due to NOTCH3 gene mutations. This gene has been identified as an up-regulator of c-FLIP, an inhibitor of Fas-ligand-induced apoptosis. The aim of this study was to assess the involvement of oxidative stress-induced apoptosis in cells from 16 Italian CADASIL patients. Peripheral blood lymphocytes (PBLs) and fibroblasts from CADASIL patients were exposed to 2-deoxy-D-ribose (dRib), which induces apoptosis by oxidative stress. Apoptosis was analyzed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy for caspase-3 activation, phosphatidylserine exposure and mitochondrial membrane depolarization. PBLs and fibroblasts from CADASIL patients showed a significantly higher response to dRib-induced apoptosis than those of controls. PBLs from CADASIL patients also showed a significantly higher percentage of apoptotic cells than PBLs from controls, even when cultured without dRib. The greater susceptibility of PBLs and fibroblasts from CADASIL patients to dRib-induced apoptosis suggests that NOTCH3 mutations are an important apoptotic trigger. Since PBLs from patients showed higher levels of apoptosis even in the absence of an apoptotic stimulus, cells from CADASIL patients appear to be physiologically prone to apoptotic cell death.

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status., ((c) 2008 Wiley-Liss, Inc.)

Mesenchymal stem cells (MSCs) participate in the wound healing process in mammalians. Adhesion of MSCs to endothelium is a key step in the homing of MSCs circulating in the bloodstream to the sites of injury and inflammation. Because endothelial cells (ECs) may become apoptotic under certain pro-inflammatory conditions, we investigated the effects of pro-inflammatory, TNF-alpha and IL-1 beta, and pro-apoptotic agents, actinomycin D, cycloheximide, okadaic acid, wortmannin, and staurosporine, on human MSCs (hMSCs) adhesion to ECs. Treatment of ECs with pro-apoptotic agents markedly increased adhesion of hMSCs to ECs. This adhesion correlated with reduction of mitochondrial membrane potential, inhibition of NADH dehydrogenases, and release of von Willebrand factor (vWF) by ECs. Treatment of ECs with exogenous vWF also stimulated hMSC adhesion. These data provide evidence that apoptosis of ECs may regulate homing of hMSCs to the sites of tissue injury. These results are consistent with the hypothesis that activation of apoptotic signaling pathways in ECs releases vWF which regulates hMSC adhesion to ECs., ((c) 2008 Wiley-Liss, Inc.)

PLK1 is essential for the maintenance of genomic stability during mitosis. In our study, we found that overexpression of PLK1 was an independent prognostic factor (RR=4.253, p=0.020) and significantly correlated with survivin, an antiapoptotic protein, in esophageal squamous cell carcinoma (ESCC). Reverse transcription-polymerase chain reaction and fluorescence in situ hybridization (FISH) revealed upregulation of PLK1 mRNA and amplification of PLK1 gene, respectively. Depletion of PLK1 activated the intrinsic apoptotic pathway, which was substantiated by loss of mitochondrial membrane potential, reduction of Mcl-1 and Bcl-2 as well as activation of caspase-9. Coimmunoprecipitation and confocal microscopy displayed that PLK1 was associated with survivin and PLK1 depletion led to downregulation of survivin. Cotransfection of survivin constructs could partially reverse PLK1-depletion-induced apoptosis. These data suggest that PLK1 might be a useful prognostic marker and a potential therapeutic target for ESCC. Survivin is probably involved in antiapoptotic function of PLK1., (Copyright (c) 2008 Wiley-Liss, Inc.)

Cells lacking mitochondrial genome (defined as rho(0)) are useful models in studies on cancer, aging, mitochondrial diseases and apoptosis, but several of their functional aspects have been poorly characterized. Using different clones of rho(0) cells derived from the human osteosarcoma line 143B, we have tested the effects of different apoptogenic molecules such as staurosporine (STS), doxorubicin, daunomycin and quercetin, and have analyzed apoptosis, mitochondrial membrane potential (MMP), levels of oxygen free radicals, reduced glutathione (GSH) content, and expression of P-glycoprotein (P-gp). When compared to parental cells, rho(0) cells resulted much less sensitive to apoptosis. MMP was well maintained in rho(0) cells, and remained unchanged after adding apoptogenic agents, and did not change after treatment with molecules able to depolarize mitochondria such as valinomycin. After adding STS, the production of reactive oxygen species was similar in both cell types, but rho(0) cells maintained higher levels of GSH. In rho(0) cells, P-gp was strongly over-expressed both at mRNA and protein level, and its functionality was higher. The resistance to apoptosis of rho(0) cells could be not only due to an increased scavenger capacity of GSH, but also due to a selection of multidrug resistant cells that hyperexpress P-gp., ((c) 2008 International Society for Advancement of Cytometry.)

Ejaculated, density purified, human spermatozoa were exposed to pulsed 900 MHz GSM mobile phone radiation at two specific absorption rate levels (SAR 2.0 and 5.7 W/kg) and compared with controls over time. Change in sperm mitochondrial membrane potential was analysed using flow cytometry. Sperm motility was determined by computer assisted sperm analysis (CASA). There was no effect of pulsed 900 MHz GSM radiation on mitochondrial membrane potential. This was also the case for all kinematic parameters assessed at a SAR of 2.0 W/kg. However, over time, the two kinematic parameters straight line velocity (VSL) and beat-cross frequency (BCF) were significantly impaired (P < 0.05) after the exposure at SAR 5.7 W/kg and no exposure by time interaction was present. This result should not be ascribed to thermal effects, due to the cooling methods employed in the RF chamber and temperature control within the incubator., ((c) 2007 Wiley-Liss, Inc.)

Mitochondrial membrane potential (Deltapsi) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Deltapsi in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluorescent cation tetramethyl rhodamine methyl ester (TMRM), which is sequestered by mitochondria according to their Deltapsi. Cells were visualized by videomicroscopy and the acquired images were processed to generate a mitochondria-specific mask. The latter was superimposed on the original image to allow quantification of TMRM fluorescence. Following validation, our approach revealed that mitochondria with different Deltapsi coexisted within the same cell. Furthermore, our method allowed reproducible detection of small (<10%) reductions in TMRM intensity induced by the complex III inhibitor antimycin A. Mitochondrial uncoupling by p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) greatly reduced mitochondrial TMRM fluorescence. Under these conditions faithful mask calculation and TMRM intensity analysis were still possible using a mitochondria-targeted green fluorescence protein (mitoAcGFP1), expressed in the cells using baculoviral transfection., ((c) 2008 International Society for Analytical Cytology)

Objective: Mutations of the mitofusin 2 gene (MFN2) may account for at least a third of the cases of Charcot-Marie-Tooth disease type 2 (CMT2). This study investigates mitochondrial cellular bioenergetics in MFN2-related CMT2A., Methods: Mitochondrial network morphology and metabolism were studied in cultures of skin fibroblasts obtained from four CMT2A patients harboring novel missense mutations of the MFN2 gene., Results: Although the mitochondrial network appeared morphologically unaltered, there was a significant defect of mitochondrial coupling associated with a reduction of the mitochondrial membrane potential., Interpretation: Our results suggest that the sharply reduced efficacy of oxidative phosphorylation in MFN2-related CMT2A may contribute to the pathophysiology of the axonal neuropathy.

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.